Beneficial Effect of Microinjected Trehalose on the Cryosurvival of Human Oocytes

Fertility and Sterility. Jan, 2002  |  Pubmed ID: 11779606

To determine the effectiveness of trehalose as an intracellular cryoprotectant for the cryopreservation of human oocytes.

Metabolic Pre-conditioning of Cultured Cells in Physiological Levels of Insulin: Generating Resistance to the Lipid-accumulating Effects of Plasma in Hepatocytes

Biotechnology and Bioengineering. Jun, 2002  |  Pubmed ID: 12001167

Understanding the regulation of hepatocyte lipid metabolism is important for several biotechnological applications involving liver cells. During exposure of hepatocytes to plasma, as is the case in extracorporeal bioartificial liver assist devices, it has been reported that hepatic-specific functions, e.g., albumin and urea synthesis and diazepam removal, are dramatically compromised and hepatocytes progressively accumulate cytoplasmic lipid droplets. We hypothesized that the composition of hepatocyte culture medium significantly affects lipid metabolism during subsequent plasma exposure. Rat hepatocytes were cultured in medium containing either physiological (50 microU/mL) or supra-physiological (500 mU/mL) insulin levels for 1 week and then exposed to human plasma supplemented with or without amino acids. We found that insulin's anabolic effects, such as stimulation of triglyceride storage, were carried over from the pre-conditioning to the plasma exposure period. While hepatocytes cultured in high insulin medium accumulated large quantities of triglycerides during subsequent plasma exposure, culture in low insulin medium largely prevented lipid accumulation. Urea and albumin secretion, as well as the ammonia removal rate, were largely unaffected by insulin but increased with amino acid supplementation. Thus, hepatocyte metabolism during plasma exposure can be modulated by medium pre-conditioning and supplements added to plasma.

Storage and Translational Issues in Reparative Medicine

Annals of the New York Academy of Sciences. Jun, 2002  |  Pubmed ID: 12081912

Storage and Translational Issues in Reparative Medicine: Breakout Session Summary

Annals of the New York Academy of Sciences. Jun, 2002  |  Pubmed ID: 12081917

Neutrophil Chemotaxis in Linear and Complex Gradients of Interleukin-8 Formed in a Microfabricated Device

Nature Biotechnology. Aug, 2002  |  Pubmed ID: 12091913

Although a wealth of knowledge about chemotaxis has accumulated in the past 40 years, these studies have been hampered by the inability of researchers to generate simple linear gradients instantaneously and to maintain them at steady state. Here we describe a device microfabricated by soft lithography and consisting of a network of microfluidic channels that can generate spatially and temporally controlled gradients of chemotactic factors. When human neutrophils are positioned within a microchannel, their migration in simple and complex interleukin-8 (IL-8) gradients can be tested. The cells exhibit strong directional migration toward increasing concentrations of IL-8 in linear gradients. Neutrophil migration halts abruptly when cells encounter a sudden drop in the chemoattractant concentration to zero ("cliff" gradient). When neutrophils are challenged with a gradual increase and decrease in chemoattractant ("hill" gradient), however, the cells traverse the crest of maximum concentration and migrate further before reversing direction. The technique described in this paper provides a robust method to investigate migratory cells under a variety of conditions not accessible to study by earlier techniques.

Antiproteolytic Action of Insulin in Burn-injured Rats

The Journal of Surgical Research. Jun, 2002  |  Pubmed ID: 12121712

Negative nitrogen balance is a typical metabolic response to burn injury resulting in decreased muscle mass and activity. Since insulin is an anabolic hormone, using insulin as a prophylactic agent in burned patients has received some attention. The present study was carried out to investigate the systemic effect of insulin on burn injury-induced muscle wasting.

Bioartificial Liver Process Monitoring and Control Systems with Integrated Systems Capability

Tissue Engineering. Jul, 2002  |  Pubmed ID: 12167233

Monitoring and control of a bioartificial liver (BAL) support system have the potential to allow for maximization of device bioactivity and protection of both patient and device from untoward consequences of a complex hemoperfused fluidic process, such as coagulation, leakage, or decreased metabolic output. In this work, an integrated embedded systems controller and associated experimental platform were developed to allow for simultaneous monitoring and control of the physical environment of the BAL support system to ensure optimal and sustained hepatic metabolic function and to allow for simplified recording of experimental data. The user interface and core embedded system kernel were developed with rapid prototyping software tools and allowed for operation of easily modified user interface panels. BAL environment monitoring consisted of real-time recording of ambient and reactor temperatures, reactor inlet pressure, the presence of bubbles in the prereactor inlet tubing, and aqueous oxygen tension. Environmental parameters under direct real-time control included reactor inlet flow rate, ambient temperature, and adaptive control of flow rate in response to changes in either inlet pressure or outlet oxygen tension. Use of embedded system integration techniques will facilitate subsequent BAL studies that are dependent on scale-up of reactor size and number, fluidic complexity, and the degree of parallelism such as large animal studies and, ultimately, human clinical studies. In addition, further studies of the effects of flow rate, shear, oxygenation and metabolic substrate on real-time cellular respiration can be pursued with the use of real-time ruthenium oxymetry, as described in this article.

A Microfabrication-based Dynamic Array Cytometer

Analytical Chemistry. Aug, 2002  |  Pubmed ID: 12199564

We have developed a microfabricated device for use in parallel luminescent single-cell assays that can sort populations upon the basis of dynamic functional responses to stimuli. This device is composed of a regular array of noncontact single-cell traps. These traps use dielectrophoresis to stably confine cells and hold them against disrupting fluid flows. Using quantitative modeling, we have designed traps with a novel asymmetric extruded-quadrupole geometry. This new trap can be physically arrayed and electrically addressed, enabling our cytometer. Situating an array of these traps in a microchannel, we have introduced cells into the array and demonstrated observation of fluorescent dynamic responses followed by sorting. Such a device has potential for use in investigating functional processes, as revealed by temporal behavior, in large numbers of single cells.

Cryopreservation of Fetal Skin is Improved by Extracellular Trehalose

Cryobiology. Jun, 2002  |  Pubmed ID: 12237087

In this study, we tested a non-permeating cryoprotectant, trehalose, in combination with dimethyl sulfoxide (Me(2)SO) in the cryopreservation of human fetal skin and compared it to Me(2)SO and glycerol, protocols that are routinely used by skin banks. The viability of fetal skin from four groups (fresh, and cryopreserved with glycerol, Me(2)SO, or trehalose/Me(2)SO) were evaluated using an in vitro membrane integrity assay and by transplantation to immunodeficient mice. The membrane integrity assay showed a 90% integrity in fresh, unfrozen fetal skin. The number of intact cells dropped to 23 and 44% in fetal skin cryopreserved with glycerol and Me(2)SO, respectively. When trehalose was added to the cryopreservation medium containing Me(2)SO, the membrane integrity rose to 65%. When transplanted to immunodeficient mice, fetal skin cryopreserved with trehalose/Me(2)SO showed a graft performance indistinguishable from fresh unfrozen fetal skin and strikingly better graft take than that of fetal skin cryopreserved with Me(2)SO or glycerol only. These results suggest that cryopreservation protocols routinely used the skin banks can be improved by combining sugars such as trehalose with a permeating cryoprotectant.

The Glass Transition Temperature of Mixtures of Trehalose and Hydroxyethyl Starch

Cryobiology. Jun, 2002  |  Pubmed ID: 12237095

Although mixtures of HES and sugars are used to preserve cells during freezing or drying, little is known about the glass transition of HES, or how mixtures of HES and sugars vitrify. These difficulties may be due to the polydispersity between HES samples or differences in preparation techniques, as well as problems in measuring the glass transition temperature (T(g)) using differential scanning calorimetry (DSC). In this report, we examine the T(g) of mixtures of HES and trehalose sugar with <1% moisture content using DSC measurements. By extrapolating these measurements to pure HES using the Gordon-Taylor and Fox equations, we were able to estimate the T(g) of our HES sample at 44 degrees C. These results were additionally confirmed by using mixtures of glucose-HES which yielded a similar extrapolated T(g) value. Our approach to estimating the glass transition temperature of HES may be useful in other cases where glass transitions are not easily identified.

Interferon Gamma Modulates Trauma-induced Muscle Wasting and Immune Dysfunction

Annals of Surgery. Nov, 2002  |  Pubmed ID: 12409672

To test the effect of burn injury in mice congenitally deficient in interferon gamma (IFN-gamma) and as well as in wild-type animals treated with IFN-gamma neutralizing antibody.

Overview of the Alliance for Cellular Signaling

Nature. Dec, 2002  |  Pubmed ID: 12478301

The Alliance for Cellular Signaling is a large-scale collaboration designed to answer global questions about signalling networks. Pathways will be studied intensively in two cells--B lymphocytes (the cells of the immune system) and cardiac myocytes--to facilitate quantitative modelling. One goal is to catalyse complementary research in individual laboratories; to facilitate this, all alliance data are freely available for use by the entire research community.

Osmometric Behavior of Mouse Oocytes in the Presence of Different Intracellular Sugars

Cryobiology. Oct, 2002  |  Pubmed ID: 12482383

In order to successfully cryopreserve oocytes using low concentrations of intracellular sugars, it is important to characterize their osmotic response in the presence of these intracellular sugars. In the present study, murine (B6D2F1) oocytes were microinjected with 0.8M glucose, trehalose or stachyose solutions to achieve an intracellular concentration equivalent to 0.1M, and then exposed to hypertonic solutions of increasing strength by supplementing an isotonic solution with 0.2, 0.3, 0.5, and 0.7 M of trehalose. Analysis of volumetric response of microinjected oocytes showed that the oocytes behaved as ideal osmometers in the presence of intracellular sugars and satisfied the Boyle van't Hoff relationship. Extrapolation of the osmotically inactive fraction (V macro b) from the Boyle van't Hoff relationship yielded values of 0.188+/-0.028, 0.212+/-0.042, 0.197+/-0.044, and 0.211+/-0.042 for control, glucose, trehalose and stachyose-injected oocytes, respectively. The present data revealing osmometric behavior of mouse oocytes in the presence of different intracellular sugars are important for the optimization of cryopreservation protocols using sugars.

Bioengineering of Liver Assist Devices

Journal of Hepato-biliary-pancreatic Surgery. 2002  |  Pubmed ID: 12658402

Over 30 000 patients die annually in the United States from liver failure. In fulminant hepatic failure, a clinical syndrome associated with high mortality, orthotopic liver transplantation is the primary therapeutic option for patients not responding to supportive therapy. However, the persistent scarcity of donor organs has limited this therapeutic modality, resulting in a continued increase in the number of patients who die waiting for a donor liver. An extracorporeal bioartificial liver device could provide vital support to a liver failure patient until a donor liver was available or until the patient's own liver regenerated. Although it is unclear which liver-specific functions must be provided by such a device to be effective, a constant challenge has been to obtain stable, well-differentiated, and normally functioning hepatocytes that can be cultured at high cell densities. Many of the devices currently undergoing clinical trials are limited by designs which are prone to substrate limitations, resulting in compromised hepatocyte function. In devices that avoid substrate limitations, hepatocyte functions can be optimized, thereby leading to increased device efficiency. In this overview, the authors describe the critical issues involved in bioartificial liver development and discuss their experiences in hepatocyte culture optimization within the context of a microchannel, flat-plate bioartificial liver device with an internal membrane oxygenator.

Quantitative Microinjection of Trehalose into Mouse Oocytes and Zygotes, and Its Effect on Development

Cryobiology. Apr, 2003  |  Pubmed ID: 12686202

Sugars such as trehalose are effectively used by various organisms as protective agents to undergo anhydrobiosis and cryobiosis. The objective of this study was first to establish a method for quantitative delivery of trehalose as a model sugar into oocytes, and then to evaluate its effect on development of mouse zygotes. To this end, a quantitative microinjection technique was developed using volumetric response of microdroplets suspended in dimethylpolysilaxene. To verify accuracy of this technique, both microdroplets and oocytes were microinjected with fluorophore-labeled dextran. Thereafter, injection volumes were calculated from fluorescence intensity, and volumetric responses of both microdroplets and oocytes. Comparison of calculated injection volumes revealed that this technique reflects microinjection into oocytes with pL-accuracy. The next series of experiments focused on toxicity of injection buffers (i.e., 10mM Tris and 15mM Hepes) and trehalose. Microinjection of Hepes and Tris buffer in the presence of 0.1M trehalose resulted in blastocyst rates of 86 and 72%, respectively, without a significant difference when compared to controls (86%). In subsequent experiments, Hepes was used as the injection buffer, and embryonic development of zygotes was studied as a function of intracellular trehalose concentrations. Microinjection of trehalose up to 0.15M resulted in development to blastocyst stage similar to controls (85 and 87%, respectively) while the blastocyst rate was significantly decreased (43%) in the presence of 0.20M intracellular trehalose. When transferred to foster mothers, trehalose-injected zygotes (0.1M) implanted and developed to day 16 fetuses similar to controls, healthy pups were born. The findings of this study suggest that trehalose at effective intracellular concentrations does not impair development of mouse zygotes.

Efficacy of an Extracorporeal Flat-plate Bioartificial Liver in Treating Fulminant Hepatic Failure

The Journal of Surgical Research. May, 2003  |  Pubmed ID: 12842448

Fulminant hepatic failure is associated with a high mortality rate. Orthotopic liver transplantation is the only established treatment for patients who do not respond to medical management. A major limitation of this treatment is a shortage of donor organs, resulting in many patients dying while waiting for a transplant. An extracorporeal bioartificial liver (BAL) has the potential to provide temporary support for patients with fulminant hepatic failure (FHF) and for patients awaiting orthotopic liver transplantation. We developed a flat-plate BAL with an internal membrane oxygenator in which porcine hepatocytes were cultured as a monolayer.

Desiccation Tolerance of Spermatozoa Dried at Ambient Temperature: Production of Fetal Mice

Biology of Reproduction. May, 2003  |  Pubmed ID: 12606475

Long-term preservation of mouse sperm by desiccation is economically and logistically attractive. The current investigation is a feasibility study of the preservation of mouse sperm by convective drying in an inert gas (nitrogen). Mouse sperm from the B6D2F1 strain isolated in an EGTA-supplemented Tris-HCl buffer were dried using three different drying rates and were stored for 18-24 h at 4 degrees C. The mean final moisture content was <5% for all the protocols. After intracytoplasmic sperm injection (ICSI), the mean blastocyst formation rates were 64%, 58%, and 35% using the rapid-, moderate-, and slow-drying protocols, respectively. The slow-drying protocol resulted in a rate of development significantly lower than that observed using rapid- and moderate-drying protocols and indicated that a slower drying rate may be detrimental to the DNA integrity of mouse sperm. The transfer of 85 two- or four-cell embryos that were produced using rapidly desiccated sperm resulted in 11 fetuses (13%) on Day 15 compared with the production of 34 fetuses (40%) produced using the transfer of 86 two- or four-cell embryos that were produced using fresh sperm (P < 0.05). The results demonstrate the feasibility of using a convective drying protocol for the successful desiccation of mouse sperm and identifies some of the important parameters required for optimization of the procedure.

Influence of Insulin Therapy on Burn Wound Healing in Rats

The Journal of Surgical Research. Feb, 2003  |  Pubmed ID: 12643849

Insulin is proposed as a therapy for suppressing muscle wasting after burn trauma although the long-term effects of this therapy on wound healing are not yet known. The present study was designed to investigate the effect of systemically administered insulin therapy on burn wound healing.

Measurement of Trehalose Loading of Mammalian Cells Porated with a Metal-actuated Switchable Pore

Biotechnology and Bioengineering. Jun, 2003  |  Pubmed ID: 12652476

Efforts to improve the tolerance of mammalian cells to desiccation have focused on the role that sugars have in protecting cells from lethal injury. Among the key determinants of desiccation tolerance is the intracellular trehalose concentration, and thus quantifying the amount and rate of trehalose accumulation has now become very critical to the success of these desiccation approaches. We introduced trehalose into 3T3 fibroblasts, human keratinocytes, and rat hepatocytes using a genetically engineered mutant of the pore-forming alpha-hemolysin from Staphylococcus aureus. Manipulating the extracellular Zn(2+) concentration selectively opens and closes this pore ( approximately 2 nm) and enables controlled loading of cells with sugars. We quantified intracellular trehalose using gas chromatography-mass spectroscopy (GC-MS) to examine the trimethylsilyl derivative of intracellular trehalose. Using the GC-MS method, we demonstrate that the switchable characteristics of H5 alpha-hemolysin permit controlled loading of the high concentrations of trehalose (up to 0.5 M) necessary for engineering desiccation tolerance in mammalian cells.

Long-term Function of Cryopreserved Rat Hepatocytes in a Coculture System

Cell Transplantation. 2004  |  Pubmed ID: 15129765

The goal of this study was to investigate postpreservation long-term function of cryopreserved primary rat hepatocytes using the hepatocyte/3T3-J2 fibroblast coculture system. The long-term function of thawed hepatocytes cocultured with fibroblasts was evaluated and compared with hepatocytes cultured without fibroblasts. Fresh isolated primary rat hepatocytes were frozen at a controlled rate (-1 degrees C/min) up to -80 degrees C, and then stored in liquid nitrogen for up to 90 days. Thawed hepatocytes were thereafter cocultured with 3T3-J2 murine fibroblasts and cocultivation was monitored for 14 days. The viability of fresh isolated hepatocytes was 91.4%, and that of cryopreserved hepatocytes was 82.1%. Cellular morphology and polarity, which were determined by the localization of actin filaments and connexin-32, were successfully maintained in cryopreserved hepatocytes following cryopreservation. Albumin and urea synthesis reached the maximum level and became stable after day 7 in coculture in both fresh and cryopreserved hepatocytes. Urea synthesis of cryopreserved hepatocytes was maintained 89.0% of nonfrozen fresh control, and albumin production of cryopreserved hepatocytes was 63.7% of control in coculture. Cytochrome P450 activity, which was measured by deethylation of ethoxyresorufin, was also maintained in cryopreserved hepatocytes at 88.6% of nonfrozen fresh control in coculture. The retention of synthetic and detoxification activities was verified to be well preserved during extended low-temperature storage (90 days). Both fresh control and cryopreserved hepatocytes cultured without fibroblast did not retain their synthetic and detoxification functions in long-term culture. These data illustrate that, through the utilization of our cryopreservation procedure, primary hepatocyte function was successfully maintained when placed into coculture configuration following thawing.

Cryopreservation of Stem Cells Using Trehalose: Evaluation of the Method Using a Human Hematopoietic Cell Line

Stem Cells and Development. Jun, 2004  |  Pubmed ID: 15186725

While stem cell cryopreservation methods have been optimized using dimethylsulfoxide (DMSO), the established techniques are not optimal when applied to unfertilized human embryonic cells. In addition, important questions remain regarding the toxicity and characteristics of DMSO for treatment of stem cells for clinical use. The objective of this study was to establish an optimal method for cryopreservation of stem cells using low concentrations (0.2 M) of trehalose, a nontoxic disaccharide of glucose, which possesses excellent protective characteristics, in place of current methods utilizing high concentrations (1-2 M) of DMSO. A human hematopoietic cell line was used in this investigation as a surrogate for human stem cells. Trehalose was loaded into cells using a genetically engineered mutant of the pore-forming protein alpha-hemolysin from Staphylococcus aureus. This method results in a nonselective pore equipped with a metal-actuated switch that is sensitive to extracellular zinc concentrations, thus permitting controlled loading of trehalose. Preliminary experiments characterized the effects of poration on TF-1 cells and established optimal conditions for trehalose loading and cell survival. TF-1 cells were frozen at 1 degrees C/min to -80 degrees C with and without intra- and extracellular trehalose. Following storage at -80 degrees C for 1 week, cells were thawed and evaluated for viability, differentiation capacity, and clonogenic activity in comparison to cells frozen with DMSO. Predictably, cells frozen without any protective agent did not survive freezing. Colony-forming units (CFU) generated from cells frozen with intra- and extracellular trehalose, however, were comparable in size, morphology, and number to those generated by cells frozen in DMSO. There was no observable alteration in phenotypic markers of differentiation in either trehalose- or DMSO-treated cells. These data demonstrate that low concentrations of trehalose can protect hematopoietic progenitors from freezing injury and support the concept that trehalose may be useful for freezing embryonic stem cells and other primitive stem cells for therapeutic and investigational use.

Dynamic Gene Expression Profiling Using a Microfabricated Living Cell Array

Analytical Chemistry. Jul, 2004  |  Pubmed ID: 15253648

We describe the development of a microfluidic platform for continuous monitoring of gene expression in live cells. This optically transparent microfluidic device integrates high-throughput molecular stimulation with nondestructive monitoring of expression events in individual living cells, hence, a living cell array (LCA). Several concentrations of a soluble molecular stimulus are generated in an upstream microfluidic network and used to stimulate downstream reporter cells, each containing a green fluorescence reporter plasmid for a gene of interest. Cellular fluorescence is continuously monitored and quantified to infer the expression dynamics of the gene being studied. We demonstrate this approach by profiling the activation of the transcription factor NF-kappaB in HeLa S3 cells in response to varying doses of the inflammatory cytokine TNF-alpha. The LCA platform offers a unique opportunity to simultaneously control dynamic inputs and measure dynamic outputs from adherent mammalian cells in a high-throughput fashion. This approach to profiling expression dynamics, in conjunction with complementary techniques such as DNA microarrays, will help provide a more complete picture of the dynamic cellular response to diverse soluble stimuli.

Use of Sugars in Cryopreserving Human Oocytes

Reproductive Biomedicine Online. Aug, 2004  |  Pubmed ID: 15333248

In the last 20 years, a worldwide effort to cryopreserve oocytes has resulted in 40 infants and approximately 50 ongoing pregnancies being reported. While the ability to freeze human embryos has become a standard of practice in assisted reproductive technologies, obtaining reliable techniques for oocyte cryopreservation has been more difficult. The unique properties of the mature oocyte, such as the meiotic stage with sensitive spindle structure as well as the large cell volume, are responsible for the limited success obtained to date. There have been two approaches to cryopreserving the oocyte: (i) slow freeze-rapid thaw, and (ii) vitrification protocols with rapid cooling-rapid warming. Both methods have incorporated sugars (sucrose) as a beneficial non-permeating extracellular cryoprotectant. Studies of organisms that survive extreme conditions of freezing/dehydration have demonstrated the ability to accumulate intracellular sugars to afford protection and survival. A novel technique using microinjection of sugars into the oocyte for cryopreservation has been developed as an alternative approach to external addition of sugars. Freezing the human oocyte has been a challenging goal; however, developing research and efforts will, in the near future, provide women with an important option for their reproductive health.

Single-cell Chemical Lysis in Picoliter-scale Closed Volumes Using a Microfabricated Device

Analytical Chemistry. Oct, 2004  |  Pubmed ID: 15481964

Investigating the intracellular contents of single cells is essential for understanding physiologic and pathologic processes at the cellular level. While existing protocols for cell lysis and sample preparation work well for larger samples, scaling to a single-cell level is challenging because of unavoidable analyte dilution and losses. Thus, we are proposing a microfabricated device for the controlled handling and mixing of picoliter cell suspension and lysis solution volumes. Cells and fluids are independently isolated in two microchambers of 25-pL volumes using the geometry of the microchannels and the coordinated action of four on-chip thermopneumatic actuators. Virtual walls formed by liquid-air interfaces in the hydrophobic capillary separate the two volumes, which are subsequently allowed to mix after drawing the air out of the capillary connecting the two microchambers. Following cell lysis, a limited and stable dilution of intracellular components is achieved, simplifying the requirements for subsequent analysis. Two assays at single-cell level, one for direct estimation of the intracellular concentration of a soluble dye and the other for indirect evaluation of intracellular quantities of insoluble actin, demonstrate the use of the microfabricated device for single-cell assays.

Hepatic Tissue Engineering for Adjunct and Temporary Liver Support: Critical Technologies

Liver Transplantation : Official Publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation Society. Nov, 2004  |  Pubmed ID: 15497161

The severe donor liver shortage, high cost, and complexity of orthotopic liver transplantation have prompted the search for alternative treatment strategies for end-stage liver disease, which would require less donor material, be cheaper, and less invasive. Hepatic tissue engineering encompasses several approaches to develop adjunct internal liver support methods, such as hepatocyte transplantation and implantable hepatocyte-based devices, as well as temporary extracorporeal liver support techniques, such as bioartificial liver assist devices. Many tissue engineered liver support systems have passed the "proof of principle" test in preclinical and clinical studies; however, they have not yet been found sufficiently reliably effective for routine clinical use. In this review we describe, from an engineering perspective, the progress and remaining challenges that must be resolved in order to develop the next generation of implantable and extracorporeal devices for adjunct or temporary liver assist.

Continuous Flow Microfluidic Device for Rapid Erythrocyte Lysis

Analytical Chemistry. Nov, 2004  |  Pubmed ID: 15516115

Leukocyte isolation from whole blood to study inflammation requires the removal of contaminating erythrocytes. Leukocytes, however, are sensitive to prolonged exposure to hyper/hypoosmotic solutions, temperature changes, mechanical manipulation, and gradient centrifugation. Even though care is taken to minimize leukocyte activation and cell loss during erythrocyte lysis, it is often not possible to completely avoid it. Most procedures for removal of contaminating erythrocytes from leukocyte preparations are designed for bulk processing of blood, where the sample is manipulated for longer periods of time than necessary at the single-cell level. Ammonium chloride-mediated lysis is the most commonly used method to obtain enriched leukocyte populations but has been shown to cause some activation and selective loss of certain cell types. The leukocyte yield and subsequent activation status of residual leukocytes after NH(4)Cl-mediated lysis have been shown to depend on the time of exposure to the lysis buffer. We have developed a microfluidic lysis device that deals with erythrocyte removal at nearly the single-cell level. We can achieve complete lysis of erythrocytes and approximately 100% recovery of leukocytes where the cells are exposed to an isotonic lysis buffer for less than 40 s, after which the leukocytes are immediately returned to physiological conditions. Theoretically, this process can be made massively parallel to process several milliliterss of whole blood to obtain a pure leukocyte population in less than 15 min.

Isothermal Desiccation and Vitrification Kinetics of Trehalose-dextran Solutions

Langmuir : the ACS Journal of Surfaces and Colloids. Jun, 2004  |  Pubmed ID: 15986695

The promise of dried state preservation is based on the hypothesis that lowering molecular mobility to halt chemical reaction and deterioration rates is the primary factor for the long-term stability of the dried specimen. In this research, the feasibility of utilizing isothermal, isobaric vitrification as an economical alternative to the preservation technologies currently in use (mainly, cryopreservation and lyophilization) is explored. Desiccation and vitrification kinetics of model trehalose and trehalose-dextran systems were examined using gravimetric analysis, modulated differential scanning calorimetry, and X-ray crystallography. It was shown that vitrification can be achieved isothermally without crystallization and that vitrification of trehalose solutions can be significantly accelerated by incorporating high-molecular-weight dextrans. Additionally, it was shown that, for the same water content, the glass transition temperature of the trehalose-dextran solution is significantly higher than that of the binary trehalose solution, making the glassy state achievable and storage feasible.

Effect of Flow and Surface Conditions on Human Lymphocyte Isolation Using Microfluidic Chambers

Langmuir : the ACS Journal of Surfaces and Colloids. Dec, 2004  |  Pubmed ID: 15595794

Phenotypically pure subpopulations of lymphocytes can provide valuable insights into the immune response to injury and disease. The isolation of these subpopulations presents unique challenges, particularly when preprocessing incubation to attach fluorescent or antibody tags is to be minimized. This paper examines the separation of T and B lymphocytes from mixtures using microfluidic chambers coated with antibodies, focusing on flow conditions and surface chemistry. The adhesion of both cell types decreases as shear stress increases irrespective of the surface chemistry. The incorporation of poly(ethylene glycol) chains along with the antibodies on the chamber surface is shown to significantly improve the reproducibility of cell adhesion and is thus an important part of the overall system design. Furthermore, this technique is shown to be an effective way of isolating highly pure subpopulations of lymphocytes from model mixtures, even when the target cell concentration is low.

Designing a Hepatocellular Microenvironment with Protein Microarraying and Poly(ethylene Glycol) Photolithography

Langmuir : the ACS Journal of Surfaces and Colloids. Apr, 2004  |  Pubmed ID: 15875819

In this study, robotic protein printing was employed as a method for designing a cellular microenvironment. Protein printing proved to be an effective strategy for creating micropatterned co-cultures of primary rat hepatocytes and 3T3 fibroblasts. Collagen spots (ca. 170 microm in diameter) were printed onto amino-silane- and glutaraldehyde-modified glass slides. Groups of 15-20 hepatocytes attached to collagen regions in a highly selective manner forming cell clusters corresponding in size to the printed collagen domains. Fibroblasts, seeded onto the same surface, adhered and spread around arrays of hepatocyte islands creating a heterotypic environment. The co-cultured hepatocytes produced and maintained high levels of liver-specific biomarkers, albumin and urea, over the course of 2 weeks. In addition, protein printing was combined with poly(ethylene glycol) photolithography to define intercellular contacts within the clusters of hepatocytes residing on individual collagen islands. Glass slides, treated with 3-acryloxypropyl trichlorosilane and imprinted with 170 m diameter collagen spots, were micropatterned with a high-density array of 30 microm x 30 microm poly(ethylene glycol) (PEG) wells. As a result, discrete groups of ca. 9 PEG microwells became functionalized with the cell-adhesive ligand. When exposed to micropatterned surfaces, hepatocytes interacted exclusively with collagen-modified regions, attaching and becoming confined at a single-cell level within the hydrogel wells. Micropatterning strategies proposed here will lead to greater insights into hepatocellular behavior and will benefit the fields of hepatic tissue engineering and liver biology.

Progressive Elimination of Microinjected Trehalose During Mouse Embryonic Development

Reproductive Biomedicine Online. Apr, 2005  |  Pubmed ID: 15901459

Recently, sugars such as trehalose have been introduced into mammalian cells by overcoming the permeability barrier of cell membranes, and have provided improved tolerance against stresses associated with freezing and drying. However, the fate of the intracellular sugars has remained an open question. To address this issue, mouse oocytes were microinjected with 0.1 mol/l trehalose, and intracellular trehalose and glucose concentrations were determined during embryonic development using a high performance liquid chromatography and pulsed amperometric detection protocol. Trehalose was not detected in non-injected controls at any stage of development. In the microinjection group, the amount of intracellular trehalose progressively decreased as embryos developed. There was a corresponding increase in intracellular glucose concentration at the two-cell stage, suggesting cleavage of trehalose to two glucose molecules. In summary, this study presents a simple, highly sensitive protocol to determine intracellular sugars. The data reveal rapid elimination of microinjected trehalose during embryonic development. These findings have implications for designing osmolarity-optimized culture media for sugar-injected oocytes.

Mouse Sperm Desiccated and Stored in Trehalose Medium Without Freezing

Biology of Reproduction. Oct, 2005  |  Pubmed ID: 15930320

Mouse sperm with and without trehalose were desiccated under nitrogen gas and stored at 4 degrees C and 22 degrees C. After rehydration, sperm were injected into oocytes using intracytoplasmic sperm injection and embryonic development was followed. Sperm were dried for 5.0, 6.25, or 7.5 min, stored at 22 degrees C for 1 wk with and without trehalose. The percentages of blastocysts that developed from sperm with trehalose were 51%, 31%, and 20%, respectively, which was significantly higher than sperm without trehalose (10%, 3%, and 5%, respectively). Desiccation and storage in medium with trehalose significantly increased sperm developmental potential compared to medium without trehalose. Sperm dried for 5 min produced more blastocysts than sperm dried for 6.25 or 7.5 min. When sperm were dried in trehalose for 5 min and stored for 1 wk, 2 wk, 1 mo, or 3 mo at 4 degrees C, the percentages of blastocysts were 73%, 84%, 63%, and 39%; whereas those stored at 22 degrees C for 1 wk, 2 wk, or 1 mo were significantly lower (53%, 17%, and 6%, respectively). Embryos from sperm partially desiccated in trehalose for 5 min and stored at 4 degrees C for 1 or 3 mo were transferred to 10 pseudopregnant recipients. Implantation rates were 81% and 48%; live fetuses were 26% and 5%, respectively. One of the recipients delivered three live fetuses. The results show that trehalose has a significant beneficial effect in preserving the developmental potential of mouse sperm following partial desiccation and storage at temperatures above freezing.

Development of a Microfabricated Cytometry Platform for Characterization and Sorting of Individual Leukocytes

Lab on a Chip. Jan, 2005  |  Pubmed ID: 15616737

Organizing leukocytes into high-density arrays makes these cells amenable to rapid optical characterization and subsequent sorting, pointing to clinical and basic science applications. The present paper describes development of a cytometry platform for creating high-density leukocyte arrays and demonstrates retrieval of single cells from the array. Poly(ethylene glycol)(PEG) photolithography was employed to fabricate arrays of microwells composed of PEG hydrogel walls and glass attachment pads 20 microm x 20 microm and 15 microm x 15 microm in size. PEG micropatterned glass surfaces were further modified with cell-adhesive ligands, poly-L-lysine, anti-CD5 and anti-CD19 antibodies, in order to engineer specific cell-surface interactions within the individual wells. Localization of the fluorescently-labeled proteins in the glass attachment pads of PEG microwells was visualized by fluorescence microscopy. Glass slides micropatterned with PEG and cell-adhesive ligands were exposed to T-lymphocytes for 30 min. These anchorage-independent cells became selectively captured in the ligand-modified microwells forming high-density cell arrays. Cell occupancy in the microwells was found to be antibody-dependent, reaching 94.6 +/- 2.3% for microwells decorated with T-cell specific anti-CD5 antibodies. Laser capture microdissection (LCM) was investigated as a method for sorting cells from the array and retrieval of single selected cells was demonstrated.

Cryopreservation of Isolated Primary Rat Hepatocytes: Enhanced Survival and Long-term Hepatospecific Function

Annals of Surgery. Jan, 2005  |  Pubmed ID: 15622000

To investigate the long-term effect of cryopreservation on hepatocyte function, as well as attempt to improve cell viability and function through the utilization of the hypothermic preservation solution, HypoThermosol (HTS), as the carrier solution.

Cryopreservation of Starfish Oocytes

Cryobiology. Feb, 2005  |  Pubmed ID: 15710368

Research from many laboratories over the past several decades indicates that invertebrate oocytes and eggs are extraordinarily difficult to freeze. Since starfish oocytes, eggs, and embryos are an important cell and developmental biology model system, there is great interest to cryopreserve these cells. Previous starfish oocyte cryopreservation studies using slow cooling protocols revealed that these cells are highly sensitive to osmotic stress and form intracellular ice at very high sub-zero temperatures, suggesting that common freezing methodologies may not prove useful. We report here that a short exposure to 1.5 M Me2SO/1 M trehalose in hypotonic salt solution followed by ultra-rapid cooling to cryogenic temperatures allows starfish oocytes to be cryopreserved with the average survival rate of 34% when normalized to control oocytes that were exposed to CPA, but not frozen. On average, 51% of the oocytes in 77% of the batches of frozen oocytes underwent meiotic maturation in response to the starfish maturation hormone, 1-methyladenine. In one experiment, eggs developing from thawed oocytes were capable of being fertilized and two developed into embryos. These data suggests that successful cryopreservation of starfish oocytes is possible, but will need further refinement to increase the numbers of fully competent embryos.

Analysis of Desiccation and Vitrification Characteristics of Carbohydrate Films by Shear-wave Resonators

Langmuir : the ACS Journal of Surfaces and Colloids. Mar, 2005  |  Pubmed ID: 15779957

Desiccated state preservation of mammalian cells and tissues in the presence of carbohydrates has started to show promise in the last two decades. Certain carbohydrates play a major role in preservation by reducing molecular mobility in the desiccated state. In this communication, the feasibility of utilizing shear-wave resonators to collect real-time molecular mobility information during desiccation and vitrification of carbohydrate based thin films was demonstrated. Simultaneous quartz crystal microbalance experimentation and optical imaging were utilized to determine the conditions for thin film formation and the vitrification kinetics of certain carbohydrate solutions of biological importance. Using the technique presented here, it was possible to gain insight into the vitrification characteristics of carbohydrate solutions establishing the basics for future research with quantitative analysis of film properties and experimentation with live mammalian cells.

Application of Genome-wide Expression Analysis to Human Health and Disease

Proceedings of the National Academy of Sciences of the United States of America. Mar, 2005  |  Pubmed ID: 15781863

The application of genome-wide expression analysis to a large-scale, multicentered program in critically ill patients poses a number of theoretical and technical challenges. We describe here an analytical and organizational approach to a systematic evaluation of the variance associated with genome-wide expression analysis specifically tailored to study human disease. We analyzed sources of variance in genome-wide expression analyses performed with commercial oligonucleotide arrays. In addition, variance in gene expression in human blood leukocytes caused by repeated sampling in the same subject, among different healthy subjects, among different leukocyte subpopulations, and the effect of traumatic injury, were also explored. We report that analytical variance caused by sample processing was acceptably small. Blood leukocyte gene expression in the same individual over a 24-h period was remarkably constant. In contrast, genome-wide expression varied significantly among different subjects and leukocyte subpopulations. Expectedly, traumatic injury induced dramatic changes in apparent gene expression that were greater in magnitude than the analytical noise and interindividual variance. We demonstrate that the development of a nation-wide program for gene expression analysis with careful attention to analytical details can reduce the variance in the clinical setting to a level where patterns of gene expression are informative among different healthy human subjects, and can be studied with confidence in human disease.

Microfabricated Grooved Substrates As Platforms for Bioartificial Liver Reactors

Biotechnology and Bioengineering. Jun, 2005  |  Pubmed ID: 15834948

An extracorporeal bioartificial liver device has the potential to provide temporary hepatic support for patients with liver failure. Our goal was to optimize the flow environment for the cultured hepatocytes in a flat-plate bioreactor, specifically focusing on oxygen delivery using high medium flow rates while reducing the detrimental effects of the resulting shear stresses. We used photolithographic techniques to fabricate microgrooves onto the underlying glass substrate. The microgrooves, perpendicular to the axial flow direction, protected the hepatocytes from the shear stress induced by the flowing medium. Using finite element analysis, we found that the velocity gradient change near the cell surface (i.e., bottom of the grooves) was smaller than that near the top surface of the flow channel, indicating that the grooves would provide protection to the attached cells from the mechanical effects of the flowing medium. We also determined that the shear stress at the cell surface could be reduced by as much as 30 times (channel height of 100 microm) in the grooved-substrate (0.5 dyn/cm(2)) bioreactor compared to the flat-substrate (15 dyn/cm(2)) bioreactor for a medium flow rate of 4.0 mL/min. Albumin and urea synthesis rates of hepatocytes cocultured with 3T3-J2 fibroblasts remained stable over 5 days of perfusion in the grooved-substrate bioreactor, whereas in the flat-substrate bioreactor they decreased over the same time period. These studies indicate that under "high" flow conditions the microgrooved-substrate in the bioreactor can decrease the detrimental effects of shear stress on the hepatocytes while providing adequate oxygenation, thereby resulting in stable liver-specific function.

Cryo-injury and Biopreservation

Annals of the New York Academy of Sciences. Dec, 2005  |  Pubmed ID: 16533923

Mammalian cells appear to be naturally tolerant to cold temperatures, but the formation of ice when cells are cooled leads to a variety of damaging effects. The study of cryo-injury, therefore, becomes the study of when and how ice is formed both inside and outside the cell during cooling. Protectant chemicals are used to control or prevent ice formation in many preservation protocols, but these chemical themselves tend to be damaging. Cooling and warming rates also strongly affect the amount and location of ice that is formed. Through careful modification of these parameters successful cold preservation techniques for many cell types have been developed, but there are many more cell types that have defied preservation techniques, and the extension of cell-based techniques to tissues and whole organs has been very limited. There are many aspects to the damaging effects of ice in cells that are still poorly understood. In this brief article we review our current understanding of cellular injury and highlight the aspects of cellular injury during cryopreservation that are still poorly understood.

Blood-on-a-chip

Annual Review of Biomedical Engineering. 2005  |  Pubmed ID: 16004567

Accurate, fast, and affordable analysis of the cellular component of blood is of prime interest for medicine and research. Yet, most often sample preparation procedures for blood analysis involve handling steps prone to introducing artifacts, whereas analysis methods commonly require skilled technicians and well-equipped, expensive laboratories. Developing more gentle protocols and affordable instruments for specific blood analysis tasks is becoming possible through the recent progress in the area of microfluidics and lab-on-a-chip-type devices. Precise control over the cell microenvironment during separation procedures and the ability to scale down the analysis to very small volumes of blood are among the most attractive capabilities of the new approaches. Here we review some of the emerging principles for manipulating blood cells at microscale and promising high-throughput approaches to blood cell separation using microdevices. Examples of specific single-purpose devices are described together with integration strategies for blood cell separation and analysis modules.

Enrichment Using Antibody-coated Microfluidic Chambers in Shear Flow: Model Mixtures of Human Lymphocytes

Biotechnology and Bioengineering. Sep, 2005  |  Pubmed ID: 16037988

Isolation of phenotypically-pure cell subpopulations from heterogeneous cell mixtures such as blood is a difficult yet fundamentally important task. Current techniques such as fluorescent activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) require pre-incubation with antibodies which lead to processing times of at least 15-60 min. In this study, we explored the use of antibody-coated microfluidic chambers to negative deplete undesired cell types, thus obtaining an enriched cell subpopulation at the outlet. We used human lymphocyte cell lines, MOLT-3 and Raji, as a model system to examine the dynamic cell binding behavior on antibody coated surfaces under shear flow. Shear stress ranging between 0.75 and 1.0 dyn/cm2 was found to provide most efficient separation. Cell adhesion was shown to follow pseudo-first order kinetics, and an anti-CD19 coated (Raji-depletion) device with approximately 2.6 min residence time was demonstrated to produce 100% pure MOLT-3 cells from 50-50 MOLT-3/Raji mixture. We have developed a mathematical model of the separation device based on the experimentally determined kinetic parameters that can be extended to design future separation modules for other cell mixtures. We conclude that we can design microfluidic devices that exploits the kinetics of dynamic cell adhesion to antibody coated surfaces to provide enriched cell subpopulations within minutes of total processing time.

Trehalose Loading Through the Mitochondrial Permeability Transition Pore Enhances Desiccation Tolerance in Rat Liver Mitochondria

Biochimica Et Biophysica Acta. Nov, 2005  |  Pubmed ID: 16242115

Trehalose has extensively been used to improve the desiccation tolerance of mammalian cells. To test whether trehalose improves desiccation tolerance of mammalian mitochondria, we introduced trehalose into the matrix of isolated rat liver mitochondria by reversibly permeabilizing the inner membrane using the mitochondrial permeability transition pore (MPTP). Measurement of the trehalose concentration inside mitochondria using high performance liquid chromatography showed that the sugar permeated rapidly into the matrix upon opening the MPTP. The concentration of intra-matrix trehalose reached 0.29 mmol/mg protein (approximately 190 mM) in 5 min. Mitochondria, with and without trehalose loaded into the matrix, were desiccated in a buffer containing 0.25 M trehalose by diffusive drying. After re-hydration, the inner membrane integrity was assessed by measurement of mitochondrial membrane potential with the fluorescent probe JC-1. The results showed that following drying to similar water contents, the mitochondria loaded with trehalose had significantly higher inner membrane integrity than those without trehalose loading. These findings suggest the presence of trehalose in the mitochondrial matrix affords improved desiccation tolerance to the isolated mitochondria.

Selective Enhancement of Cytochrome P-450 Activity in Rat Hepatocytes by in Vitro Heat Shock

Tissue Engineering. Sep-Oct, 2005  |  Pubmed ID: 16259607

We investigated the effect of heat shock on cytochrome P-450 activity in rat hepatocytes and report a significant, selective, and time-dependent enhancement of cytochrome P-450 activity in heatshocked hepatocytes. Stable long-term cultures of rat hepatocytes were heat shocked (42.5 degrees C) for 1 to 3 h and allowed to recover at 37 degrees C. Cytochrome P-450-dependent ethoxyresorufin O-dealkylase (EROD) and benzyloxyresorufin O-dealkylase (BROD) activities were measured up to 48 h after heat shock treatment. In general, the optimal heat shock exposure time was between 2 and 3 h. BROD activity (induced by sodium phenobarbital) increased approximately 6-fold in hepatocytes heat shocked for 3 h in comparison with hepatocytes maintained at 37 degrees C. EROD activity (induced by 3-methylcholanthrene) increased 2-fold on exposure to heat shock for 2 h. The expression of inducible heat shock proteins Hsp70 and Hsp32 was verified by Western immunoblot analyses. In the absence of the appropriate inducer, heat shock treatment did not enhance cytochrome P-450 activity. Furthermore, enhanced P-450 enzyme activity was delayed for heat-shocked hepatocytes. It is hypothesized that heat shock treatment attenuates the negative effects triggered by the addition of the toxic inducers and possibly stabilizes the levels of cytochrome P-450 proteins. These results suggest that heat shock treatment may be used to enhance the functionality of hepatocytes, specifically, in bioartificial liver assist devices.

The Influence of Microtextured Basal Lamina Analog Topography on Keratinocyte Function and Epidermal Organization

Journal of Biomedical Materials Research. Part A. Jan, 2005  |  Pubmed ID: 15543632

The rational design of future bioengineered skin substitutes requires an understanding of the mechanisms by which the three-dimensional microarchitecture of tissue scaffolds modulates keratinocyte function. Microtextured basal lamina analogs were developed to investigate the relationship between the characteristic topography at the dermal-epidermal interface of native skin and keratinocyte function. Microfabrication techniques were used to create master patterns, negative replicates, and collagen membranes with ridges and channels of length scales (e.g., grooves of 50-200 microm in depth and width) similar to the invaginations found in basal lamina at the dermal-epidermal junction of native skin. Keratinocytes were seeded on the surfaces of basal lamina analogs, and histological analyses were performed after 7 days of tissue culture at the air-liquid interface. The keratinocytes formed a differentiated and stratified epidermis that conformed to the features of the microtextured membranes. Morphometric analyses of immunostained skin equivalents suggest that keratinocyte stratification and differentiation increases as channel depth increases and channel width decreases. This trend was most pronounced in channels with the highest depth-to-width ratios (i.e., 200 microm deep, 50 microm wide). It is anticipated that the findings from these studies will elucidate design parameters to enhance the performance of future bioengineered skin substitutes.

Thermal Characterization of Nakagata's Mouse Sperm Freezing Protocol

Cryobiology. Feb, 2006  |  Pubmed ID: 16332363

This paper reports the results of an experimental study of the warming and cooling rates achieved using the popular Nakagata Protocol for murine sperm cryopreservation. Problems with the storage and maintenance of the huge number of genetically engineered mouse strains have led to an increased need for murine sperm preservation. Recent studies have begun to focus on optimizing the cryopreservation of murine sperm by carefully studying the effects of cooling and warming rates on sperm survival. In current practice, however, the Nakagata protocol is widely used. The actual cooling and warming rates achieved using the Nakagata protocol have not previously been determined; and the Nakagata protocol has a number of unspecified parameters which we have found can significantly affect cooling rates, warming rates and sperm survival. A detailed study of the thermal response of samples frozen and thawed using the Nakagata protocol reveals that the cooling rates range from 30 to almost 300 degrees C per minute depending on the exact manner in which the Nakagata protocol is implemented. Warming rates range from 160 degrees C/min to about 1000 degrees C/min. Sperm survival depended significantly on the particular cooling rate achieved, and less strongly on the warming rates. Overall, it was found that the particular manner in which the Nakagata protocol was implemented could strongly affect cooling rates and sperm survival; and, consistent with the findings of Mazur and Koshimoto, an optimal cooling rate appears to exist in the range of cooling rates that can be achieved using the Nakagata protocol.

Trehalose Uptake Through P2X7 Purinergic Channels Provides Dehydration Protection

Cryobiology. Feb, 2006  |  Pubmed ID: 16338230

The tetra-anionic form of ATP (ATP4-) is known to induce monovalent and divalent ion fluxes in cells that express purinergic P2X7 receptors and with sustained application of ATP it has been shown that dyes as large as 831 Da can permeate the cell membrane. The current study explores the kinetics of loading alpha,alpha-trehalose (342 Da) into ATP stimulated J774.A1 cells, which are known to express the purinergic P2X7 receptor. Cells that were incubated at 37 degrees C in a 50 mM phosphate buffer (pH 7.0) containing 225 mM trehalose and 5 mM ATP, were shown to load trehalose linearly over time. Concentrations of approximately 50 mM were reached within 90 min of incubation. Cells incubated in the same solution at 4 degrees C loaded minimally, consistent with the inactivity of the receptor at low temperatures. However, extended incubation at 37 degrees C (>60 min) resulted in zero next-day survival, with adverse effects appearing even with incubation periods as short as 30 min. By using a two-step protocol with a short time period at 37 degrees C to allow pore formation, followed by an extended loading period on ice, cells could be loaded with up to 50 mM trehalose while maintaining good next day recovery (49 +/- 12% by Trypan blue exclusion, 56 +/- 20% by alamarBlue assay). Cells porated by this method and allowed an overnight recovery period exhibited improved dehydration tolerance suggesting a role for ATP poration in the anhydrous preservation of cells.

Microfluidic Diffusive Filter for Apheresis (leukapheresis)

Lab on a Chip. Jan, 2006  |  Pubmed ID: 16372073

Apheresis is a procedure used to fractionate whole blood into its individual components. Following fractionation, the desired component is isolated and the remaining blood in many cases is returned to the donor. Leukapheresis is one type of apheresis where leukocytes (white blood cells) are selectively removed. This procedure is commonly used for blood transfusions to remove donor leukocytes from being transferred to the recipient. Apheresis also has several therapeutic applications. In this manuscript we discuss the design, fabrication and testing of a continuous flow diffusive filter, fabricated using simple soft lithographic techniques for depletion of leukocytes. This device employs micro sieves that exploit the size and shape difference between the different cell types to obtain depletion of leukocytes from whole blood. Currently, conventional apheresis methods like centrifugation or fiber mesh filtration are commonly used. A theoretical model was developed to determine the optimal shape of the diffuser to ensure that the volumetric flow through individual sieve elements is equal. This device was designed to serve as a passive device that does not require any external manipulation. Results show that for the given device design, isolation of approximately 50% of the inlet erythrocytes (red blood cells), along with depletion of >97% of the inlet leukocytes is possible at a flow rate of 5 microl min(-1). Simple modifications to the geometry and dimensions of the sieves can be made to obtain isolation of plasma.

Microfluidic System for Measuring Neutrophil Migratory Responses to Fast Switches of Chemical Gradients

Lab on a Chip. Feb, 2006  |  Pubmed ID: 16450027

Experimental systems that provide temporal and spatial control of chemical gradients are required for probing into the complex mechanisms of eukaryotic cell chemotaxis. However, no current technique can simultaneously generate stable chemical gradients and allow fast gradient changes. We developed a microfluidic system with microstructured membranes for exposing neutrophils to fast and precise changes between stable, linear gradients of the known chemoattractant Interleukin-8 (IL-8). We observed that rapidly lowering the average concentration of IL-8 within a gradient, while preserving the direction of the gradient, resulted in temporary neutrophil depolarization. Fast reversal of the gradient direction while increasing or decreasing the average concentration also resulted in temporary depolarization. Neutrophils adapted and maintained their directional motility, only when the average gradient concentration was increased and the direction of the gradient preserved. Based on these observations we propose a two-component temporal sensing mechanism that uses variations of chemokine concentration averaged over the entire cell surface and localized at the leading edge, respectively, and directs neutrophil responses to changes in their chemical microenvironment.

Murine B16 Melanomas Expressing High Levels of the Chemokine Stromal-derived Factor-1/CXCL12 Induce Tumor-specific T Cell Chemorepulsion and Escape from Immune Control

Journal of Immunology (Baltimore, Md. : 1950). Mar, 2006  |  Pubmed ID: 16493048

The chemokine, stromal-derived factor-1/CXCL12, is expressed by normal and neoplastic tissues and is involved in tumor growth, metastasis, and modulation of tumor immunity. T cell-mediated tumor immunity depends on the migration and colocalization of CTL with tumor cells, a process regulated by chemokines and adhesion molecules. It has been demonstrated that T cells are repelled by high concentrations of the chemokine CXCL12 via a concentration-dependent and CXCR4 receptor-mediated mechanism, termed chemorepulsion or fugetaxis. We proposed that repulsion of tumor Ag-specific T cells from a tumor expressing high levels of CXCL12 allows the tumor to evade immune control. Murine B16/OVA melanoma cells (H2b) were engineered to constitutively express CXCL12. Immunization of C57BL/6 mice with B16/OVA cells lead to destruction of B16/OVA tumors expressing no or low levels of CXCL12 but not tumors expressing high levels of the chemokine. Early recruitment of adoptively transferred OVA-specific CTL into B16/OVA tumors expressing high levels of CXCL12 was significantly reduced in comparison to B16/OVA tumors, and this reduction was reversed when tumor-specific CTLs were pretreated with the specific CXCR4 antagonist, AMD3100. Memory OVA-specific CD8+ T cells demonstrated antitumor activity against B16/OVA tumors but not B16/OVA.CXCL12-high tumors. Expression of high levels of CXCL12 by B16/OVA cells significantly reduced CTL colocalization with and killing of target cells in vitro in a CXCR4-dependent manner. The repulsion of tumor Ag-specific T cells away from melanomas expressing CXCL12 confirms the chemorepellent activity of high concentrations of CXCL12 and may represent a novel mechanism by which certain tumors evade the immune system.

Cell Handling Using Microstructured Membranes

Lab on a Chip. Mar, 2006  |  Pubmed ID: 16511616

Gentle and precise handling of cell suspensions is essential for scientific research and clinical diagnostic applications. Although different techniques for cell analysis at the micro-scale have been proposed, many still require that preliminary sample preparation steps be performed off the chip. Here we present a microstructured membrane as a new microfluidic design concept, enabling the implementation of common sample preparation procedures for suspensions of eukaryotic cells in lab-on-a-chip devices. We demonstrate the novel capabilities for sample preparation procedures by the implementation of metered sampling of nanoliter volumes of whole blood, concentration increase up to three orders of magnitude of sparse cell suspension, and circumferentially uniform, sequential exposure of cells to reagents. We implemented these functions by using microstructured membranes that are pneumatically actuated and allowed to reversibly decouple the flow of fluids and the displacement of eukaryotic cells in suspensions. Furthermore, by integrating multiple structures on the same membrane, complex sequential procedures are possible using a limited number of control steps.

Nonmetabolizable Glucose Compounds Impart Cryotolerance to Primary Rat Hepatocytes

Tissue Engineering. Mar, 2006  |  Pubmed ID: 16579691

We herein report a novel method for the cryopreservation of hepatocytes using a non-metabolizable glucose derivative in an attempt to mimic the natural cryoprotective adaptations observed in freeze-tolerant frogs. Primary rat hepatocytes were loaded with 3-O-methyl glucose (3OMG) through endogenous glucose transporters without evident toxicity. The 3OMG-loaded hepatocytes were then frozen in a controlled rate freezer down to -80 degrees C and stored in liquid nitrogen at -196 degrees C. Hepatocytes cryopreserved with a relatively small amount of intracellular 3OMG (<0.2 M) showed high post-thaw viability and maintained long-term hepatospecific functions, including synthesis, metabolism, and detoxification. Metabolite uptake and secretion rates were also largely preserved in the cryopreserved hepatocytes. This is the first study to demonstrate the use of the non-metabolizable glucose derivative 3OMG in hepatocyte cryopreservation.

Antiproteolytic Action of Orally Delivered Insulin Using PH-responsive Hydrogels in a Rat Burn Model

The Journal of Surgical Research. Sep, 2006  |  Pubmed ID: 16616764

Subcutaneously delivered small doses of insulin has shown beneficial effects on burn injury-induced muscle wasting and wound healing. To improve the method of insulin treatment for clinical settings, this study investigated the effect of insulin delivered orally using pH-responsive poly(methacrylic-g-ethylene glycol) (P(MAA-g-EG)) hydrogels in a 20% total burn surface area rat burn injury model. P(MAA-g-EG) were synthesized in-house and insulin release characteristics were performed in vitro. Young rats weighing 80-150 g were subjected to 15-20% total body surface area burn injury and treated with insulin-containing hydrogels enclosed in gelatin capsules for 3 days. The dosage was adjusted to match 0.25 U (day 1), 0.5U (day 2), and 1.0 U (day 3) per 100 grams of body weight. All animals were housed in metabolic cages and their physical activity, body weight, food consumption, water uptake, circulating glucose levels, and urinary tyrosine content were monitored for 4 to 15 days after burn. Results show that the orally delivered insulin restored the body weight of burned rats and influenced wound healing, similar to subcutaneous delivery. Measured glucose levels showed significantly less perturbation, suggesting the possibility of increasing the dosage. In conclusion, muscle wasting can be significantly inhibited by the oral administration of insulin using pH-responsive hydrogels.

Universal Microfluidic Gradient Generator

Analytical Chemistry. May, 2006  |  Pubmed ID: 16689552

The study of cellular responses to chemical gradients in vitro would greatly benefit from experimental systems that can generate precise and stable gradients comparable to chemical nonhomogeneities occurring in vivo. Recently, microfluidic devices have been demonstrated for linear gradient generation for biological applications with unmatched accuracy and stability. However, no systematic approach exists at this time for generating other gradients of target spatial configuration. Here we demonstrate experimentally and provide mathematical proof for a systematic approach to generating stable gradients of any profile by the controlled mixing of two starting solutions.

Magnetically and Biologically Active Bead-patterned Hydrogels

Langmuir : the ACS Journal of Surfaces and Colloids. May, 2006  |  Pubmed ID: 16700603

We present a new approach to the direct patterning of biologically and magnetically active microbeads in nonbiofouling polymer scaffolds for use in microfluidic devices. Briefly, the process involves treatment of a glass substrate, conformal contact bonding of a PDMS microchannel on the substrate, filling of the channel with beads and prepolymer solution, and UV-initiated photopolymerization of a mask-defined pattern using a standard inverted microscope. This versatile and simple method allows for the rapid fabrication of dispersed or packed bead patterns in poly(ethylene glycol) (PEG) hydrogels that are covalently linked to glass surfaces. By exploiting the relative opacity of the microbeads used, we are able to create both partially exposed and fully encapsulated bead patterns. To demonstrate the utility of this new technology, we separated magnetic bead-bound B lymphocytes from T lymphocytes on a PEG-encapsulated magnetic filtration platform and also captured B cells directly on patterned, protein-decorated beads in a flow-through microfluidic device. Beyond cell sorting, the accurate patterning of industrially standardized, chemically diverse microbeads may have significant implications for microchip-based analyte detection.

Size-based Microfluidic Enrichment of Neonatal Rat Cardiac Cell Populations

Biomedical Microdevices. Sep, 2006  |  Pubmed ID: 16732418

Native heart consists of myocytes and non-myocytes. We demonstrate here the feasibility of a size-based microfluidic separation of myocytes and non-myocytes from the neonatal rat myocardium. The device consists of a middle channel (50 microm wide, 200 microm tall, and 4 cm long) connected to adjacent side channels by microsieves (80 microm wide, 5 microm tall and 40 microm in length). The side channels increase in width in a flared shape along the length of the device to ensure constant pressure gradient across all sieves. In the first step, non-myocytes were removed from the myocytes by a conventional pre-plating method for 75 min. Subsequently, the non-myocytes were further enriched in a microfluidic device at 20 microl/min. We demonstrated that the cells in the middle and side channels maintained viability during sorting and the ability to attach and grow in culture. Upon culture for 48 h cardiomyocytes from the reservoir (control) and middle channel stained positive for cardiac Troponin I, exhibited a well developed contractile apparatus and contracted spontaneously and in response to electrical field stimulation. Most of the cells in the side channel expressed a non-myocyte marker vimetin. Fluorescent activated cell sorting indicated significant enrichment in the side channel (p < 0.001) for non-myocytes. Original cell suspension had a bimodal cell size distribution with the peaks in the range from 7-9 microm and 15-17 microm. Upon cell sorting the distribution was Gaussian in both side channel and middle channel with the peaks in the range 7-9 microm and 9-11 microm respectively, indicating that the separation by size occurred.

Treatment of Fulminant Hepatic Failure in Rats Using a Bioartificial Liver Device Containing Porcine Hepatocytes Producing Interleukin-1 Receptor Antagonist

Tissue Engineering. May, 2006  |  Pubmed ID: 16771644

Fulminant hepatic failure (FHF) is a serious clinical condition that is associated with high mortality. There is evidence that FHF is an inflammatory disease, which is supported clinically by elevated serum levels of cytokines. In an effort to develop hepatocytes with additional functions for use in our bioartificial liver (BAL) device, we focused on interleukin-1 (IL-1) blockade as a therapeutic modality. Primary porcine hepatocytes were isolated from the livers of miniature swine and then transfected with an adenoviral vector encoding human interleukin-1 receptor antagonist (AdIL-1Ra). The transfected hepatocytes secreted human IL-1Ra. These transfected hepatocytes were incorporated into a flat-plate BAL device to evaluate their efficacy in treating D-galactosamine (GalN)- induced FHF in a rat model. After extracorporeal perfusion with the BAL device containing the transfected hepatocytes, there were significant reductions in the plasma levels of hepatic enzymes (aspartate aminotransferase and alanine aminotransferase) and cytokines (IL-1 and IL-6), indicating a beneficial effect. Animal survival was significantly improved in the treated group compared to the control group. These experiments demonstrate that combining inflammatory cytokine blockade with a functional BAL device may be an effective therapeutic option in the treatment of FHF.

Liver-specific Functional Studies in a Microfluidic Array of Primary Mammalian Hepatocytes

Analytical Chemistry. Jul, 2006  |  Pubmed ID: 16808435

Nearly half a billion dollars in resources are lost each time a drug candidate is withdrawn from the market by the Food and Drug Administration (FDA) for reasons of liver toxicity. The number of late-phase drug developmental failures due to liver toxicity could potentially be reduced through the use of hepatocyte-based systems capable of modeling the response of in vivo liver tissue to toxic insults. With this article, we report progress toward the goal of realizing an array of primary hepatocytes for use in high-throughput liver toxicity studies. Described herein is the development of a 64 (8 x 8) element array of microfluidic wells capable of supporting micropatterned primary rat hepatocytes in coculture with 3T3-J2 fibroblasts. Each of the wells within the array was continuously perfused with medium and oxygen in a nonaddressable format. The key features of the system design and fabrication are described, including the use of two microfluidic perfusion networks to provide the coculture with an independent and continuous supply of cell culture medium and oxygen. Also described are the fabrication techniques used to selectively pattern hepatocytes and 3T3-J2 fibroblasts within the wells of the array. The functional studies used to demonstrate the synthetic and metabolic capacity of the array are outlined in this article. These studies demonstrate that the hepatocytes contained within the array are capable of continuous, steady-state albumin synthesis (78.4 microg/day, sigma = 3.98 microg/day, N = 8) and urea production (109.8 microg/day, sigma = 11.9 microg/day, N = 8). In the final section of the article, these results are discussed as they relate to the final goal of this research effort, the development of an array of primary hepatocytes for use in physiologically relevant toxicology studies.

Panning of Multiple Subsets of Leukocytes on Antibody-decorated Poly(ethylene) Glycol-coated Glass Slides

Journal of Immunological Methods. Jun, 2006  |  Pubmed ID: 16822521

The antibody (Ab) array format provides a unique opportunity to pan and characterize multiple leukocyte subsets in parallel. However, the questions of reproducibility and robustness of leukocyte panning on Ab arrays need to be answered for this technology to become an immunophenotyping tool. The present study sought to address several of these questions, including: (1) purity of leukocyte subsets captured on Ab regions, (2) dynamics of leukocyte binding, (3) elimination of non-specific cell adhesion, and (4) standardization of cell washing conditions. Abs for CD4 T-cells, CD8 T-cells, CD36 monocytes, and CD16b neutrophils were dispensed onto standard glass slides containing a thin film of poly(ethylene glycol) (PEG) hydrogel. PEG gel coating was highly effective in eliminated non-specific cell adhesion on the surface. Incubation of the Ab arrays with red blood cell (RBC) depleted whole blood resulting in antigen-specific panning of leukocyte subsets on the respective Ab domains. A flow through chamber was employed to determine optimal shear stress conditions for removal of non-specifically attached cells. The purity of the four subsets remaining on the surface after washing was determined by Wright staining and immunofluorescence, and was found to be as follows: CD4 T-cells (99.2+/-0.3%), CD8 T-cells (98.7+/-0.3%), CD36 monocytes (97.2+/-0.9%), and CD16b neutrophils (99.1+/-0.6%). In conclusion, the methods described in this study allow to separate whole blood into pure leukocyte subsets with minimal sample preparation and handling. These approaches will be valuable in the future development of Ab arrays as tools for quantitative immunophenotyping of leukocytes.

Polyelectrolyte Nano-scaffolds for the Design of Layered Cellular Architectures

Tissue Engineering. Jun, 2006  |  Pubmed ID: 16846351

The design of in vitro multilayered cellular architectures that resemble the stratified, lattice-like structure in tissues poses a significant challenge for tissue engineering. There is currently no generally applicable methodology to design multilayered cellular constructs that mimic the structure of tissues in vivo. We report a novel and generalizable approach to create multilayered cellular constructs that addresses these issues. These in vitro constructs comprise alternating layers of cells and nano-scale biocompatible polyelectrolyte (PE) scaffolds. We apply this methodology to address two specific problems in hepatic tissue engineering: the design of in vitro liver sinusoidal structures and the critical need to increase viable cell mass in extracorporeal liver-assist devices. We assembled ultrathin polymer scaffolds on the top of a confluent monolayer of cells by the sequential deposition of oppositely charged PEs. The thickness of the PE scaffold lies in the nanometer range. The PE scaffold plays a dual role. First, it is a technique to culture hepatocytes in vitro that maintains their morphology, cytoskeletal structure, and liver-specific functions. Second, the nano-scaffold provides a cell-adhesive surface on which a second layer of cells can be cultured, resulting in layered architectures. We have used this approach to design layered three-dimensional hepatocyte-PE-hepatocyte constructs, hepatocyte-PE-endothelial cell constructs, and hepatocyte-PE-fibroblast constructs. As a result of its versatility, this approach can, in principle, be used to design layered cellular constructs of any tissue type, and therefore has potentially wide applications in tissue engineering, bioreactor devices, and in drug delivery. This methodology has the potential to generate realistic in vitro constructs of any tissue type.

Microfluidic Isolation of Leukocytes from Whole Blood for Phenotype and Gene Expression Analysis

Analytical Chemistry. Aug, 2006  |  Pubmed ID: 16878882

Technologies that enable the isolation of cell subtypes from small samples of complex populations will greatly facilitate the implementation of proteomics and genomics to human diseases. Transcriptome analysis of blood requires the depletion of contaminating erythrocytes. We report an automated microfluidic device to rapidly deplete erythrocytes from whole blood via deionized water lysis and to collect enriched leukocytes for phenotype and genomic analyses. Starting with blood from healthy subjects, we demonstrate the utility of this microfluidic cassette and lysis protocol to prepare unstimulated leukocytes, and leukocytes stimulated ex vivo with Staphylococcal enterotoxin B, which mimics some of the cellular effects seen in patients with severe bacterial infections. Microarrays are used to assess the global gene expression response to enterotoxin B. The results demonstrate that this system can isolate unactivated leukocytes from small blood samples without any significant loss, which permits more information to be obtained from subsequent analysis, and will be readily applicable to clinical settings.

3-O-methyl-D-glucose Improves Desiccation Tolerance of Keratinocytes

Tissue Engineering. Jul, 2006  |  Pubmed ID: 16889517

Transplantation of autologous skin grafts and tissue engineered skin replacements for the treatment of burns, trauma, and ulcerative wounds has been shown to restore a protective barrier to infection and fluid loss, reduce heat loss, provide mechanical strength, diminish pain, and dampen the hypermetabolic stress response to thermal injury. Patencies of these grafts depend mainly on the high viability and sustained function of the enmeshed keratinocytes. With growing demand in tissue replacement therapies, development of successful and economical preservation techniques for skin grafts and replacements becomes essential. In this regard, if attained, desiccated state storage offers an economical solution to availability, storage, and transportation problems. Recent studies indicate that carbohydrates are very efficient in stabilizing mammalian cells against various types of stresses, including those associated with cryopreservation and desiccation. In this study we introduce the use of 3-O-methyl-D-glucose (3-OMG), a nonmetabolizable glucose derivative, as a new means of providing protection for keratinocytes undergoing desiccation. We show that with decreasing water contents, viability of the cells decreases; however, at the same water content the immediate post-rehydration viability and long-term survival of the cells exposed to 3-OMG are much higher than those of controls.

Elevated Hepatocyte-specific Functions in Fetal Rat Hepatocytes Co-cultured with Adult Rat Hepatocytes

Tissue Engineering. Oct, 2006  |  Pubmed ID: 17518664

Fetal hepatocytes (FHEPs) are a potential source of highly proliferative transplantable cells but express low levels of liver-specific functions. We hypothesized that the microenvironment of adult hepatocytes (AHEPs) may upregulate these functions. Primary FHEPs were seeded on top of collagen-sandwiched AHEPs directly or separated by a porous transwell membrane insert. In direct co-cultures, albumin (ALB) secretion, urea synthesis, and cytochrome P450 (CytP450) activity were all approximately 2 times as high as the sum of the corresponding monocultures. Using a transwell porous insert led to similar results, suggesting a major role for soluble factors. When AHEPs and FHEPs were separated after co-culture, they both initially showed significantly higher ALB secretion than control monocultures, whereas urea synthesis was significantly lower for the FHEPs only. Functions of previously co-cultured FHEPs normalized over the course of a week, but AHEP function remained high even after separation. In conclusion, co-culturing AHEPs with FHEPs increases expression of liver-specific functions in both cell types. The effect on FHEPs, but not AHEPs, was reversible. Unraveling the underlying mechanisms and optimizing this phenomenon will be useful in making fetal liver cells a potential cell source for hepatic tissue-engineering applications.

Engineering Complex Tissues

Tissue Engineering. Dec, 2006  |  Pubmed ID: 17518671

This article summarizes the views expressed at the third session of the workshop "Tissue Engineering--The Next Generation," which was devoted to the engineering of complex tissue structures. Antonios Mikos described the engineering of complex oral and craniofacial tissues as a "guided interplay" between biomaterial scaffolds, growth factors, and local cell populations toward the restoration of the original architecture and function of complex tissues. Susan Herring, reviewing osteogenesis and vasculogenesis, explained that the vascular arrangement precedes and dictates the architecture of the new bone, and proposed that engineering of osseous tissues might benefit from preconstruction of an appropriate vasculature. Jennifer Elisseeff explored the formation of complex tissue structures based on the example of stratified cartilage engineered using stem cells and hydrogels. Helen Lu discussed engineering of tissue interfaces, a problem critical for biological fixation of tendons and ligaments, and the development of a new generation of fixation devices. Rita Kandel discussed the challenges related to the re-creation of the cartilage-bone interface, in the context of tissue engineered joint repair. Frederick Schoen emphasized, in the context of heart valve engineering, the need for including the requirements derived from "adult biology" of tissue remodeling and establishing reliable early predictors of success or failure of tissue engineered implants. Mehmet Toner presented a review of biopreservation techniques and stressed that a new breakthrough in this field may be necessary to meet all the needs of tissue engineering. David Mooney described systems providing temporal and spatial regulation of growth factor availability, which may find utility in virtually all tissue engineering and regeneration applications, including directed in vitro and in vivo vascularization of tissues. Anthony Atala offered a clinician's perspective for functional tissue regeneration, and discussed new biomaterials that can be used to develop new regenerative technologies.

Cell Detection and Counting Through Cell Lysate Impedance Spectroscopy in Microfluidic Devices

Lab on a Chip. Jun, 2007  |  Pubmed ID: 17538717

Cell-based microfluidic devices have attracted interest for a wide range of applications. While optical cell counting and flow cytometry-type devices have been reported extensively, sensitive and efficient non-optical methods to detect and quantify cells attached over large surface areas within microdevices are generally lacking. We describe an electrical method for counting cells based on the measurement of changes in conductivity of the surrounding medium due to ions released from surface-immobilized cells within a microfluidic channel. Immobilized cells are lysed using a low conductivity, hypotonic media and the resulting change in impedance is measured using surface patterned electrodes to detect and quantify the number of cells. We found that the bulk solution conductance increases linearly with the number of isolated cells contributing to solution ion concentration. The method of cell lysate impedance spectroscopy is sensitive enough to detect 20 cells microL(-1), and offers a simple and efficient method for detecting and enumerating cells within microfluidic devices for many applications including measurement of CD4 cell counts in HIV patients in resource-limited settings. To our knowledge, this is the most sensitive approach using non-optical setups to enumerate immobilized cells. The microfluidic device, capable of isolating specific cell types from a complex bio-fluidic and quantifying cell number, can serve as a single use cartridge for a hand-held instrument to provide simple, fast and affordable cell counting in point-of-care settings.

Long-term Storage of Mouse Spermatozoa After Evaporative Drying

Reproduction (Cambridge, England). May, 2007  |  Pubmed ID: 17616722

To determine if mouse spermatozoa could be preserved long-term without using liquid nitrogen, mouse spermatozoa in trehalose-EGTA solution were partially evaporatively dried under nitrogen gas (5 min at flow rate10 l/min) and stored for 1 week and 5 months at 4, -20, and -80 degrees C before intracytoplasmic sperm injection. Fertilization rates were neither different with spermatozoa stored at 4, -20, or -80 degrees C for 1 week or 1, 3, and 5 months respectively, nor blastocyst formation rates with spermatozoa stored for 1 week and 1 month. However, spermatozoa stored at 4 and -20 degrees C for 3 months resulted in fewer blastocysts (35.1 and 54.3% respectively) when compared with spermatozoa stored at -80 degrees C (74.4%). Blastocyst formation rates using spermatozoa stored for 5 months at -20 degrees C (57.4%) or -80 degrees C (74.5%) were not significantly different from those stored for 3 months at the same temperatures respectively, but were significantly better than those stored for 5 months at 4 degrees C (10.2%). Blastocysts derived from spermatozoa stored for 3 and 5 months at -20 and -80 degrees C respectively, were then transferred to pseudopregnant mothers to develop into healthy liveborn offspring. No significant differences were found in embryo transfer rates (number of pups born/number of embryos transferred), weaning rates, or sex ratios of resultant pups, which were healthy and reproductively sound. These results demonstrate for the first time that partially evaporatively dried mouse spermatozoa in trehalose-EGTA solution can be preserved for long term at -20 and -80 degrees C. The possibility that the storage temperature must be less than the glass transition temperature is discussed.

Microfabrication-based Modulation of Embryonic Stem Cell Differentiation

Lab on a Chip. Aug, 2007  |  Pubmed ID: 17653344

Embryonic stem (ES) cells form spontaneous aggregates during differentiation, and cell-cell communication in the aggregates plays an important role in differentiation. The development of a controlled differentiation scheme for ES cells has been hindered by the lack of a reliable method to produce uniform aggregate sizes. Conventional techniques, such as hanging drop and suspension cultures, do not allow precise control over size of ES cell aggregates. To surmount this problem, we microfabricated adhesive stencils to make mouse ES (mES) cell aggregates of specific sizes ranging from 100 microm to 500 microm in diameter. With this technique, we studied the effect of the initial aggregate size on ES cell differentiation. After 20 days of induction of differentiation, we analyzed the stem cell populations using gene and protein expression assays as well as biochemical functions. Notably, we found that germ layer differentiation depends on the initial size of the ES cell aggregate. Among the ES cell aggregate sizes tested, the aggregates with 300 microm diameter showed similar differentiation profiles of three germ layers as embryoid bodies made using the "hanging drop" technique. The smaller (100 microm) aggregates showed the increased expression of ectodermal markers compared to the larger (500 microm) aggregates, while the 500 microm aggregates showed the increased expression of mesodermal and endodermal markers compared to the 100 microm aggregates. These results indicate that the initial size of the aggregate is an important factor for ES cell differentiation, and can affect germ layer selection as well as the extent of differentiation.

Osmotic Selection of Human Mesenchymal Stem/progenitor Cells from Umbilical Cord Blood

Tissue Engineering. Oct, 2007  |  Pubmed ID: 17665999

The isolation of undifferentiated adult stem/progenitor cells remains a challenging task primarily due to the rare quantity of these cells in biological samples and the lack of unique markers. Herein, we report a relatively straightforward method for isolation of human mesenchymal stem cells (MSCs) based on their unusual resistance to osmotic lysis, which we term "osmotic selection" (OS). MSCs can remarkably withstand significant exposure to hypotonic conditions (> 30 min) with only a reversible impairment in cell proliferation and with no loss of stem cell potential after exposure. Comparison of MSCs to other circulating nonhematopoietic cells revealed a time regime, by which purification of these cells would be attainable without considerable cell loss. OS showed a 50-fold enrichment of fibroblast colony-forming units from umbilical cord blood samples when compared to commonly employed techniques. After upstream processing, isolated cells using OS were immunophenotyped to be CD14-, CD34-, CD45-, CD44+, CD105+, and CD106+, and displayed multipotent differentiation. Preliminary investigations to determine mechanisms responsible for osmolytic resistance revealed MSCs to have an ineffective volume of 59%, with the ability to double cell volume at infinite dilution. Disruption of filamentous actin polymerization by cytochalasin D sensitized MSCs to osmotic lysis, which suggests a cytoskeletal element involved in osmolytic resistance.

Thermal Performance of Quartz Capillaries for Vitrification

Cryobiology. Dec, 2007  |  Pubmed ID: 17919532

In this paper we report the thermal behavior of a new approach for vitrification. Thermal performance of traditional open pulled straws is compared with a new technique based on the combined use of quartz capillaries with slush nitrogen. This new method of vitrification achieved ultrafast cooling rates of 250,000 degrees C/min. As a result, a much lower concentration of cryoprotectant was needed to reach vitrification. In fact, a cryoprotectant solution typically used in oocyte slow freezing protocols was shown to remain transparent after cooling to liquid nitrogen temperatures indicating apparent "vitrification". This approach offers a new and very promising technique for vitrification of cells using low levels of cryoprotectants.

Phenotypic Analysis of C57BL/6J and FVB/NJ Mice Generated Using Evaporatively Dried Spermatozoa

Comparative Medicine. Oct, 2007  |  Pubmed ID: 17974129

Combination of evaporative drying and frozen storage at -80 degrees C has been used successfully to preserve hybrid B6D2F1 mouse spermatozoa. To determine whether this method can be applied equally well to inbred mice, spermatozoa of C57BL/6J and FVB/ NJ mice were evaporatively dried and stored for 1 mo at -80 degrees C before being used for intracytoplasmic sperm injection (ICSI) to produce live offspring. After weaning, 1 male and 1 female mouse from each litter were randomly selected at 8 wk of age for natural mating to produce live offspring. Results showed that spermatozoa from both inbred strains that had been evaporatively dried and subsequently stored at -80 degrees C could be used successfully to derive live, healthy, and reproductively sound offspring by ICSI. No significant differences were found in embryo transfer rate (number of pups born/number of embryos transferred), litter size, weaning rate, body weight, number of pathologic lesions, and amount of contamination by pathogens of mice produced by ICSI using evaporatively dried spermatozoa compared with mice produced by natural mating or by ICSI using fresh (that is, nonpreserved) spermatozoa. Progeny produced by mating mice generated from ICSI using evaporatively dried spermatozoa were normal. Therefore, spermatozoa from inbred mouse strains C57BL/6J and FVB/NJ can be preserved successfully after evaporative drying and frozen storage at -80 degrees C.

Role of Trehalose in Prevention of Giant Vesicle Adsorption and Encapsulated Solute Leakage in Anhydrobiotic Preservation

Langmuir : the ACS Journal of Surfaces and Colloids. Dec, 2007  |  Pubmed ID: 17983248

Anhydrobiotic preservation has the potential to allow the processing and storage of mammalian cells in a state of suspended animation at ambient conditions in trehalose glasses; however, stresses--particularly to the lipid bilayer--during desiccation and rehydration have thus far prevented the full realization of the promise of this technique. Giant gel-phase 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and liquid-crystalline-phase 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) vesicles provide a model cell system with which to elucidate the role of trehalose in surface-lipid bilayer interactions, as well as the part played by lipid phase. In the absence of trehalose, DSPC liposomes adsorbed to polystyrene, producing irreversible structural changes and apparent leakage of all intravesicular solute upon drying and rehydration. Addition of trehalose significantly reduced vesicle adsorption with only transient intravesicular solute leakage for the rehydrated vesicles; however, at very low moisture contents, the vesicles underwent permanent structural changes. In contrast to the results with DSPC vesicles, DLPC vesicles largely avoided adsorption and exhibited high intravesicular solute retention when dried and rehydrated even in the absence of trehalose, despite significant internal structural changes.

Continuous Inertial Focusing, Ordering, and Separation of Particles in Microchannels

Proceedings of the National Academy of Sciences of the United States of America. Nov, 2007  |  Pubmed ID: 18025477

Under laminar flow conditions, when no external forces are applied, particles are generally thought to follow fluid streamlines. Contrary to this perspective, we observe that flowing particles migrate across streamlines in a continuous, predictable, and accurate manner in microchannels experiencing laminar flows. The migration is attributed to lift forces on particles that are observed when inertial aspects of the flow become significant. We identified symmetric and asymmetric channel geometries that provide additional inertial forces that bias particular equilibrium positions to create continuous streams of ordered particles precisely positioned in three spatial dimensions. We were able to order particles laterally, within the transverse plane of the channel, with >80-nm accuracy, and longitudinally, in regular chains along the direction of flow. A fourth dimension of rotational alignment was observed for discoidal red blood cells. Unexpectedly, ordering appears to be independent of particle buoyant direction, suggesting only minor centrifugal contributions. Theoretical analysis indicates the physical principles are operational over a range of channel and particle length scales. The ability to differentially order particles of different sizes, continuously, at high rates, and without external forces in microchannels is expected to have a broad range of applications in continuous bioparticle separation, high-throughput cytometry, and large-scale filtration systems.

Polar Stimulation and Constrained Cell Migration in Microfluidic Channels

Lab on a Chip. Dec, 2007  |  Pubmed ID: 18030401

Asymmetrical delivery of stimuli to moving cells for perturbing spatially-heterogeneous intracellular signaling is an experimental challenge not adequately met by existing technologies. Here, we report a robust microfluidic platform allowing localized treatment of the front and/or back of moving cells which crawl through narrow channels that they completely occlude. The enabling technical element for this study is a novel design for precise, passive balancing of flow inside the microfluidic device by contacting two fluid streams before splitting them again. The microchannels constrain cell morphology and induce qualitative and quantitative changes in neutrophil chemotaxis that mimic cells crawling through tissues.

Oxygen Uptake Rates and Liver-specific Functions of Hepatocyte and 3T3 Fibroblast Co-cultures

Biotechnology and Bioengineering. May, 2007  |  Pubmed ID: 17054120

Bioartificial liver (BAL) devices have been developed to treat patients undergoing acute liver failure. One of the most important parameters to consider in designing these devices is the oxygen consumption rate of the seeded hepatocytes which are known to have oxygen consumption rates 10 times higher than most other cell types. Hepatocytes in various culture configurations have been tested in BAL devices including those formats that involve co-culture of hepatocytes with other cell types. In this study, we investigated, for the first time, oxygen uptake rates (OUR)s of hepatocytes co-cultured with 3T3-J2 fibroblasts at various hepatocyte to fibroblast seeding ratios. OURs were determined by measuring the rate of oxygen disappearance using a ruthenium-coated optical probe after closing and sealing the culture dish. Albumin and urea production rates were measured to assess hepatocyte function. Lower hepatocyte density co-cultures demonstrated significantly higher OURs (2 to 3.5-fold) and liver- specific functions (1.6-fold for albumin and 4.5-fold for urea production) on a per cell basis than those seeded at higher densities. Increases in OUR correlated well with increased liver-specific functions. OURs (V(m)) were modeled by fitting Michaelis-Menten kinetics and the model predictions closely correlated with the experimental data. This study provides useful information for predicting BAL design parameters that will avoid oxygen limitations, as well as maximize metabolic functions.

A Bioartificial Liver Device Secreting Interleukin-1 Receptor Antagonist for the Treatment of Hepatic Failure in Rats

The Journal of Surgical Research. Jan, 2007  |  Pubmed ID: 17081566

Liver transplantation is the treatment of choice for many patients with fulminant hepatic failure (FHF). A major limitation of this treatment is the lack of available donors. An optimally functioning bio-artificial liver (BAL) device has the potential to provide critical hepatic support to patients with FHF. In this study, we examined the efficacy of combining interleukin-1 (IL-1) receptor blockade with the synthetic function of hepatocytes in a BAL device for the treatment of FHF.

A High-throughput Microfluidic Real-time Gene Expression Living Cell Array

Lab on a Chip. Jan, 2007  |  Pubmed ID: 17180208

The dynamics of gene expression are fundamental to the coordination of cellular responses. Measurement of temporal gene expression patterns is currently limited to destructive low-throughput techniques such as northern blotting, reverse transcription polymerase chain reaction (RT-PCR), and DNA microarrays. We report a scalable experimental platform that combines microfluidic addressability with quantitative live cell imaging of fluorescent protein transcriptional reporters to achieve real-time characterization of gene expression programs in living cells. Integrated microvalve arrays control row-seeding and column-stimulation of 256 nanoliter-scale bioreactors to create a high density matrix of stimulus-response experiments. We demonstrate the approach in the context of hepatic inflammation by acquiring approximately 5000 single-time-point measurements in each automated and unattended experiment. Experiments can be assembled in hours and perform the equivalent of months of conventional experiments. By enabling efficient investigation of dynamic gene expression programs, this technology has the potential to make significant impacts in basic science, drug development, and clinical medicine.

A Microfluidic Device for Practical Label-free CD4(+) T Cell Counting of HIV-infected Subjects

Lab on a Chip. Feb, 2007  |  Pubmed ID: 17268618

Practical HIV diagnostics are urgently needed in resource-limited settings. While HIV infection can be diagnosed using simple, rapid, lateral flow immunoassays, HIV disease staging and treatment monitoring require accurate counting of a particular white blood cell subset, the CD4(+) T lymphocyte. To address the limitations of current expensive, technically demanding and/or time-consuming approaches, we have developed a simple CD4 counting microfluidic device. This device uses cell affinity chromatography operated under differential shear flow to specifically isolate CD4(+) T lymphocytes with high efficiency directly from 10 microliters of unprocessed, unlabeled whole blood. CD4 counts are obtained under an optical microscope in a rapid, simple and label-free fashion. CD4 counts determined in our device matched measurements by conventional flow cytometry among HIV-positive subjects over a wide range of absolute CD4 counts (R(2) = 0.93). This CD4 counting microdevice can be used for simple, rapid and affordable CD4 counting in point-of-care and resource-limited settings.

Multifunctional Encoded Particles for High-throughput Biomolecule Analysis

Science (New York, N.Y.). Mar, 2007  |  Pubmed ID: 17347435

High-throughput screening for genetic analysis, combinatorial chemistry, and clinical diagnostics benefits from multiplexing, which allows for the simultaneous assay of several analytes but necessitates an encoding scheme for molecular identification. Current approaches for multiplexed analysis involve complicated or expensive processes for encoding, functionalizing, or decoding active substrates (particles or surfaces) and often yield a very limited number of analyte-specific codes. We present a method based on continuous-flow lithography that combines particle synthesis and encoding and probe incorporation into a single process to generate multifunctional particles bearing over a million unique codes. By using such particles, we demonstrate a multiplexed, single-fluorescence detection of DNA oligomers with encoded particle libraries that can be scanned rapidly in a flow-through microfluidic channel. Furthermore, we demonstrate with high specificity the same multiplexed detection using individual multiprobe particles.

Patterned Co-culture of Primary Hepatocytes and Fibroblasts Using Polyelectrolyte Multilayer Templates

Macromolecular Bioscience. Mar, 2007  |  Pubmed ID: 17370273

This paper describes the formation of patterned cell co-cultures using the layer-by-layer deposition of synthetic ionic polymers and without the aid of adhesive proteins/ligands such as collagen or fibronectin. In this study, we used synthetic polymers, namely poly(diallyldimethylammonium chloride) (PDAC) and sulfonated polystyrene (SPS) as the polycation and polyanion, respectively, to build the multilayer films. We formed SPS patterns on polyelectrolyte multilayer (PEM) surfaces either by microcontact printing PDAC onto SPS surfaces or vice-versa. To create patterned co-cultures on PEMs, we capitalize on the preferential attachment and spreading of primary hepatocytes on SPS as opposed to PDAC surfaces. In contrast, fibroblasts readily attached to both PDAC and SPS surfaces, and as a result, we were able to obtain patterned co-cultures of fibroblast and primary hepatocytes on synthetic PEM surfaces. We characterized the morphology and hepatic-specific functions of the patterned cell co-cultures with microscopy and biochemical assays. Our results suggest an alternative approach to fabricating controlled co-cultures with specified cell-cell and cell-surface interactions; this approach provides flexibility in designing cell-specific surfaces for tissue engineering applications.

A Microchip Approach for Practical Label-free CD4+ T-cell Counting of HIV-infected Subjects in Resource-poor Settings

Journal of Acquired Immune Deficiency Syndromes (1999). Jul, 2007  |  Pubmed ID: 17414933

Simple affordable CD4 cell counting is urgently needed to stage and monitor HIV-infected patients in resource-limited settings. To address the limitations of current approaches, we designed a simple, label-free, and cost-effective CD4 cell counting device using microfluidic technology. We previously described the fabrication of a microfluidic system for high-efficiency isolation of pure populations of CD4+ T cells based on cell affinity chromatography operated under controlled flow. Here, we compare the performance of a microfluidic CD4 cell counting device against standard flow cytometry in 49 HIV-positive subjects over a wide range of absolute CD4 cell counts. We observed a close correlation between CD4 cell counts from the microchip device and measurements by flow cytometry, using unprocessed whole blood from HIV-positive adult subjects. Sensitivities for distinguishing clinically relevant thresholds of 200, 350, and 500 cells/microL are 0.86, 0.90, and 0.97, respectively. Specificity is 0.94 or higher at all thresholds. This device can serve as a functional cartridge for fast, accurate, affordable, and simple CD4 cell counting in resource-limited settings.

Isolation of Rare Circulating Tumour Cells in Cancer Patients by Microchip Technology

Nature. Dec, 2007  |  Pubmed ID: 18097410

Viable tumour-derived epithelial cells (circulating tumour cells or CTCs) have been identified in peripheral blood from cancer patients and are probably the origin of intractable metastatic disease. Although extremely rare, CTCs represent a potential alternative to invasive biopsies as a source of tumour tissue for the detection, characterization and monitoring of non-haematologic cancers. The ability to identify, isolate, propagate and molecularly characterize CTC subpopulations could further the discovery of cancer stem cell biomarkers and expand the understanding of the biology of metastasis. Current strategies for isolating CTCs are limited to complex analytic approaches that generate very low yield and purity. Here we describe the development of a unique microfluidic platform (the 'CTC-chip') capable of efficient and selective separation of viable CTCs from peripheral whole blood samples, mediated by the interaction of target CTCs with antibody (EpCAM)-coated microposts under precisely controlled laminar flow conditions, and without requisite pre-labelling or processing of samples. The CTC-chip successfully identified CTCs in the peripheral blood of patients with metastatic lung, prostate, pancreatic, breast and colon cancer in 115 of 116 (99%) samples, with a range of 5-1,281 CTCs per ml and approximately 50% purity. In addition, CTCs were isolated in 7/7 patients with early-stage prostate cancer. Given the high sensitivity and specificity of the CTC-chip, we tested its potential utility in monitoring response to anti-cancer therapy. In a small cohort of patients with metastatic cancer undergoing systemic treatment, temporal changes in CTC numbers correlated reasonably well with the clinical course of disease as measured by standard radiographic methods. Thus, the CTC-chip provides a new and effective tool for accurate identification and measurement of CTCs in patients with cancer. It has broad implications in advancing both cancer biology research and clinical cancer management, including the detection, diagnosis and monitoring of cancer.

Invention, Innovation, Entrepreneurship in Academic Medical Centers

Surgery. Feb, 2008  |  Pubmed ID: 18242329

Equilibrium Separation and Filtration of Particles Using Differential Inertial Focusing

Analytical Chemistry. Mar, 2008  |  Pubmed ID: 18275222

Rapid separation and filtration of particles in solution has a wide range of applications including blood cell separation, ultrasound contrast agent preparation, and purification of fermentation products. However, current techniques that provide quick processing rates are high in complexity. We present a rapid microfluidic filtration technology capable of separating particles based on size, with purities from 90 to 100% and high-volume throughputs of 1 mL/min. Data for separation of rigid particles, deformable emulsions, and platelets from whole blood are presented. The system is based upon differential inertial focusing of particles of varying sizes and allows continuous separation based only on intrinsic hydrodynamic forces developed in a flow through an asymmetrically curved channel. A theoretical description of the underlying forces is developed, and in combination with data determining a size cutoff for separation, a semiempirical relationship describing how channel geometry is related to this cutoff is shown. Cascading separations in series is shown to be useful for increasing purity and yield. This type of microfluidic system can filter deformable particles, is largely independent of particle density, and can provide throughputs typical of macroscale filtration in a compact format, enabling applications in blood filtration and particle concentration.

Microfluidic Leukocyte Isolation for Gene Expression Analysis in Critically Ill Hospitalized Patients

Clinical Chemistry. May, 2008  |  Pubmed ID: 18375483

Microarray technology is becoming a powerful tool for diagnostic, therapeutic, and prognostic applications. There is at present no consensus regarding the optimal technique to isolate nucleic acids from blood leukocyte populations for subsequent expression analyses. Current collection and processing techniques pose significant challenges in the clinical setting. Here, we report the clinical validation of a novel microfluidic leukocyte nucleic acid isolation technique for gene expression analysis from critically ill, hospitalized patients that can be readily used on small volumes of blood.

Elastomeric Microchip Electrospray Emitter for Stable Cone-jet Mode Operation in the Nanoflow Regime

Analytical Chemistry. May, 2008  |  Pubmed ID: 18419138

Despite widespread interest in combining laboratory-on-a-chip technologies with mass spectrometry (MS)-based analyses, the coupling of microfluidics to electrospray ionization (ESI)-MS remains challenging. We report a robust, integrated poly(dimethylsiloxane) microchip interface for ESI-MS using simple and widely accessible microfabrication procedures. The interface uses an auxiliary channel to provide electrical contact for the stable cone-jet electrospray without sample loss or dilution. The electric field at the channel terminus is enhanced by two vertical cuts that cause the interface to taper to a line rather than to a point, and the formation of a small Taylor cone at the channel exit ensures subnanoliter postcolumn dead volumes. Cone-jet mode electrospray was demonstrated for up to 90% aqueous solutions and for extended durations. Comparable ESI-MS sensitivities were achieved using both microchip and conventional fused silica capillary emitters, but stable cone-jet mode electrosprays could be established over a far broader range of flow rates (from 50-1000 nL/min) and applied potentials using the microchip emitters. This attribute of the microchip emitter should simplify electrospray optimization and make the stable electrospray more resistant to external perturbations.

Rotationally Oscillating Drill (Ros-Drill) for Mouse ICSI Without Using Mercury

Molecular Reproduction and Development. Dec, 2008  |  Pubmed ID: 18437690

Intracytoplasmic sperm injection (ICSI) is an important assisted reproductive technology (ART). Due to deployment difficulties and low efficiency of the earlier (conventional) version of ICSI, especially in the mouse, a piezo-assisted ICSI technique had evolved as a popular ART methodology in recent years. An important and remaining problem with this technique, however, is that it requires small amounts of mercury to stabilize the pipette tip when piezoelectric force pulses are applied. To eliminate this problem we developed and tested a completely different and mercury-free technology, called the "Ros-Drill" (rotationally oscillating drill). The technique uses microprocessor-controlled rotational oscillations on a spiked micropipette without mercury or piezo. Preliminary experimental results show that this new microinjection technology gives high survival rate (>70% of the injected oocytes) and fertilization rate (>80% of the survived oocytes), and blastocyst formation rates in early trials (approximately 50% of the survived oocytes). Blastocysts created by Ros-Drill ICSI were transferred into the uteruses of pseudopregnant surrogate mothers and healthy pups were born and weaned. The Ros-Drill ICSI technique is automated and therefore; it requires a very short preliminary training for the specialists, as evidenced in many successful biological trials. These advantages of Ros-Drill ICSI over conventional and piezo-assisted ICSI are clearly demonstrated and it appears to have resolved an important problem in reproductive biology.

Vitrification by Ultra-fast Cooling at a Low Concentration of Cryoprotectants in a Quartz Micro-capillary: a Study Using Murine Embryonic Stem Cells

Cryobiology. Jun, 2008  |  Pubmed ID: 18462712

Conventional cryopreservation protocols for slow-freezing or vitrification involve cell injury due to ice formation/cell dehydration or toxicity of high cryoprotectant (CPA) concentrations, respectively. In this study, we developed a novel cryopreservation technique to achieve ultra-fast cooling rates using a quartz micro-capillary (QMC). The QMC enabled vitrification of murine embryonic stem (ES) cells using an intracellular cryoprotectant concentration in the range used for slowing freezing (1-2M). The cryoprotectants used included 2M 1,2-propanediol (PROH, cell membrane permeable) and 0.5M extracellular trehalose (cell membrane impermeable). More than 70% of the murine ES cells post-vitrification attached with respect to non-frozen control cells, and the proliferation rates of the two groups were similar. Preservation of undifferentiated properties of the pluripotent murine ES cells post-vitrification cryopreservation was verified using three different types of assays: the expression of transcription factor Oct-4, the presentation of the membrane surface glycoprotein SSEA-1, and the elevated expression of the intracellular enzyme alkaline phosphatase. These results indicate that vitrification at a low concentration (2M) of intracellular cryoprotectants is a viable and effective approach for the cryopreservation of murine embryonic stem cells.

Cell-cell Interaction Modulates Neuroectodermal Specification of Embryonic Stem Cells

Neuroscience Letters. Jun, 2008  |  Pubmed ID: 18467031

The controlled differentiation of embryonic stem (ES) cells is of utmost interest to their clinical, biotechnological, and basic science use. Many investigators have combinatorially assessed the role of specific soluble factors and extracellular matrices in guiding ES cell fate, yet the interaction between neighboring cells in these heterogeneous cultures has been poorly defined due to a lack of conventional tools to specifically uncouple these variables. Herein, we explored the role of cell-cell interactions during neuroectodermal specification of ES cells using a microfabricated cell pair array. We tracked differentiation events in situ, using an ES cell line expressing green fluorescent protein (GFP) under the regulation of the Sox1 gene promoter, an early marker of neuroectodermal germ cell commitment in the adult forebrain. We observed that a previously specified Sox1-GFP+ cell could induce the specification of an undifferentiated ES cell. This induction was modulated by the two cells being in contact and was dependent on the age of previously specified cell prior to coculture. A screen of candidate cell adhesion molecules revealed that the expression of connexin (Cx)-43 correlated with the age-dependent effect of cell contact in cell pair experiments. ES cells deficient in Cx-43 showed aberrant neuroectodermal specification and lineage commitment, highlighting the importance of gap junctional signaling in the development of this germ layer. Moreover, this study demonstrates the integration of microscale culture techniques to explore the biology of ES cells and gain insight into relevant developmental processes otherwise undefined due to bulk culture methods.

Desiccation Kinetics and Biothermodynamics of Glass Forming Trehalose Solutions in Thin Films

Annals of Biomedical Engineering. Aug, 2008  |  Pubmed ID: 18500553

In this study, the desiccation kinetics of aqueous trehalose solutions were investigated numerically by solving the coupled heat and mass transfer problem with a moving interface using the finite element method. The free volume models for vapor pressure and mutual diffusion coefficient were incorporated into the model to account for the effect of glass transition on the heat and mass transport process that ultimately determines the desiccation kinetics. It was found that the temperature in the film could drop significantly upon the initiation of drying due to the absorption of latent heat associated with water evaporation although the spatial distribution of temperature in the solution is very homogeneous. On the contrary, the spatial distribution of water content in the solution is non-homogeneous, particularly at the solution-vapor interface where an extremely thin layer of skin with extremely low molecular mobility usually forms during drying. The solution film can be dried to approximately 6-10 wt.% residual water within minutes for thin films; but drying times depends strongly on the initial film thickness, initial solution concentration, temperature, and convective coefficient. Desiccation to below 6 wt.% residual water is very slow due to the retarded water mobility in the extremely thin skin where the solution is in the glassy state. Since the water mobility in a trehalose solution or glass with 6-10% residual water is still high enough to allow degradative reactions to occur in a relatively short time at room temperature, it is important that the samples should be kept at a temperature around 0 degrees C or lower for storage after drying. Furthermore, approaches that might enable further quick reduction of the residual water to less than 6-10 wt.% are also proposed so that a sample could be preserved at super-zero or even room temperature. The established models and the reported results will be useful for the development of effective protocols for lyopreservation of biomaterials including living cells using trehalose as the excipient.

Enhanced Differentiation of Embryonic Stem Cells Using Co-cultivation with Hepatocytes

Biotechnology and Bioengineering. Dec, 2008  |  Pubmed ID: 18571804

We examined the effects of co-cultivated hepatocytes on the hepatospecific differentiation of murine embryonic stem (ES) cells. Utilizing an established mouse ES cell line expressing high or low levels of E-cadherin, that we have previously shown to be responsive to hepatotrophic growth factor stimulation (Dasgupta et al., 2005. Biotechnol Bioeng 92(3):257-266), we compared co-cultures of cadherin-expressing ES (CE-ES) cells with cultured rat hepatocytes, allowing for either paracrine interactions (indirect co-cultures) or both juxtacrine and paracrine interactions (direct co-cultures, random and patterned). Hepatospecific differentiation of ES cells was evaluated in terms of hepatic-like cuboidal morphology, heightened gene expression of late maturation marker, glucose-6-phosphatase in relation to early marker, alpha-fetoprotein (AFP), and the intracellular localization of albumin. Hepatocytes co-cultured with growth factor primed CE-ES cells markedly enhanced ES cell differentiation toward the hepatic lineage, an effect that was reversed through E-cadherin blockage and inhibited in control ES cells with reduced cadherin expression. Comparison of single ES cell cultures versus co-cultures show that direct contact co-cultures of hepatocytes and CE-ES cells maximally promoted ES cell commitment towards hepatodifferentiation, suggesting cooperative effects of cadherin-based juxtacrine and paracrine interactions. In contrast, E-cadherin deficient mouse ES (CD-ES) cells co-cultured with hepatocytes failed to show increased G6P expression, confirming the role of E-cadherin expression. To establish whether albumin expression in CE-ES cells was spatially regulated by co-cultured hepatocytes, we co-cultivated CE-ES cells around micropatterned, pre-differentiated rat hepatocytes. Albumin localization was enhanced "globally" within CE-ES cell colonies and was inhibited through E-cadherin antibody blockage in all but an interfacial band of ES cells. Thus, stem cell based cadherin presentation may be an effective tool to induce hepatotrophic differentiation by leveraging both distal/paracrine and contact/juxtacrine interactions with primary cells of the liver.

'Living Cantilever Arrays' for Characterization of Mass of Single Live Cells in Fluids

Lab on a Chip. Jul, 2008  |  Pubmed ID: 18584076

The size of a cell is a fundamental physiological property and is closely regulated by various environmental and genetic factors. Optical or confocal microscopy can be used to measure the dimensions of adherent cells, and Coulter counter or flow cytometry (forward scattering light intensity) can be used to estimate the volume of single cells in a flow. Although these methods could be used to obtain the mass of single live cells, no method suitable for directly measuring the mass of single adherent cells without detaching them from the surface is currently available. We report the design, fabrication, and testing of 'living cantilever arrays', an approach to measure the mass of single adherent live cells in fluid using silicon cantilever mass sensor. HeLa cells were injected into microfluidic channels with a linear array of functionalized silicon cantilevers and the cells were subsequently captured on the cantilevers with positive dielectrophoresis. The captured cells were then cultured on the cantilevers in a microfluidic environment and the resonant frequencies of the cantilevers were measured. The mass of a single HeLa cell was extracted from the resonance frequency shift of the cantilever and was found to be close to the mass value calculated from the cell density from the literature and the cell volume obtained from confocal microscopy. This approach can provide a new method for mass measurement of a single adherent cell in its physiological condition in a non-invasive manner, as well as optical observations of the same cell. We believe this technology would be very valuable for single cell time-course studies of adherent live cells.

Detection of Mutations in EGFR in Circulating Lung-cancer Cells

The New England Journal of Medicine. Jul, 2008  |  Pubmed ID: 18596266

The use of tyrosine kinase inhibitors to target the epidermal growth factor receptor gene (EGFR) in patients with non-small-cell lung cancer is effective but limited by the emergence of drug-resistance mutations. Molecular characterization of circulating tumor cells may provide a strategy for noninvasive serial monitoring of tumor genotypes during treatment.

Microflow and Crack Formation Patterns in Drying Sessile Droplets of Liposomes Suspended in Trehalose Solutions

Langmuir : the ACS Journal of Surfaces and Colloids. Aug, 2008  |  Pubmed ID: 18613701

Anhydrobiotic preservation potentially provides a means of long-term storage of mammalian cells in carbohydrate glasses under ambient conditions. During desiccation, sessile droplets of glass-forming carbohydrate solutions exhibit complex phenomena, including fluid flow, droplet deformation, and crack formation, all of which may alter the cell preservation efficacy. Cell-sized liposomes were employed as a model system to explore these phenomena in diffusively dried sessile droplets of trehalose solutions. Two factors were identified that strongly influenced the features of the desiccated droplets: the underlying surface and the liposomes themselves. In particular, the surface altered the droplet shape as well as the microflow pattern and, in turn, the moisture conditions encountered by the liposomes during desiccation. A ring deposit formed when the droplets were dried on polystyrene, as would be expected owing to the capillary flow that generally occurs in pinned droplets. In contrast, when dried on the more hydrophilic glass slide, the resulting droplets were thinner, and the liposomes accumulated near their centers, which was an unexpected result likely owing to the glass-forming nature of trehalose solutions. As might be anticipated given the variations in liposome distribution, the choice of surface also influenced crack formation upon continued drying. In addition to providing a preferential path for drying, such cracks are relevant because they could inflict mechanical damage on cells. The liposomes themselves had an even more profound effect on crack formation; indeed, whereas cracks were found in all droplets containing liposomes, in their absence few of the droplets cracked at all, regardless of the surface type. These complex drying dynamics merit further investigation in the development of anhydrobiotic preservation protocols, particularly with regard to the role therein of surface hydrophobicity and the cells themselves.

Controlled Encapsulation of Single-cells into Monodisperse Picolitre Drops

Lab on a Chip. Aug, 2008  |  Pubmed ID: 18651066

Encapsulation of cells within picolitre-size monodisperse drops provides new means to perform quantitative biological studies on a single-cell basis for large cell populations. Variability in the number of cells per drop due to stochastic cell loading is a major barrier to these techniques. We overcome this limitation by evenly spacing cells as they travel within a high aspect-ratio microchannel; cells enter the drop generator with the frequency of drop formation.

Microvortex for Focusing, Guiding and Sorting of Particles

Lab on a Chip. Dec, 2008  |  Pubmed ID: 19023476

We report a microvortex manipulator (MVM) that is a passive, scalable system with great potential for the manipulation and separation of particulate samples in microfluidic environments. The movement of particles is determined by a unique combination of helical flow, buoyant, and gravitational forces. Helical flows are induced by topographically patterned microchannel surfaces, which have previously been used for molecular mixing in microfluidic devices. We illustrate the mechanism of MVM and its applications in passive focusing of beads and cells into parallel streams and guiding of particles and cells. We also explore the application of the unique density-selectivity of microvortex focusing and successfully sort a mixture of two bead populations whose density difference is as small as 0.1 g cm(-3).

Rapid Appearance of Resolvin Precursors in Inflammatory Exudates: Novel Mechanisms in Resolution

Journal of Immunology (Baltimore, Md. : 1950). Dec, 2008  |  Pubmed ID: 19050288

Resolution of inflammation is essential. Although supplementation of omega-3 fatty acids is widely used, their availability at sites of inflammation is not known. To this end, a multidisciplinary approach was taken to determine the relationship of circulating omega-3 to inflammatory exudates and the generation of resolution signals. In this study, we monitored resolvin precursors in evolving exudates, which initially paralleled increases in edema and infiltrating neutrophils. We also prepared novel microfluidic chambers to capture neutrophils from a drop of blood within minutes that permitted single-cell monitoring. In these, docosahexaenoic acid-derived resolvin D1 rapidly stopped neutrophil migration, whereas precursor docosahexaenoic acid did not. In second organ injury via ischemia-reperfusion, resolvin metabolically stable analogues were potent organ protectors reducing neutrophils. Together, these results indicate that circulating omega-3 fatty acids rapidly appear in inflammatory sites that require conversion to resolvins that control excessive neutrophil infiltration, protect organs, and foster resolution.

Neutrophil Migration Assay from a Drop of Blood

Lab on a Chip. Dec, 2008  |  Pubmed ID: 19023468

Neutrophil directional migration in response to chemical gradients, also known as chemotaxis, is one of the key phenomena in the immune responses against bacterial infection. To better study neutrophils chemotaxis, several in vitro assays have been developed that replicate chemotactic gradients around neutrophils isolated from whole blood. One drawback for most of these assays is the lengthy processing of blood required for neutrophils isolation, which can alter the responsiveness of neutrophils compared to the in vivo conditions. To address this limitation, we have designed a microfluidic chip for chemotaxis studies which can use neutrophils isolated on the chip, directly from whole blood. We have tested three different cell adhesion molecules as substrates for neutrophil isolation (P-selectin, E-selectin and fibronectin) and found average capture efficiencies of 20-40 neutrophils/mm2 at optimized concentrations. Subsequent analysis of neutrophil migration in chemoattractant gradients of N-formyl-methyl-leucyl-phenylalanine (fMLP) or Interleukin-8 (IL-8) shows higher average velocities over E-selectin as compared to the P-selectin. Our microfluidic assay uses just a drop of whole blood (<10 microL) for neutrophil isolation and provides a robust platform to perform chemotaxis assays in the competing environment of different chemokines.

Microfluidic Flow-encoded Switching for Parallel Control of Dynamic Cellular Microenvironments

Lab on a Chip. Jan, 2008  |  Pubmed ID: 18094768

The temporal pattern of a biological stimulus is an important determinant of the resulting cellular response. We present a microfluidic parallel perfusion culture system for controlling the dynamics of soluble cell microenvironments while simultaneously performing live-cell imaging of cellular responses. A "Flow-encoded Switching" (FES) design strategy is developed to simultaneously deliver many different temporal profiles of stimuli, including pulse train widths, lengths, and frequencies, to downstream adherent cells using a single input control. The design strategy uses principles of laminar flow and diffusion-limited mixing to encode the state of the network (the instantaneous stimulus concentrations in each channel) into the ratio of two flow rates, which is controlled by a single differential pressure. To demonstrate the utility of this experimental system, we investigated the effect of dynamic stimuli on NFkappaB transcriptional activation and cell fate determination. Our results illustrate that transcriptional responses and cell fate decisions depend both quantitatively and qualitatively on the timing of the stimulus. In summary, by encoding dynamic stimuli in a single input pressure, microfluidic flow-encoded switching offers a scalable experimental method for systematically probing the functional significance of temporally patterned cellular environments.

Radial Flow Hepatocyte Bioreactor Using Stacked Microfabricated Grooved Substrates

Biotechnology and Bioengineering. Feb, 2008  |  Pubmed ID: 17626294

Bioartificial liver (BAL) devices with fully functioning hepatocytes have the potential to provide temporary hepatic support for patients with liver failure. The goal of this study was to optimize the flow environment for the cultured hepatocytes in a stacked substrate, radial flow bioreactor. Photolithographic techniques were used to microfabricate concentric grooves onto the underlying glass substrates. The microgrooves served to protect the seeded hepatocytes from the high shear stresses caused by the volumetric flow rates necessary for adequate convective oxygen delivery. Finite element analysis was used to analyze the shear stresses and oxygen concentrations in the bioreactor. By employing high volumetric flow rates, sufficient oxygen supply to the hepatocytes was possible without an integrated oxygen permeable membrane. To implement this concept, 18 microgrooved glass substrates, seeded with rat hepatocytes cocultured with 3T3-J2 fibroblasts, were stacked in the bioreactor, creating a channel height of 100 microm between each substrate. In this bioreactor configuration, liver-specific functions (i.e., albumin and urea synthesis rates) of the hepatocytes remained stable over 5 days of perfusion, and were significantly increased compared to those in the radial flow bioreactor with stacked substrates without microgrooves. This study suggests that this radial flow bioreactor with stacked microgrooved substrates is scalable and may have potential as a BAL device in the treatment of liver failure.

Differential Inertial Focusing of Particles in Curved Low-aspect-ratio Microchannels

New Journal of Physics. Jul, 2009  |  Pubmed ID: 20862272

Microfluidic-based manipulation of particles is of great interest due to the insight it provides into the physics of hydrodynamic forces. Here, we study a particle-size-dependent phenomenon based on differential inertial focusing that utilizes the flow characteristics of curved, low aspect ratio (channel width ≫ height), microfluidic channels. We report the emergence of two focusing points along the height of the channel (z-plane), where different sized particles are focused and ordered in evenly spaced trains at correspondingly different lateral positions within the channel cross-section. We applied the system for continuous ordering and separation of suspension particles.

Stop-flow Lithography for the Production of Shape-evolving Degradable Microgel Particles

Journal of the American Chemical Society. Apr, 2009  |  Pubmed ID: 19215127

Microgel particles capable of bulk degradation have been synthesized from a solution of diacrylated triblock copolymer composed of poly(ethylene glycol) and poly(lactic acid) in a microfluidic device using stop-flow lithography (SFL). It has been previously demonstrated that SFL can be used to fabricate particles with precise control over particle size and shape. Here, we have fabricated hydrogel particles of varying size and shape and examined their mass-loss and swelling behavior histologically and mechanically. We report that these features, as well as degradation behavior of the hydrogel particles may be tailored with SFL. By reducing the applied UV dose during fabrication, hydrogel particles can be made to exhibit a distinct deviation from the classical erosion profiles of bulk-degrading hydrogels. At higher UV doses, a saturation in cross-linking density occurs and bulk-degrading behavior is observed. Finally, we synthesized multifunctional composite particles, providing unique features not found in homogeneous hydrogels.

Occurrence of Mitochondria-targeted Late Embryogenesis Abundant (LEA) Gene in Animals Increases Organelle Resistance to Water Stress

The Journal of Biological Chemistry. Apr, 2009  |  Pubmed ID: 19228698

Anhydrobiotic animals survive virtually complete loss of cellular water. The mechanisms that explain this phenomenon are not fully understood but often include the accumulation of low molecular weight solutes such as trehalose and macromolecules like Late Embryogenesis Abundant (LEA) proteins. Here we report for the first time the occurrence of a mitochondria-targeted LEA gene (Afrlea3m) product in an animal species. The deduced molecular mass of the 307-amino acid polypeptide from the brine shrimp Artemia franciscana is 34 kDa. Bioinformatic analyses reveal features typical of a Group 3 LEA protein, and subcellular localization programs predict targeting of the mature peptide to the mitochondrial matrix, based on an N-terminal, amphipathic presequence. Real-time quantitative PCR shows that Afralea3m mRNA is expressed manyfold higher in desiccation-tolerant embryonic stages when compared with intolerant nauplius larvae. Mitochondrial localization of the protein was confirmed by transfection of human hepatoma cells (HepG2/C3A) with a nucleotide construct encoding the first 70 N-terminal amino acids of AfrLEA3m in-frame with the nucleotide sequence for green fluorescence protein. The chimeric protein was readily incorporated into mitochondria of these cells. Successful targeting of a protein to human mitochondria by use of an arthropod signaling sequence clearly reveals the highly conserved nature of such presequences, as well as of the import machinery. Finally, mitochondria isolated from A. franciscana embryos, which naturally contain AfrLEA3m and trehalose, exhibit resistance to water stress (freezing) as evidenced by an unchanged capacity for oxidative phosphorylation on succinate + rotenone, a resistance that is absent in mammalian mitochondria lacking AfrLEA3m.

The CTC-chip: an Exciting New Tool to Detect Circulating Tumor Cells in Lung Cancer Patients

Journal of Thoracic Oncology : Official Publication of the International Association for the Study of Lung Cancer. Mar, 2009  |  Pubmed ID: 19247082

Circulating tumor cells (CTCs) are rare cells that originate from a malignancy and circulate freely in the peripheral blood. The ability to capture and study CTCs is an emerging field with implications for early detection, diagnosis, determining prognosis and monitoring of cancer, as well as for understanding the fundamental biology of the process of metastasis. Here, we review the development and initial clinical studies with a novel microfluidic platform for isolating these cells, the CTC-chip, and discuss its potential uses in the study of lung cancer.

High-throughput Single Cell Arrays As a Novel Tool in Biopreservation

Cryobiology. Jun, 2009  |  Pubmed ID: 19303403

Microwell array cytometry is a novel high-throughput experimental technique that makes it possible to correlate pre-stress cell phenotypes and post-stress outcomes with single cell resolution. Because the cells are seeded in a high density grid of cell-sized microwells, thousands of individual cells can be tracked and imaged through manipulations as extreme as freezing or drying. Unlike flow cytometry, measurements can be made at multiple time points for the same set of cells. Unlike conventional image cytometry, image analysis is greatly simplified by arranging the cells in a spatially defined pattern and physically separating them from one another. To demonstrate the utility of microwell array cytometry in the field of biopreservation, we have used it to investigate the role of mitochondrial membrane potential in the cryopreservation of primary hepatocytes. Even with optimized cryopreservation protocols, the stress of freezing almost always leads to dysfunction or death in part of the cell population. To a large extent, cell fate is dominated by the stochastic nature of ice crystal nucleation, membrane rupture, and other biophysical processes, but natural variation in the initial cell population almost certainly plays an important and under-studied role. Understanding why some cells in a population are more likely to survive preservation will be invaluable for the development of new approaches to improve preservation yields. For this paper, primary hepatocytes were seeded in microwell array devices, imaged using the mitochondrial dyes Rh123 or JC-1, cryopreserved for up to a week, rapidly thawed, and checked for viability after a short recovery period. Cells with a high mitochondrial membrane potential before freezing were significantly less likely to survive the freezing process, though the difference in short term viability was fairly small. The results demonstrate that intrinsic cell factors do play an important role in cryopreservation survival, even in the short term where extrinsic biophysical factors would be expected to dominate. We believe that microwell array cytometry will be an important tool for a wide range of studies in biopreservation and stress biology.

Desiccation Tolerance in Bovine Sperm: a Study of the Effect of Intracellular Sugars and the Supplemental Roles of an Antioxidant and a Chelator

Cryobiology. Jun, 2009  |  Pubmed ID: 19318090

Desiccation preservation holds promise as a simplified alternative to cryopreservation for the long term storage of cells. We report a study on the protective effects of intracellular and extracellular sugars during bovine sperm desiccation and the supplemental effects of the addition of an antioxidant (catalase) or a chelator (desferal). The goal of the study was to preserve mammalian sperm in a partially or completely desiccated state. Sperm loaded intracellularly with two different types of sugars, trehalose or sucrose, were dried with and without catalase and desferal and evaluated for motility and membrane integrity immediately after rehydration. Intracellular sugars were loaded using ATP induced poration. Drying was performed in desiccator boxes maintained at 11% relative humidity (RH). Results indicated that sperm exhibited improved desiccation tolerance if they were loaded with either intracellular trehalose or sucrose. Survival was further enhanced by the addition of 1mM desferal to the desiccation buffer. Though sperm motility after drying to low dry basis water fractions (DBWF) did not show significant improvement under any of the tested conditions, there was an increase in the sperm membrane integrity that could be retained after partial desiccation through the use of intracellular sugars and desferal.

Comparison Between Ideal and Nonideal Solution Models for Single-cell Cryopreservation Protocols

The Journal of Physical Chemistry. B. Apr, 2009  |  Pubmed ID: 19338369

Models for cell dehydration during a cryopreservation protocol are usually based on the hypothesis of ideal dilute solution. The strong electrolyte character of NaCl makes us revisit these models. The case of nonideal solution is analyzed by computing the dehydration curves without this additional hypothesis. The conclusion is that, in general, while the application of the ideal dilute solution hypothesis is convenient in many cases, for some specific cooling rates there exist important differences in the degree of dehydration predicted by these two models in the studied cases of mouse sperm and hepatocyte. It is shown how this finding has relevant implications for the design and optimization of cryopreservation protocols.

Further Optimization of Mouse Spermatozoa Evaporative Drying Techniques

Cryobiology. Aug, 2009  |  Pubmed ID: 19375415

It has been shown in the past that mouse spermatozoa could be dried under a stream of nitrogen gas at ambient temperature and stored at 4 degrees C or 22 degrees C for up to 3 months and was capable of generating live-born offspring. In previous desiccation work, dried sperm were stored in a vacuum-sealed plastic bag placed in a vacuum-packed Mylar bag. However, dried specimens stored in this way often lost moisture, particularly in samples stored at higher temperatures (22 degrees C) compared to lower temperatures (4 degrees C). The present report describes a method which minimizes this water loss from the dried sperm samples. Its use is described in a preliminary study on the effect of supplementing the trehalose with glycerol. The results have demonstrated that mouse sperm can be stored at 4 degrees C over saturated NaBr without the uptake of water which occurs when they are stored in Mylar packages. In addition, we were able to get some survival of sperm (9-15%) at room temperature storage after 3 months. The addition of glycerol to trehalose had little effect on the survival of dried mouse sperm stored over NaBr for 1 and 3 months.

Particle Segregation and Dynamics in Confined Flows

Physical Review Letters. Mar, 2009  |  Pubmed ID: 19392526

Nonlinearity in finite-Reynolds-number flow results in particle migration transverse to fluid streamlines, producing the well-known "tubular pinch effect" in cylindrical pipes. Here we investigate these nonlinear effects in highly confined systems where the particle size approaches the channel dimensions. Experimental and numerical results reveal distinctive dynamics, including complex scaling of lift forces with channel and particle geometry. The unique behavior described in this Letter has broad implications for confined particulate flows.

Enhancing the Performance of a Point-of-care CD4+ T-cell Counting Microchip Through Monocyte Depletion for HIV/AIDS Diagnostics

Lab on a Chip. May, 2009  |  Pubmed ID: 19417901

CD4+ T cell counts are important tests used to stage HIV-positive patients, enabling clinicians to make informed antiretroviral treatment decisions and to monitor the therapeutic outcomes. However, state-of-the-art CD4 counting methods based on flow cytometry are not applicable in resource-limited settings, due to their high cost and technical requirements. In previous work, we reported the development of a cell isolation microchip that can be used at the point of care for CD4 counts. In that microfluidic chip, CD4+ T cells were separated from 10 microL of whole blood, and enumerated via either light microscopy or impedance sensing. The microchip counts matched flow cytometry results in the intermediate CD4 count range, between 200-800 cells/microL, but displayed a positive bias at absolute CD4 counts below 200 cells/microL, due largely to monocyte contamination. To enhance the performance in the low CD4 count range, we report here an improved design of a two-stage microfluidic device to deplete monocytes from whole blood, followed by CD4+ T cell capture. Using the double-stage device combined with a high viscosity rinsing solution, we obtained microchip CD4 counts comparable to flow cytometry results in the full clinically relevant range. In addition to CD4 counting, the strategy of contaminant depletion prior to target cell isolation can be easily adapted to immunoaffinity capture of other cell types that lack a unique surface marker from a complex biological fluid.

Adaptive-control Model for Neutrophil Orientation in the Direction of Chemical Gradients

Biophysical Journal. May, 2009  |  Pubmed ID: 19450463

Neutrophils have a remarkable ability to detect the direction of chemoattractant gradients and move directionally in response to bacterial infections and tissue injuries. For their role in health and disease, neutrophils have been extensively studied, and many of the molecules involved in the signaling mechanisms of gradient detection and chemotaxis have been identified. However, the cellular-scale mechanisms of gradient sensing and directional neutrophil migration have been more elusive, and existent models provide only limited insight into these processes. Here, we propose a what we believe is a novel adaptive-control model for the initiation of cell polarization in response to gradients. In this model, the neutrophils first sample the environment by extending protrusions in random directions and subsequently adapt their sensitivity depending on localized, temporal changes in stimulation levels. Our results suggest that microtubules may play a critical role in integrating all the sensing events from the cellular periphery through their redistribution inside the neutrophils, and may also be involved in modulating local signaling. An unexpected finding was that model neutrophils exhibit significant randomness in timing and directionality of activation, comparable to our experimental observations in microfluidic devices. Moreover, their responses are robust against alterations of the rate and amplitude of the signaling reactions, and for a broad range in chemoattractant concentrations and spatial gradients.

Nucleation and Solidification in Static Arrays of Monodisperse Drops

Lab on a Chip. Jul, 2009  |  Pubmed ID: 19532960

The precise measurement of nucleation and non-equilibrium solidification are vital to fields as diverse as atmospheric science, food processing, cryopreservation and metallurgy. The emulsion technique, where the phase under study is partitioned into many droplets suspended within an immiscible continuous phase, is a powerful method for uncovering rates of nucleation and dynamics of phase changes as it isolates nucleation events to single droplets. However, averaging the behavior of many drops in a bulk emulsion leads to the loss of any drop-specific information, and drop polydispersity clouds the analysis. Here we adapt a microfluidic technique for trapping monodisperse drops in planar arrays to characterize solidification of highly supercooled aqueous solutions of glycerol. This system measured rates of nucleation between 10(-5) and 10(-2) pL(-1) s(-1), yielded an ice-water interfacial energy of 33.4 mJ m(-2) between -38 and -35 degrees C, and enabled the specific dynamics of solidification to be observed for over a hundred drops in parallel without any loss of specificity. In addition to the physical insights gained, the ability to observe the time and temperature of nucleation and subsequent growth of the solid phase in static arrays of uniform drops provides a powerful tool to discover thermodynamic protocols that generate desirable crystal structures.

Chemical Gradient-mediated Melting Curve Analysis for Genotyping of SNPs

Electrophoresis. Jul, 2009  |  Pubmed ID: 19593749

This report describes a microfluidic solid-phase chemical gradient-mediated melting curve analysis method for SNP analysis. The method is based on allele-specific denaturation to discriminate mismatched (MM) from perfectly matched (PM) DNA duplexes upon exposure to linear chemical gradient. PM and MM DNA duplexes conjugated on beads are captured in a microfluidic gradient generator device designed with dams, keeping the beads trapped perpendicular to a gradient generating channel. Two denaturants, formamide and urea, were tested for their ability to destabilize the DNA duplex by competing with Watson-Crick pairing. Upon exposure to the chemical gradient, rapid denaturing profile was monitored in real time using fluorescence microscopy. The results show that the two duplexes exhibit different kinetics of denaturation profiles, enabling discrimination of MM from PM DNA duplexes to score SNP.

Dynamic Effect of Heat Shock Pretreatment on Apoptotic Responses to TNF-alpha in Liver Cells

Journal of Biomechanical Engineering. Jul, 2009  |  Pubmed ID: 19640128

The heat shock (HS) response is a protective mechanism for cells to protect themselves against subsequent lethal stress. HS upregulated heat shock protein (HSP) expression reduced apoptosis following tumor necrosis factor-alpha (TNF-alpha) stimulation. However, vector-mediated overexpression of HSP70 failed to provide similar protection but rather sensitized cells to TNF-alpha induced apoptosis. This may be due to the fact that the kinetics of vector-mediated HSP overexpression is totally different from that of HSP upregulation by HS. We hypothesized that the response depends on the timing of TNF-alpha challenge relative to HSP expression dynamics after HS. Therefore, we investigated the correlation between the dynamic change of HSP expression and the levels of apoptosis induced by TNF-alpha after HS. Hepatoma cells were subjected to mild heat shock at 42 degrees C for 2 h followed by varied recovery times and then treated with TNF-alpha to induce apoptosis. The results from quantitative apoptosis assays using the TUNEL reaction reveal an optimal HS protection window centered around 5 h post-HS against TNF-alpha induced apoptosis. In addition, we found a window extending up to 2 h after HS where HS sensitized cells to TNF-alpha stress. Importantly, the correlation between apoptosis and HSP expression kinetics demonstrates that both high levels of HSPs and proper timing between HS and TNF-alpha stress were critical for optimal protection. Our study establishes a dynamic experimental model for further investigation of HS as a potential clinical approach to target tissue survival or death.

High Throughput Single Cell Bioinformatics

Biotechnology Progress. Nov-Dec, 2009  |  Pubmed ID: 19830811

Advances in systems biology and bioinformatics have highlighted that no cell population is truly uniform and that stochastic behavior is an inherent property of many biological systems. As a result, bulk measurements can be misleading even when particular care has been taken to isolate a single cell type, and measurements averaged over multiple cell populations in a tissue can be as misleading as the average height at an elementary school. There is a growing need for experimental techniques that can provide a combination of single cell resolution, large cell populations, and the ability to track cells over multiple time points. In this article, a microwell array cytometry platform was developed to meet this need and investigate the heterogeneity and stochasticity of cell behavior on a single cell basis. The platform consisted of a microfabricated device with high-density arrays of cell-sized microwells and custom software for automated image processing and data analysis. As a model experimental system, we used primary hepatocytes labeled with fluorescent probes sensitive to mitochondrial membrane potential and free radical generation. The cells were exposed to oxidative stress and the responses were dynamically monitored for each cell. The resulting data was then analyzed using bioinformatics techniques such as hierarchical and k-means clustering to visualize the data and identify interesting features. The results showed that clustering of the dynamic data not only enhanced comparisons between the treatment groups but also revealed a number of distinct response patterns within each treatment group. Heatmaps with hierarchical clustering also provided a data-rich complement to survival curves in a dose response experiment. The microwell array cytometry platform was shown to be powerful, easy to use, and able to provide a detailed picture of the heterogeneity present in cell responses to oxidative stress. We believe that our microwell array cytometry platform will have general utility for a wide range of questions related to cell population heterogeneity, biological stochasticity, and cell behavior under stress conditions.

A Robust Electrical Microcytometer with 3-dimensional Hydrofocusing

Lab on a Chip. Nov, 2009  |  Pubmed ID: 19865723

In this paper, we present a device to electrically count blood cell populations using an AC impedance interrogation technique in a microfabricated cytometer (microcytometer). Specifically, we direct our attention to obtaining the concentration of human CD4+ T lymphocytes (helper T cells), which is a necessary method to diagnose patients for HIV/AIDS and to give an accurate prognosis on the effectiveness of ARV (anti-retroviral) drug treatments. We study the effectiveness of a simple-to-fabricate 3-dimensional (3D) hydrodynamic focusing mechanism through fluidic simulations and corresponding experiments to increase the signal-to-noise ratio of impedance pulses caused by particle translocation and ensure lower variance in particle translocation height through the electrical sensing region. We found that the optimal 3D sheath flow settings result in a 44.4% increase in impedance pulse signal-to-noise ratio in addition to giving a more accurate representation of particle size distribution. Our microcytometer T cell counts closely with those found using an industry-standard flow cytometer for the concentration range of over three orders of magnitude and using a sample volume approximately the size of a drop of blood (approximately 20 microL). In addition, our device displayed the capability to differentiate between live and dead/dying lymphocyte populations. This microcytometer can be the basis of a portable, rapid, inexpensive solution to obtaining live/dead blood cell counts even in the most resource-poor regions of the world.

Successful Cryopreservation of Mouse Oocytes by Using Low Concentrations of Trehalose and Dimethylsulfoxide

Biology of Reproduction. Jan, 2009  |  Pubmed ID: 18815355

Sugars such as trehalose, sucrose, and glucose are effectively used by a variety of animals (e.g., brine shrimp, tardigrades, some frogs, and insects), as well as by bacteria, yeasts, and plant seeds to survive freezing and extreme drying. The objective of this study was to examine the potential application of sugars to mammalian oocyte cryopreservation. To this end, we used trehalose, a nonreducing disaccharide, and mouse metaphase II oocytes as models. Our experiments show that extracellular trehalose alone affords some protection at high subzero temperatures (e.g., -15 degrees C), which diminishes with further cooling of the oocytes to -30 degrees C and below. When present both intracellularly and extracellularly, trehalose dramatically improves the cryosurvival with increasing extracellular concentrations to 0.5 M, even after cooling to -196 degrees C. Furthermore, the combination of intracellular and extracellular trehalose with small amounts of a conventional penetrating cryoprotectant (i.e., 0.5 M dimethylsulfoxide) provide high survival, fertilization, and embryonic development rates statistically similar to untreated controls. When transferred to foster mothers, cryopreserved oocytes give rise to healthy offspring showing the proof of principle. Our experiments with differential scanning calorimetry indicate that when cooled using the same cryopreservation protocol, the mixture of 0.5 M trehalose and cryopreservation medium undergoes glass transition at high subzero temperatures, which further substantiates the use of sugars as intracellular and extracellular cryoprotectants. Taken together, our results are in agreement with the survival schemes in nature and demonstrate the successful use of sugars in cryopreservation of mammalian oocytes.

Spontaneous Migration of Cancer Cells Under Conditions of Mechanical Confinement

Integrative Biology : Quantitative Biosciences from Nano to Macro. Sep, 2009  |  Pubmed ID: 20023765

When cancer cells spread away from the primary tumor, they often follow the trajectories of lymphatic vessels, nerves, white matter tracts, or other heterogeneous structures in tissues. To better understand this form of guided cell migration we designed a series of microfluidic devices that mechanically constrain migrating cancer cells inside microchannels with cross-section comparable to cell size. We observed unexpectedly fast and persistent movement in one direction for several hours of cancer cells of different types. The persistent motility occurs spontaneously, in the absence of external gradients, suggesting the presence of intrinsic mechanisms driving cancer cell motility that are induced in conditions of mechanical confinement. To probe the mechanisms responsible for this behavior, we exposed cancer cells inside channels to drugs targeting the microtubules, and measured a significant reduction in the average migration speed. Surprisingly, a small number of cells appeared not to be affected by the treatment and displayed fast and persistent migration, comparable to the untreated cells. The new matrix-free, 3D-confined motility assay replicates critical interactions that cancer cells would normally have inside tissues, is compatible with high-content, high-throughput analysis of cellular motility at single cell level, and could provide useful insights into the biology of cancer cell migratory phenotype.

Genome-wide Transcriptome Analysis of 150 Cell Samples

Integrative Biology : Quantitative Biosciences from Nano to Macro. Jan, 2009  |  Pubmed ID: 20023796

A major challenge in molecular biology is interrogating the human transcriptome on a genome wide scale when only a limited amount of biological sample is available for analysis. Current methodologies using microarray technologies for simultaneously monitoring mRNA transcription levels require nanogram amounts of total RNA. To overcome the sample size limitation of current technologies, we have developed a method to probe the global gene expression in biological samples as small as 150 cells, or the equivalent of approximately 300 pg total RNA. The new method employs microfluidic devices for the purification of total RNA from mammalian cells and ultra-sensitive whole transcriptome amplification techniques. We verified that the RNA integrity is preserved through the isolation process, accomplished highly reproducible whole transcriptome analysis, and established high correlation between repeated isolations of 150 cells and the same cell culture sample. We validated the technology by demonstrating that the combined microfluidic and amplification protocol is capable of identifying biological pathways perturbed by stimulation, which are consistent with the information recognized in bulk-isolated samples.

Surface Topography Induces 3D Self-orientation of Cells and Extracellular Matrix Resulting in Improved Tissue Function

Integrative Biology : Quantitative Biosciences from Nano to Macro. Feb, 2009  |  Pubmed ID: 20023803

The organization of cells and extracellular matrix (ECM) in native tissues plays a crucial role in their functionality. However, in tissue engineering, cells and ECM are randomly distributed within a scaffold. Thus, the production of engineered-tissue with complex 3D organization remains a challenge. In the present study, we used contact guidance to control the interactions between the material topography, the cells and the ECM for three different tissues, namely vascular media, corneal stroma and dermal tissue. Using a specific surface topography on an elastomeric material, we observed the orientation of a first cell layer along the patterns in the material. Orientation of the first cell layer translates into a physical cue that induces the second cell layer to follow a physiologically consistent orientation mimicking the structure of the native tissue. Furthermore, secreted ECM followed cell orientation in every layer, resulting in an oriented self-assembled tissue sheet. These self-assembled tissue sheets were then used to create 3 different structured engineered-tissue: cornea, vascular media and dermis. We showed that functionality of such structured engineered-tissue was increased when compared to the same non-structured tissue. Dermal tissues were used as a negative control in response to surface topography since native dermal fibroblasts are not preferentially oriented in vivo. Non-structured surfaces were also used to produce randomly oriented tissue sheets to evaluate the impact of tissue orientation on functional output. This novel approach for the production of more complex 3D tissues would be useful for clinical purposes and for in vitro physiological tissue model to better understand long standing questions in biology.

Layered Patterning of Hepatocytes in Co-culture Systems Using Microfabricated Stencils

BioTechniques. Jan, 2010  |  Pubmed ID: 20078427

Microfabrication and micropatterning techniques in tissue engineering offer great potential for creating and controlling microenvironments in which cell behavior can be observed. Here we present a novel approach to generate layered patterning of hepatocytes on micropatterned fibroblast feeder layers using microfabricated polydimethylsiloxane (PDMS) stencils. We fabricated PDMS stencils to pattern circular holes with diameters of 500 microm. Hepatocytes were co-cultured with 3T3-J2 fibroblasts in two types of patterns to evaluate and characterize the cellular interactions in the co-culture systems. Results of this study demonstrated uniform intracellular albumin staining and E-cadherin expression, increased liver-specific functions, and active glycogen synthesis in the hepatocytes when the heterotypic interface between hepatocytes and fibroblasts was increased by the layered patterning technique. This patterning technique can be a useful experimental tool for applications in basic science, drug screening, and tissue engineering, as well as in the design of bioartificial liver devices.

Concentration of Glycerol in Aqueous Microdroplets by Selective Removal of Water

Analytical Chemistry. Feb, 2010  |  Pubmed ID: 20102162

A major roadblock to the vitrification of cells is the requirement of high concentrations of cryoprotectant (CPA) chemicals and the damage caused by prolonged exposure of cells to these high concentrations above the glass transition temperature. These effects are minimized with controlled CPA loading. Certain organic oils, such as soybean oil, are made of triacylglycerols and are capable of dissolving small amounts of water, a property which is enhanced significantly as temperature is increased. This phenomenon was exploited here to accomplish temperature-controlled concentration of glycerol in single water droplets dispersed in the organic phase. Emulsions of aqueous solutions of glycerol in soybean oil were made and subjected to a temperature increase of 10 degrees C from room temperature. Upon increasing temperature, water dissolved into the oil, rendering the 15-20 microm droplets concentrated an average of 3.6 times and 2.6 times for 1 and 2 M starting concentrations, respectively, with the oil-insoluble glycerol in 90-110 s. This phenomenon could be used to dynamically concentrate CPAs within cell-containing droplets which may then be vitrified before being exposed to high temperatures for fatally long times.

Ultra-rapid Vitrification of Mouse Oocytes in Low Cryoprotectant Concentrations

Reproductive Biomedicine Online. Feb, 2010  |  Pubmed ID: 20113958

The ideal cryopreservation protocol would combine the benefits of slow freezing with the benefits of vitrification. This report describes a method for the ultra-rapid vitrification of oocytes using slush nitrogen in quartz capillaries. The approach minimizes the thermal mass of the vitrification vessel by using open microcapillaries made of highly conductive quartz and achieves cooling rates of 250,000 degrees C/min. The process of vitrification can be optimized by maximizing the rate at which the sample is cooled, which allows for the use of lower cryoprotectant concentrations. Mouse oocytes can be successfully vitrified using 1.5 mol/l 1,2-propanediol and 0.5 mol/l trehalose and achieve survival rates of 90.0%(36/40). Fertilization and blastocyst formation rates of vitrified-warmed and fresh oocytes were not significantly different. A total of 120 blastocysts from each of the vitrified-warmed and fresh oocytes were transferred to surrogate mothers and 23 and 27 offspring were born respectively. All offspring in both groups were healthy, grew and bred normally and gave rise to a second generation of pups. Thus, an ultra-rapid vitrification technique has been developed for mouse oocytes that uses low concentrations of cryoprotectants and slush nitrogen in quartz capillaries, which combines the benefits of slow freezing and vitrification.

Microfluidic Isolation and Transcriptome Analysis of Serum Microvesicles

Lab on a Chip. Feb, 2010  |  Pubmed ID: 20126692

Microvesicles (exosomes) shed from both normal and cancerous cells may serve as means of intercellular communication. These microvesicles carry proteins, lipids and nucleic acids derived from the host cell. Their isolation and analysis from blood samples have the potential to provide information about state and progression of malignancy and should prove of great clinical importance as biomarkers for a variety of disease states. However, current protocols for isolation of microvesicles from blood require high-speed centrifugation and filtration, which are cumbersome and time consuming. In order to take full advantage of the potential of microvesicles as biomarkers for clinical applications, faster and simpler methods of isolation will be needed. In this paper, we present an easy and rapid microfluidic immunoaffinity method to isolate microvesicles from small volumes of both serum from blood samples and conditioned medium from cells in culture. RNA of high quality can be extracted from these microvesicles providing a source of information about the genetic status of tumors to serve as biomarkers for diagnosis and prognosis of cancer.

Particle Focusing in Staged Inertial Microfluidic Devices for Flow Cytometry

Analytical Chemistry. May, 2010  |  Pubmed ID: 20373755

Microfluidic inertial focusing has been demonstrated to be an effective method for passively positioning microparticles and cells without the assistance of sheath fluid. Because inertial focusing produces well-defined lateral equilibrium particle positions in addition to highly regulated interparticle spacing, its value in flow cytometry has been suggested. Particle focusing occurs in straight channels and can be manipulated through cross sectional channel geometry by the introduction of curvature. Here, we present a staged channel design consisting of both curved and straight sections that combine to order particles into a single streamline with longitudinal spacing. We have evaluated the performance of these staged inertial focusing channels using standard flow cytometry methods that make use of calibration microspheres. Our analysis has determined the measurement precision and resolution, as a function of flow velocity and particle concentration that is provided by these channels. These devices were found to operate with increasing effectiveness at higher flow rates and particle concentrations, within the examined ranges, which is ideal for high throughput analysis. Further, the prototype flow cytometer equipped with an inertial focusing microchannel matched the resolution provided by a commercial hydrodynamic focusing flow cytometer. Most notably, our analysis indicates that the inertial focusing channels virtually eliminated particle coincidence at the analysis point. These properties suggest a potentially significant role for inertial focusing in the development of inexpensive flow cytometry-based diagnostics and in applications requiring the analysis of high particle concentrations.

Isolation and Characterization of Circulating Tumor Cells from Patients with Localized and Metastatic Prostate Cancer

Science Translational Medicine. Mar, 2010  |  Pubmed ID: 20424012

Rare circulating tumor cells (CTCs) are present in the blood of patients with metastatic epithelial cancers but have been difficult to measure routinely. We report a quantitative automated imaging system for analysis of prostate CTCs, taking advantage of prostate-specific antigen (PSA), a unique prostate tumor-associated marker. The specificity of PSA staining enabled optimization of criteria for baseline image intensity, morphometric measurements, and integration of multiple signals in a three-dimensional microfluidic device. In a pilot analysis, we detected CTCs in prostate cancer patients with localized disease, before surgical tumor removal in 8 of 19 (42%) patients (range, 38 to 222 CTCs per milliliter). For 6 of the 8 patients with preoperative CTCs, a precipitous postoperative decline (<24 hours) suggests a short half-life for CTCs in the blood circulation. Other patients had persistent CTCs for up to 3 months after prostate removal, suggesting early but transient disseminated tumor deposits. In patients with metastatic prostate cancer, CTCs were detected in 23 of 36 (64%) cases (range, 14 to 5000 CTCs per milliliter). In previously untreated patients followed longitudinally, the numbers of CTCs declined after the initiation of effective therapy. The prostate cancer-specific TMPRSS2-ERG fusion was detectable in RNA extracted from CTCs from 9 of 20 (45%) patients with metastatic disease, and dual staining of captured CTCs for PSA and the cell division marker Ki67 indicated a broad range for the proportion of proliferating cells among CTCs. This method for analysis of CTCs will facilitate the application of noninvasive tumor sampling to direct targeted therapies in advanced prostate cancer and warrants the initiation of long-term clinical studies to test the importance of CTCs in invasive localized disease.

Concentration and Purification of Human Immunodeficiency Virus Type 1 Virions by Microfluidic Separation of Superparamagnetic Nanoparticles

Analytical Chemistry. Jan, 2010  |  Pubmed ID: 19954210

The low concentration and complex sample matrix of many clinical and environmental viral samples presents a significant challenge in the development of low cost, point-of-care viral assays. To address this problem, we investigated the use of a microfluidic passive magnetic separator combined with on-chip mixer to both purify and concentrate whole-particle human immunodeficiency virus type 1 (HIV-1) virions. Virus-containing plasma samples are first mixed to allow specific binding of the viral particles with antibody-conjugated superparamagnetic nanoparticles, and several passive mixer geometries were assessed for their mixing efficiencies. The virus-nanoparticle complexes are then separated from the plasma in a novel magnetic separation chamber, where packed micrometer-sized ferromagnetic particles serve as high magnetic gradient concentrators for an externally applied magnetic field. Thereafter, a viral lysis buffer was flowed through the chip and the released HIV proteins were assayed off-chip. Viral protein extraction efficiencies of 62% and 45% were achieved at 10 and 30 muL/min throughputs, respectively. More importantly, an 80-fold concentration was observed for an initial sample volume of 1 mL and a 44-fold concentration for an initial sample volume of 0.5 mL. The system is broadly applicable to microscale sample preparation of any viral sample and can be used for nucleic acid extraction as well as 40-80-fold enrichment of target viruses.

Squishy Non-spherical Hydrogel Microparticles

Macromolecular Rapid Communications. Jan, 2010  |  Pubmed ID: 21590884

Recent advances in the synthesis of polymeric colloids have opened the doors to new advanced materials. There is strong interest in using these new techniques to produce particles that mimic and/or interact with biological systems. An important characteristic of biological systems that has not yet been exploited in synthetic polymeric colloids is their wide range of deformability. A canonical example of this is the human red blood cell (RBC) which exhibits extreme reversible deformability under flow. Here we report the synthesis of soft polymeric colloids with sizes and shapes that mimic those of the RBC. Additionally, we demonstrate that the mechanical flexibility of the colloids can be reproducibly varied over a large range resulting in RBC-like deformability under physiological flow conditions. These materials have the potential to impact the interaction between biological and synthetic systems.

Microfluidic Proportional Flow Controller

Journal of Micromechanics and Microengineering : Structures, Devices, and Systems. Oct, 2010  |  Pubmed ID: 21874096

Precise flow control in microfluidic chips is important for many biochemical assays and experiments at microscale. While several technologies for controlling fluid flow have been implemented either on- or off-chip, these can provide either high-speed or high-precision control, but seldom could accomplish both at the same time. Here we describe a new on-chip, pneumatically activated flow controller that allows for fast and precise control of the flow rate through a microfluidic channel. Experimental results show that the new proportional flow controllers exhibited a response time of approximately 250 ms, while our numerical simulations suggest that faster actuation down to approximately 50 ms could be achieved with alternative actuation schemes.

MYC Regulation of a "poor-prognosis" Metastatic Cancer Cell State

Proceedings of the National Academy of Sciences of the United States of America. Feb, 2010  |  Pubmed ID: 20133671

Gene expression signatures are used in the clinic as prognostic tools to determine the risk of individual patients with localized breast tumors developing distant metastasis. We lack a clear understanding, however, of whether these correlative biomarkers link to a common biological network that regulates metastasis. We find that the c-MYC oncoprotein coordinately regulates the expression of 13 different "poor-outcome" cancer signatures. In addition, functional inactivation of MYC in human breast cancer cells specifically inhibits distant metastasis in vivo and invasive behavior in vitro of these cells. These results suggest that MYC oncogene activity (as marked by "poor-prognosis" signature expression) may be necessary for the translocation of poor-outcome human breast tumors to distant sites.

A Study of the Effect of Sorbitol on Osmotic Tolerance During Partial Desiccation of Bovine Sperm

Cryobiology. Jun, 2010  |  Pubmed ID: 20233588

The goal of the study was to improve the partial desiccation survival of bovine sperm by decreasing the dehydration induced osmotic injury. The protective role of sorbitol, a polyol, was investigated by (i) studying the osmotic behavior of sperm in hypertonic Tyrode's buffer in the presence of sorbitol and trehalose, (ii) studying the effect of sorbitol and trehalose on sperm motility following partial dehydration. The osmotic behavior studies included the assessment of motility and volumetric responses in the presence of the additives. For the drying experiments, motility was assayed after drying the samples to different end water content followed by immediate rehydration. Compared to the effect of "intracellular+extracellular" trehalose alone, results showed a much improved motility in the presence of sorbitol and trehalose. While the drying results suggest an enhanced osmotolerance in the presence of sorbitol, the study of motility under hypertonic conditions combined with the sperm volume excursion experiments suggest that sorbitol imparts the enhancement by permeating into the cell cytoplasm.

Analysis of Factorial Time-course Microarrays with Application to a Clinical Study of Burn Injury

Proceedings of the National Academy of Sciences of the United States of America. Jun, 2010  |  Pubmed ID: 20479259

Time-course microarray experiments are capable of capturing dynamic gene expression profiles. It is important to study how these dynamic profiles depend on the multiple factors that characterize the experimental condition under which the time course is observed. Analytic methods are needed to simultaneously handle the time course and factorial structure in the data. We developed a method to evaluate factor effects by pooling information across the time course while accounting for multiple testing and nonnormality of the microarray data. The method effectively extracts gene-specific response features and models their dependency on the experimental factors. Both longitudinal and cross-sectional time-course data can be handled by our approach. The method was used to analyze the impact of age on the temporal gene response to burn injury in a large-scale clinical study. Our analysis reveals that 21% of the genes responsive to burn are age-specific, among which expressions of mitochondria and immunoglobulin genes are differentially perturbed in pediatric and adult patients by burn injury. These new findings in the body's response to burn injury between children and adults support further investigations of therapeutic options targeting specific age groups. The methodology proposed here has been implemented in R package "TANOVA" and submitted to the Comprehensive R Archive Network at http://www.r-project.org/. It is also available for download at http://gluegrant1.stanford.edu/TANOVA/.

Metabolic Preconditioning of Cells with AICAR-riboside: Improved Cryopreservation and Cell-type Specific Impacts on Energetics and Proliferation

Cryobiology. Aug, 2010  |  Pubmed ID: 20510224

In species whose evolutionary history has provided natural tolerance to dehydration and freezing, metabolic depression is often a pre-requisite for survival. We tested the hypothesis that preconditioning of mammalian cells with 5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside (AICAR) to achieve metabolic depression will promote greater survivorship during cryopreservation. AICAR is used extensively to stimulate AMP-activated protein kinase (AMPK), which can result in downregulation of biosynthetic processes. We showed that the metabolic interconversion of AICAR was cell-type dependent. Accumulation of 5-aminoimidazole-4-carboxamide-1b-D-ribofuranosyl-5'-monophosphate (ZMP), as well as other metabolites that possess multiple phosphates (i.e., ZDP, ZTP), varied approximately 3.5-fold across the cell lines tested. AICAR treatment also significantly influenced the concentrations of cellular adenylates (ATP, ADP, and AMP). Depression of cell metabolism and proliferation with AICAR treatment differed among cell lines. Proliferation for a given cell line was negatively correlated with the fold-increase achieved in the 'effective adenylate ratio' ([AMP]+[ZMP])/[ATP]) after AICAR treatment. Metabolic preconditioning with AICAR promoted a significant increase in viability post-freezing in J774.A1 macrophages, HepG2/C3A cells and primary hepatocytes but not in NIH/3T3 fibroblasts or OMK cells. The effect of AICAR on viability after freezing was positively correlated (r(2)=0.94) with the fold-increase in the 'effective adenylate ratio'. Thus for each cell line, the greater the depression of metabolism and proliferation due to preconditioning with AICAR, the greater was the survivorship post-freezing.

Controlled Induction of Distributed Microdeformation in Wounded Tissue Via a Microchamber Array Dressing

Journal of Biomedical Materials Research. Part A. Nov, 2010  |  Pubmed ID: 20607869

Mechanical stimuli are known to play an important role in determining the structure and function of living cells and tissues. Recent studies have highlighted the role of mechanical signals in mammalian dermal wound healing. However, the biological link between mechanical stimulation of wounded tissue and the subsequent cellular response has not been fully determined. The capacity for researchers to study this link is partially limited by the lack of instrumentation capable of applying controlled mechanical stimuli to wounded tissue. The studies outlined here tested the hypothesis that it was possible to control the magnitude of induced wound tissue deformation using a microfabricated dressing composed of an array of open-faced, hexagonally shaped microchambers rendered in a patch of silicone rubber. By connecting the dressing to a single vacuum source, the underlying wounded tissue was drawn up into each of the microchambers, thereby inducing tissue deformation. For these studies, the dressings were applied to full-thickness murine dermal wounds with 200 mmHg vacuum for 12 h. These studies demonstrated that the dressing was capable of inducing wound tissue deformation with values ranging from 11 to 29%. Through statistical analysis, the magnitude of the induced deformation was shown to be a function of both microchamber height and width. These results demonstrated that the dressing was capable of controlling the amount of deformation imparted in the underlying tissue. By allowing the application of mechanical stimulation with varying intensities, such a dressing will enable the performance of sophisticated mechanobiology studies in dermal wound healing.

Directional Decisions During Neutrophil Chemotaxis Inside Bifurcating Channels

Integrative Biology : Quantitative Biosciences from Nano to Macro. Nov, 2010  |  Pubmed ID: 20676444

The directional migration of human neutrophils in classical chemotaxis assays is often described as a "biased random walk" implying significant randomness in speed and directionality. However, these experiments are inconsistent with in vivo observations, where neutrophils can navigate effectively through complex tissue microenvironments towards their targets. Here, we demonstrate a novel biomimetic assay for neutrophil chemotaxis using enclosed microfluidic channels. Remarkably, under these enclosed conditions, neutrophils recapitulate the highly robust and efficient navigation observed in vivo. In straight channels, neutrophils undergo sustained, unidirectional motion towards a chemoattractant source. In more complex maze-like geometries, neutrophils are able to select the most direct route over 90% of the time. Finally, at symmetric bifurcations, neutrophils split their leading edge into two sections and a "tug of war" ensues. The competition between the two new leading edges is ultimately resolved by stochastic, symmetry-breaking behavior. This behavior is suggestive of directional decision-making localized at the leading edge and a signaling role played by the cellular cytoskeleton.

Burn Injury Reduces Neutrophil Directional Migration Speed in Microfluidic Devices

PloS One. 2010  |  Pubmed ID: 20689600

Thermal injury triggers a fulminant inflammatory cascade that heralds shock, end-organ failure, and ultimately sepsis and death. Emerging evidence points to a critical role for the innate immune system, and several studies had documented concurrent impairment in neutrophil chemotaxis with these post-burn inflammatory changes. While a few studies suggest that a link between neutrophil motility and patient mortality might exist, so far, cumbersome assays have prohibited exploration of the prognostic and diagnostic significance of chemotaxis after burn injury. To address this need, we developed a microfluidic device that is simple to operate and allows for precise and robust measurements of chemotaxis speed and persistence characteristics at single-cell resolution. Using this assay, we established a reference set of migration speed values for neutrophils from healthy subjects. Comparisons with samples from burn patients revealed impaired directional migration speed starting as early as 24 hours after burn injury, reaching a minimum at 72-120 hours, correlated to the size of the burn injury and potentially serving as an early indicator for concurrent infections. Further characterization of neutrophil chemotaxis using this new assay may have important diagnostic implications not only for burn patients but also for patients afflicted by other diseases that compromise neutrophil functions.

Clinical Microfluidics for Neutrophil Genomics and Proteomics

Nature Medicine. Sep, 2010  |  Pubmed ID: 20802500

Neutrophils have key roles in modulating the immune response. We present a robust methodology for rapidly isolating neutrophils directly from whole blood with 'on-chip' processing for mRNA and protein isolation for genomics and proteomics. We validate this device with an ex vivo stimulation experiment and by comparison with standard bulk isolation methodologies. Last, we implement this tool as part of a near-patient blood processing system within a multi-center clinical study of the immune response to severe trauma and burn injury. The preliminary results from a small cohort of subjects in our study and healthy controls show a unique time-dependent gene expression pattern clearly demonstrating the ability of this tool to discriminate temporal transcriptional events of neutrophils within a clinical setting.

Isolation of Circulating Tumor Cells Using a Microvortex-generating Herringbone-chip

Proceedings of the National Academy of Sciences of the United States of America. Oct, 2010  |  Pubmed ID: 20930119

Rare circulating tumor cells (CTCs) present in the bloodstream of patients with cancer provide a potentially accessible source for detection, characterization, and monitoring of nonhematological cancers. We previously demonstrated the effectiveness of a microfluidic device, the CTC-Chip, in capturing these epithelial cell adhesion molecule (EpCAM)-expressing cells using antibody-coated microposts. Here, we describe a high-throughput microfluidic mixing device, the herringbone-chip, or "HB-Chip," which provides an enhanced platform for CTC isolation. The HB-Chip design applies passive mixing of blood cells through the generation of microvortices to significantly increase the number of interactions between target CTCs and the antibody-coated chip surface. Efficient cell capture was validated using defined numbers of cancer cells spiked into control blood, and clinical utility was demonstrated in specimens from patients with prostate cancer. CTCs were detected in 14 of 15 (93%) patients with metastatic disease (median = 63 CTCs/mL, mean = 386 ± 238 CTCs/mL), and the tumor-specific TMPRSS2-ERG translocation was readily identified following RNA isolation and RT-PCR analysis. The use of transparent materials allowed for imaging of the captured CTCs using standard clinical histopathological stains, in addition to immunofluorescence-conjugated antibodies. In a subset of patient samples, the low shear design of the HB-Chip revealed microclusters of CTCs, previously unappreciated tumor cell aggregates that may contribute to the hematogenous dissemination of cancer.

A Microfabricated Electrical Differential Counter for the Selective Enumeration of CD4+ T Lymphocytes

Lab on a Chip. Apr, 2011  |  Pubmed ID: 21283908

We have developed a microfabricated biochip that enumerates CD4+ T lymphocytes from leukocyte populations obtained from human blood samples using electrical impedance sensing and immunoaffinity chromatography. T cell counts are found by obtaining the difference between the number of leukocytes before and after depleting CD4+ T cells with immobilized antibodies in a capture chamber. This differential counting technique has been validated to analyze physiological concentrations of leukocytes with an accuracy of ∼9 cells per µL by passivating the capture chamber with bovine serum albumin. In addition, the counter provided T cell counts which correlated closely with an optical control (R(2) = 0.997) for CD4 cell concentrations ranging from approximately 100 to 700 cells per µL. We believe that this approach can be a promising method for bringing quantitative HIV/AIDS diagnostics to resource-poor regions in the world.

A Spin-drying Technique for Lyopreservation of Mammalian Cells

Annals of Biomedical Engineering. May, 2011  |  Pubmed ID: 21293974

Stabilization of cellular material in the presence of glass-forming sugars at ambient temperatures is a viable approach that has many potential advantages over current cryogenic strategies. Experimental evidence indicates the possibility to preserve biomolecules in glassy matrices of low-molecular mobility using "glass-forming" sugars like trehalose at ambient temperatures. However, when cells are desiccated in trehalose solution using passive drying techniques, a glassy skin is formed at the liquid/vapor interface of the sample. This glassy skin prevents desiccation of the sample beyond a certain level of dryness and induces non-uniformities in the final water content. Cells trapped underneath this glassy skin may degrade due to a relatively high molecular mobility in the sample. This undesirable result underscores the need for development of a uniform, fast drying technique. In the present study, we report a new technique based on the principles of "spin drying" that can effectively address these problems. Forced convective evaporation of water along with the loss of solution due to centrifugal force leads to rapid vitrification of a thin layer of trehalose containing medium that remains on top of cells attached to the spinning glass substrate. The glassy layer produced has a consistent thickness and a small "surface-area-to-volume" ratio that minimizes any non-homogeneity. Thus, the chance of entrapping cells in a high-mobility environment decreases substantially. We compared numerical predictions to experimental observations of the drying time of 0.2-0.6 M trehalose solutions at a variety of spinning speeds ranging from 1000 to 4000 rpm. The model developed here predicts the formation of sugar films with thicknesses of 200-1000 nm, which was in good agreement with experimental results. Preliminary data suggest that after spin drying cells to about 0.159 ± 0.09 gH₂O/gdw (n = 11, ±SE), more than 95% of cells were able to preserve their membrane integrity. Membrane integrity after spin drying is therefore considerably higher than what is achieved by conventional drying methods; where about 90% of cells lose membrane integrity at 0.4 gH₂O/gdw (Acker et al. Cell Preserv. Technol. 1(2):129-140, 2002; Elliott et al. Biopreserv. Biobank. 6(4):253-260, 2009).

Circulating Tumor Cells: Approaches to Isolation and Characterization

The Journal of Cell Biology. Feb, 2011  |  Pubmed ID: 21300848

Circulating tumor cells (CTCs) shed from primary and metastatic cancers are admixed with blood components and are thus rare, making their isolation and characterization a major technological challenge. CTCs hold the key to understanding the biology of metastasis and provide a biomarker to noninvasively measure the evolution of tumor genotypes during treatment and disease progression. Improvements in technologies to yield purer CTC populations amenable to better cellular and molecular characterization will enable a broad range of clinical applications, including early detection of disease and the discovery of biomarkers to predict treatment responses and disease progression.

Metabolic Restructuring During Energy-limited States: Insights from Artemia Franciscana Embryos and Other Animals

Journal of Insect Physiology. May, 2011  |  Pubmed ID: 21335009

Many life history stages of animals that experience environmental insults enter developmental arrested states that are characterized by reduced cellular proliferation, with or without a concurrent reduction in overall metabolism. In the case of the most profound metabolic arrest reported in invertebrates, i.e., anaerobic quiescence in Artemia franciscana embryos, acidification of the intracellular milieu is a major factor governing catabolic and anabolic downregulation. Release of ions from intracellular compartments is the source for approximately 50% of the proton equivalents needed for the 1.5 unit acidification that is observed. Recovery from the metabolic arrest requires re-sequestration of the protons with a vacuolar-type ATPase (V-ATPase). The remarkable facet of this mechanism is the ability of embryonic cells to survive the dissipation of intracellular ion gradients. Across many diapause-like states, the metabolic reduction and subsequent matching of energy demand is accomplished by shifting energy metabolism from oxidative phosphorylation to aerobic glycolysis. Molecular pathways that are activated to induce these resilient hypometabolic states include stimulation of the AMP-activated protein kinase (AMPK) and insulin signaling via suite of daf (dauer formation) genes for diapause-like states in nematodes and insects. Contributing factors for other metabolically depressed states involve hypoxia-inducible factor-1 and downregulation of the pyruvate dehydrogenase complex. Metabolic similarities between natural states of stasis and some cancer phenotypes are noteworthy. Reduction of flux through oxidative phosphorylation helps prevent cell death in certain cancer types, similar to the way it increases viability of dauer stages in Caenorhabditis elegans. Mechanisms that underlie natural stasis are being used to pre-condition mammalian cells prior to cell biostabilization and storage.

Nanoporous Elements in Microfluidics for Multiscale Manipulation of Bioparticles

Small (Weinheim an Der Bergstrasse, Germany). Apr, 2011  |  Pubmed ID: 21413145

Solid materials, such as silicon, glass, and polymers, dominate as structural elements in microsystems including microfluidics. Porous elements have been limited to membranes sandwiched between microchannel layers or polymer monoliths. This paper reports the use of micropatterned carbon-nanotube forests confined inside microfluidic channels for mechanically and/or chemically capturing particles ranging over three orders of magnitude in size. Nanoparticles below the internanotube spacing (80 nm) of the forest can penetrate inside the forest and interact with the large surface area created by individual nanotubes. For larger particles (>80 nm), the ultrahigh porosity of the nanotube elements reduces the fluid boundary layer and enhances particle-structure interactions on the outer surface of the patterned nanoporous elements. Specific biomolecular recognition is demonstrated using cells (≈10 μm), bacteria (≈1 μm), and viral-sized particles (≈40 nm) using both effects. This technology can provide unprecedented control of bioseparation processes to access bioparticles of interest, opening new pathways for both research and point-of-care diagnostics.

A 3D Aligned Microfibrous Myocardial Tissue Construct Cultured Under Transient Perfusion

Biomaterials. Aug, 2011  |  Pubmed ID: 21570112

The goal of this study was to design and develop a myocardial patch to use in the repair of myocardial infarctions or to slow down tissue damage and improve long-term heart function. The basic 3D construct design involved two biodegradable macroporous tubes, to allow transport of growth media to the cells within the construct, and cell seeded, aligned fiber mats wrapped around them. The microfibrous mat housed mesenchymal stem cells (MSCs) from human umbilical cord matrix (Wharton's Jelly) aligned in parallel to each other in a similar way to cell organization in native myocardium. Aligned micron-sized fiber mats were obtained by electrospinning a polyester blend (PHBV (5% HV), P(L-D,L)LA (70:30) and poly(glycerol sebacate) (PGS)). The micron-sized electrospun parallel fibers were effective in Wharton's Jelly (WJ) MSCs alignment and the cells were able to retract the mat. The 3D construct was cultured in a microbioreactor by perfusing the growth media transiently through the macroporous tubing for two weeks and examined by fluorescence microscopy for cell distribution and preservation of alignment. The fluorescence images of thin sections of 3D constructs from static and perfused cultures confirmed enhanced cell viability, uniform cell distribution and alignment due to nutrient provision from inside the 3D structure.

Computer Aided Diagnostic Tools Aim to Empower Rather Than Replace Pathologists: Lessons Learned from Computational Chess

Journal of Pathology Informatics. 2011  |  Pubmed ID: 21773056

Controlled Loading of Cryoprotectants (CPAs) to Oocyte with Linear and Complex CPA Profiles on a Microfluidic Platform

Lab on a Chip. Oct, 2011  |  Pubmed ID: 21887438

Oocyte cryopreservation has become an essential tool in the treatment of infertility by preserving oocytes for women undergoing chemotherapy. However, despite recent advances, pregnancy rates from all cryopreserved oocytes remain low. The inevitable use of the cryoprotectants (CPAs) during preservation affects the viability of the preserved oocytes and pregnancy rates either through CPA toxicity or osmotic injury. Current protocols attempt to reduce CPA toxicity by minimizing CPA concentrations, or by minimizing the volume changes via the step-wise addition of CPAs to the cells. Although the step-wise addition decreases osmotic shock to oocytes, it unfortunately increases toxic injuries due to the long exposure times to CPAs. To address limitations of current protocols and to rationally design protocols that minimize the exposure to CPAs, we developed a microfluidic device for the quantitative measurements of oocyte volume during various CPA loading protocols. We spatially secured a single oocyte on the microfluidic device, created precisely controlled continuous CPA profiles (step-wise, linear and complex) for the addition of CPAs to the oocyte and measured the oocyte volumetric response to each profile. With both linear and complex profiles, we were able to load 1.5 M propanediol to oocytes in less than 15 min and with a volumetric change of less than 10%. Thus, we believe this single oocyte analysis technology will eventually help future advances in assisted reproductive technologies and fertility preservation.

Microfluidics-based Capture of Human Neutrophils for Expression Analysis in Blood and Bronchoalveolar Lavage

Laboratory Investigation; a Journal of Technical Methods and Pathology. Dec, 2011  |  Pubmed ID: 21931299

Gene expression analysis can be a powerful tool in predicting patient outcomes and identifying patients who may benefit from targeted therapies. However, isolating human blood polymorphonuclear cells (PMNs) for genomic analysis has been challenging. We used a novel microfluidic technique that isolates PMNs by capturing CD66b(+) cells and compared it with dextran-Ficoll gradient isolation. We also used microfluidic isolation techniques for blood and bronchoalveolar lavage (BAL) samples of patients with acute respiratory distress syndrome (ARDS) to evaluate PMN genomic alterations secondary to pulmonary sequestration. PMNs obtained from ex vivo lipopolysaccharide (LPS)-stimulated or -unstimulated whole blood from five healthy volunteers were isolated by either dextran-Ficoll gradient, microfluidics capture, or a combination of the two techniques. Blood and BAL fluid PMNs were also isolated using microfluidics from seven hospitalized patients with ARDS. Gene expression was inferred from extracted RNA using Affymetrix U133 Plus 2.0 GeneChips. All methods of PMN isolation produced similar quantities of high-quality RNA, when adjusted for recovered cell number. Unsupervised analysis and hierarchical clustering indicated that LPS stimulation was the primary factor affecting gene expression patterns among all ex vivo samples. Patterns of gene expression from blood and BAL PMNs differed significantly from each other in the patients with ARDS. Isolation of PMNs by microfluidics can be applied to both blood and BAL specimens from critically ill, hospitalized patients. Unique genomic expression patterns are obtained from the blood and BAL fluid of critically ill patients with ARDS, and these differ significantly from genomic patterns seen after ex vivo LPS stimulation.

Cryopreservation of Spin-dried Mammalian Cells

PloS One. 2011  |  Pubmed ID: 21966385

This study reports an alternative approach to achieve vitrification where cells are pre-desiccated prior to cooling to cryogenic temperatures for storage. Chinese Hamster Ovary (CHO) cells suspended in a trehalose solution were rapidly and uniformly desiccated to a low moisture content (<0.12 g of water per g of dry weight) using a spin-drying technique. Trehalose was also introduced into the cells using a high-capacity trehalose transporter (TRET1). Fourier Transform Infrared Spectroscopy (FTIR) was used to examine the uniformity of water concentration distribution in the spin-dried samples. 62% of the cells were shown to survive spin-drying in the presence of trehalose following immediate rehydration. The spin-dried samples were stored in liquid nitrogen (LN(2)) at a vitrified state. It was shown that following re-warming to room temperature and re-hydration with a fully complemented cell culture medium, 51% of the spin-dried and vitrified cells survived and demonstrated normal growth characteristics. Spin-drying is a novel strategy that can be used to improve cryopreservation outcome by promoting rapid vitrification.

Specific Pathogen Detection Using Bioorthogonal Chemistry and Diagnostic Magnetic Resonance

Bioconjugate Chemistry. Dec, 2011  |  Pubmed ID: 22043803

The development of faster and more sensitive detection methods capable of identifying specific bacterial species and strains has remained a longstanding clinical challenge. Thus to date, the diagnosis of bacterial infections continues to rely on the performance of time-consuming microbiological cultures. Here, we demonstrate the use of bioorthogonal chemistry for magnetically labeling specific pathogens to enable their subsequent detection by nuclear magnetic resonance. Antibodies against a bacterial target of interest were first modified with trans-cyclooctene and then coupled to tetrazine-modified magnetic nanoprobes, directly on the bacteria. This labeling method was verified by surface plasmon resonance as well as by highly specific detection of Staphylococcus aureus using a miniaturized diagnostic magnetic resonance system. Compared to other copper-free bioorthogonal chemistries, the cycloaddition reaction reported here displayed faster kinetics and yielded higher labeling efficiency. Considering the short assay times and the portability of the necessary instrumentation, it is feasible that this approach could be adapted for clinical use in resource-limited settings.

On a Chip

IEEE Pulse. Nov, 2011  |  Pubmed ID: 22147065

The future of clinical and POC BioMEMS is very bright. With an increasing emphasis on the personalization of medicine and the rising costs of health care, early detection and diagnostics at the POC will be even more important. Early detection implies early intervention, resulting in the saving of lives and reducing overall spending. The potential impact of these technologies on the early diagnosis and management of both communicable and noncommunicable diseases is very high. Many grand challenges applications are possible, e.g., routine tests such as complete blood cell count on a chip that an individual can perform at home; detection of cardiac markers from blood after a perceived heart attack; detection of cancer markers such as exosomes, CTCs from blood, or protein biomarkers in serum; and detection of infectious agents such as virus and bacteria for public health. These applications are expected to result in new diagnostic assays for home, doctor's office, clinical laboratories, and various POC settings.

LEA Proteins During Water Stress: Not Just for Plants Anymore

Annual Review of Physiology. Mar, 2011  |  Pubmed ID: 21034219

Late embryogenesis abundant (LEA) proteins are extremely hydrophilic proteins that were first identified in land plants. Intracellular accumulation is tightly correlated with acquisition of desiccation tolerance, and data support their capacity to stabilize other proteins and membranes during drying, especially in the presence of sugars like trehalose. Exciting reports now show that LEA proteins are not restricted to plants; multiple forms are expressed in desiccation-tolerant animals from at least four phyla. We evaluate here the expression, subcellular localization, biochemical properties, and potential functions of LEA proteins in animal species during water stress. LEA proteins are intrinsically unstructured in aqueous solution, but surprisingly, many assume their native conformation during drying. They are targeted to multiple cellular locations, including mitochondria, and evidence supports that LEA proteins stabilize vitrified sugar glasses thought to be important in the dried state. More in vivo experimentation will be necessary to fully unravel the multiple functional properties of these macromolecules during water stress.

Spatially Invariant Vector Quantization: A Pattern Matching Algorithm for Multiple Classes of Image Subject Matter Including Pathology

Journal of Pathology Informatics. 2011  |  Pubmed ID: 21383936

HISTORICALLY, EFFECTIVE CLINICAL UTILIZATION OF IMAGE ANALYSIS AND PATTERN RECOGNITION ALGORITHMS IN PATHOLOGY HAS BEEN HAMPERED BY TWO CRITICAL LIMITATIONS: 1) the availability of digital whole slide imagery data sets and 2) a relative domain knowledge deficit in terms of application of such algorithms, on the part of practicing pathologists. With the advent of the recent and rapid adoption of whole slide imaging solutions, the former limitation has been largely resolved. However, with the expectation that it is unlikely for the general cohort of contemporary pathologists to gain advanced image analysis skills in the short term, the latter problem remains, thus underscoring the need for a class of algorithm that has the concurrent properties of image domain (or organ system) independence and extreme ease of use, without the need for specialized training or expertise.

Trehalose Transporter from African Chironomid Larvae Improves Desiccation Tolerance of Chinese Hamster Ovary Cells

Cryobiology. Apr, 2012  |  Pubmed ID: 22155480

Dry preservation has been explored as an energy-efficient alternative to cryopreservation, but the high sensitivity of mammalian cells to desiccation stress has been one of the major hurdles in storing cells in the desiccated state. An important strategy to reduce desiccation sensitivity involves use of the disaccharide trehalose. Trehalose is known to improve desiccation tolerance in mammalian cells when present on both sides of the cell membrane. Because trehalose is membrane impermeant the development of desiccation strategies involving this promising sugar is hindered. We explored the potential of using a high-capacity trehalose transporter (TRET1) from the African chironomid Polypedilum vanderplanki[21] to introduce trehalose into the cytoplasm of mammalian cells and thereby increase desiccation tolerance. When Chinese hamster ovary cells (CHO) were stably transfected with TRET1 (CHO-TRET1 cells) and incubated with 0.4M trehalose for 4h at 37°C, a sevenfold increase in trehalose uptake was observed compared to the wild-type CHO cells. Following trehalose loading, desiccation tolerance was investigated by evaporative drying of cells at 14% relative humidity. After desiccation to 2.60g of water per gram dry weight, a 170% increase in viability and a 400% increase in growth (after 7days) was observed for CHO-TRET1 relative to control CHO cells. Our results demonstrate the beneficial effect of intracellular trehalose for imparting tolerance to partial desiccation.

Asymmetry and Aging of Mycobacterial Cells Lead to Variable Growth and Antibiotic Susceptibility

Science (New York, N.Y.). Jan, 2012  |  Pubmed ID: 22174129

Cells use both deterministic and stochastic mechanisms to generate cell-to-cell heterogeneity, which enables the population to better withstand environmental stress. Here we show that, within a clonal population of mycobacteria, there is deterministic heterogeneity in elongation rate that arises because mycobacteria grow in an unusual, unipolar fashion. Division of the asymmetrically growing mother cell gives rise to daughter cells that differ in elongation rate and size. Because the mycobacterial cell division cycle is governed by time, not cell size, rapidly elongating cells do not divide more frequently than slowly elongating cells. The physiologically distinct subpopulations of cells that arise through asymmetric growth and division are differentially susceptible to clinically important classes of antibiotics.

Preservation of Mouse Sperm by Convective Drying and Storing in 3-o-methyl-d-glucose

PloS One. 2012  |  Pubmed ID: 22272261

With the fast advancement in the genetics and bio-medical fields, the vast number of valuable transgenic and rare genetic mouse models need to be preserved. Preservation of mouse sperm by convective drying and subsequent storing at above freezing temperatures could dramatically reduce the cost and facilitate shipping. Mouse sperm were convectively dried under nitrogen gas in the Na-EGTA solution containing 100 mmol/L 3-O-methyl-D-glucose and stored in LiCl sorption jars (Relative Humidity, RH, 12%) at 4°C and 22°C for up to one year. The functionality of these sperm samples after storage was tested by intracytoplasmic injection into mouse oocytes. The percentages of blastocysts produced from sperm stored at 4°C for 1, 2, 3, 6, and 12 months were 62.6%, 53.4%, 39.6%, 33.3%, and 30.4%, respectively, while those stored at 22°C for 1, 2, and 3 months were 28.8%, 26.6%, and 12.2%, respectively. Transfer of 38 two- to four-cell embryos from sperm stored at 4°C for 1 year produced two live pups while 59 two- to four-cell embryos from sperm stored at 22°C for 3 months also produced two live pups. Although all the pups looked healthy at 3 weeks of age, normality of offspring produced using convectively dried sperm needs further investigation. The percentages of blastocyst from sperm stored in the higher relative humidity conditions of NaBr and MgCl(2) jars and driest condition of P(2)O(5) jars at 4°C and 22°C were all lower. A simple method of mouse sperm preservation is demonstrated. Three-O-methyl-D-glucose, a metabolically inactive derivative of glucose, offers significant protection for dried mouse sperm at above freezing temperatures without the need for poration of cell membrane.

Epithelial Cell Guidance by Self-generated EGF Gradients

Integrative Biology : Quantitative Biosciences from Nano to Macro. Feb, 2012  |  Pubmed ID: 22314635

Cancer epithelial cells often migrate away from the primary tumor to invade into the surrounding tissues. Their migration is commonly assumed to be directed by pre-existent spatial gradients of chemokines and growth factors in the target tissues. Unexpectedly however, we found that the guided migration of epithelial cells is possible in vitro in the absence of pre-existent chemical gradients. We observed that both normal and cancer epithelial cells can migrate persistently and reach the exit along the shortest path from microscopic mazes filled with uniform concentrations of media. Using microscale engineering techniques and biophysical models, we uncovered a self-guidance strategy during which epithelial cells generate their own guiding cues under conditions of biochemical confinement. The self-guidance strategy depends on the balance between three interdependent processes: epidermal growth factor (EGF) uptake by the cells (U), the restricted transport of EGF through the structured microenvironment (T), and cell chemotaxis toward the resultant EGF gradients (C). The UTC self-guidance strategy can be perturbed by inhibition of signalling through EGF-receptors and appears to be independent from chemokine signalling. Better understanding of the UTC self-guidance strategy could eventually help devise new ways for modulating epithelial cell migration and delaying cancer cell invasion or accelerating wound healing.