Dissociation in Italian Conjugations: a Single-route Account

Brain and Language. Apr-Jun, 2002  |  Pubmed ID: 12081400

Say and Clahsen (S and C) report an experiment involving assignment of past participle suffixes to nonce words in Italian. Their evidence suggests a dual-route model that assigns the theme vowel of the 1st conjugation, while storing it lexically in other conjugations. However, these nonce words were assigned suffixes by a computer algorithm that determined the past participle of the nonce items on the basis of phonological similarity to existing inflected forms. The outcome of the simulation mirrors that of the subjects closely, suggesting that the dissociations found by S and C are not adequate evidence for a dual-route model of Italian stem formation.

Flow Control with Hydrogels

Advanced Drug Delivery Reviews. Feb, 2004  |  Pubmed ID: 14741116

With the advent of the genomic revolution and the sequencing of the human genome complete, the majority of pharmaceuticals under development are proteins. Consequently, new techniques to more effectively administer these new protein therapeutics need to be developed. One method that is gaining popularity in the research community involves the use of responsive hydrogel actuators for flow control in drug delivery devices. Responsive hydrogels are materials able to undergo a volume change in response to a stimulus from their local environment. The following paper overviews recent advances made using hydrogel actuators for flow control such as resistance based valves, hydrogel jacket valves, hybrid hydrogel membrane valve, electrically triggered valves, and biomimetic valves. Also reviewed are several hydrogel flow control systems such as a flow sorter and pH-regulation system. The chemistry of the hydrogel actuators can be tweaked to allow physiological variables to trigger the volume expansion of the hydrogel actuators as demonstrated by several glucose sensitive hydrogel valves reviewed below. Therefore, the door to physiological feedback controlling the infusion rate in a drug delivery device is opened and has the potential to revolutionize protein pharmaceutical drug delivery.

Microfluidic Tectonics Platform: A Colorimetric, Disposable Botulinum Toxin Enzyme-linked Immunosorbent Assay System

Electrophoresis. Jun, 2004  |  Pubmed ID: 15188260

A fabrication platform for realizing integrated microfluidic devices is discussed. The platform allows for creating specific microsystems for multistep assays in an ad hoc manner as the components that perform the assay steps can be created at any location inside the device via in situ fabrication. The platform was utilized to create a prototype microsystem for detecting botulinum neurotoxin directly from whole blood. Process steps such as sample preparation by filtration, mixing and incubation with reagents was carried out on the device. Various microfluidic components such as channel network, valves and porous filter were fabricated from prepolymer mixture consisting of monomer, cross-linker and a photoinitiator. For detection of the toxoid, biotinylated antibodies were immobilized on streptavidin-functionalized agarose gel beads. The gel beads were introduced into the device and were used as readouts. Enzymatic reaction between alkaline phosphatase (on secondary antibody) and substrate produced an insoluble, colored precipitate that coated the beads thus making the readout visible to the naked eye. Clinically relevant amounts of the toxin can be detected from whole blood using the portable enzyme-linked immunosorbent assay (ELISA) system. Multiple layers can be realized for effective space utilization and creating a three-dimensional (3-D) chaotic mixer. In addition, external materials such as membranes can be incorporated into the device as components. Individual components that were necessary to perform these steps were characterized, and their mutual compatibility is also discussed.

Development of a Disposable Infusion System for the Delivery of Protein Therapeutics

Biomedical Microdevices. Sep, 2005  |  Pubmed ID: 16133810

This paper describes the development and optimization of a low flow open-loop infusion device for continuous delivery of protein therapeutics. Specifically, a non-electronic polymer device is actuated with responsive hydrogels to infuse at 2 microL/hr for 12 hours. Hydrogel actuators transduce a chemical signal (change in pH of the local environment) into a mechanical response (swelling) generating the pressure to drive the infusion. The hydrogel actuators are separated from the drug reservoir by an elastomeric impermeable membrane. As the hydrogel actuators expand, the expansion deflects the flexible membrane down and reduces the volume of the drug reservoir causing the infusion of drug through the needle that is the only outlet for the reservoir.

Estrogen Effects on the Expression of Brx in the Brain and Pituitary of the Mouse

Brain Research Bulletin. Apr, 2006  |  Pubmed ID: 16624676

A member of the Dbl family of oncoproteins was discovered in breast cancer tissue extracts. This novel protein, designated Brx, contains an estrogen-receptor binding motif and is highly expressed in hormone-responsive breast tissue. Due to its ability to augment ligand-dependent activation of estrogen receptors, we analyzed the expression of Brx in the adult mouse brain and pituitary. Results indicated that Brx was expressed in specific regions of the brain and pituitary. Furthermore, the results indicate that differences exist in both brain and pituitary tissue of male and female mice with greater expression in the female. However, estrogen did not influence Brx expression in ovariectomized mice. The anatomical studies support a role for Brx in its association with the estrogen receptor and that Brx may be involved in neuronal and pituitary function in a sexually dimorphic manner.

Rapid Prototyping for Neuroscience and Neural Engineering

Journal of Neuroscience Methods. Jul, 2008  |  Pubmed ID: 18565590

Rapid prototyping (RP) is a useful method for designing and fabricating a wide variety of devices used for neuroscience research. The present study confirms the utility of using fused deposition modeling, a specific form of RP, to produce three devices commonly used for basic science experimentation. The accuracy and precision of the RP method varies according to the type and quality of the printer as well as the thermoplastic substrate. The printer was capable of creating device channels with a minimum diameter of 0.4 or 0.6mm depending on the orientation of fabrication. RP enabled the computer-aided design and fabrication of three custom devices including a cortical recording/stroke induction platform capable of monitoring electrophysiological function during ischemic challenge. In addition to the recording platform, two perfusion chambers and a cranial window device were replicated with sub-millimeter precision. The ability to repeatedly modify the design of each device with minimal effort and low turn-around time is helpful for oft-unpredictable experimental conditions. Results obtained from validation studies using both the cortical recording platform and perfusion chamber did not vary from previous results using traditional hand-fabricated or commercially available devices. Combined with computer-aided design, rapid prototyping is an excellent alternative for developing and fabricating custom devices for neuroscience research.

Microfluidic Add-on for Standard Electrophysiology Chambers

Lab on a Chip. Jul, 2008  |  Pubmed ID: 18584078

We have developed a microfluidic brain slice device (microBSD) that marries an off-the shelf brain slice perfusion chamber with an array of microfluidic channels set into the bottom surface of the chamber substrate. As this device is created through rapid prototyping, once optimized, it is trivial to replicate and share the devices with other investigators. The device integrates seamlessly into standard physiology and imaging chambers and it is immediately available to the whole slice physiology community. With this technology we can address the flow of neurochemicals and any other soluble factors to precise locations in the brain slice with the temporal profile we choose. Dopamine (DA) was chosen as a model neurotransmitter and we have quantified delivery in brain tissue using cyclic voltammetry (CV) and fluorescence imaging.

The Phonetic Context of American English Flapping: Quantitative Evidence

Language and Speech. 2008  |  Pubmed ID: 19626926

The phonetic context in which word-medial flaps occur (in contrast to [th]) in American English is explored. The analysis focuses on stress placement, following phone, and syllabification. In Experiment 1, subjects provided their preference for [th] or [[symbol: see text]] in bisyllabic nonce words. Consistent with previous studies, flaps were preferred before stressless syllables and [th] before stressed syllables, but the following phone also exerted a small degree of influence. Experiments 2 and 3 tested whether [th] or [[symbol: see text]] are associated with a particular syllable position in bisyllabic words. They demonstrate that [th] is favored in onsets, while [[symbol: see text]] is not consistently placed in either the onset or coda, nor is it generally ambisyllabic. These findings contradict analyses that posit syllable division as a conditioning factor in the appearance of [th] versus [[symbol: see text]]. Experiment 4 examined the pronunciation of 480 multisyllabic words from the TIMIT corpus. VCV was seen to favor [[symbol: see text]], while VCV favored [th]. In addition, flaps tend to be followed by syllabic sonorants and [th] by tense vowels. Because the following phones that influenced [th] and [[symbol: see text]] in Experiment 4 differ from those that were significant in Experiment 1, more research is necessary into the effect that following phones have on the appearance of [th] and [[symbol: see text]].

Modulating Temporal and Spatial Oxygenation over Adherent Cellular Cultures

PloS One. 2009  |  Pubmed ID: 19727397

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical research. A microfabricated insert for multiwell plates has been developed to more effectively control the temporal and spatial oxygen concentration to better model physiological phenomena found in vivo. The platform consists of a polydimethylsiloxane insert that nests into a standard multiwell plate and serves as a passive microfluidic gas network with a gas-permeable membrane aimed to modulate oxygen delivery to adherent cells. Equilibration time is on the order of minutes and a wide variety of oxygen profiles can be attained based on the device design, such as the cyclic profile achieved in this study, and even oxygen gradients to mimic those found in vivo. The proper biological consequences of the device's oxygen delivery were confirmed in cellular models via a proliferation assay and western analysis of the upregulation of hypoxia inducible transcription factor-1alpha. These experiments serve as a demonstration for the platform as a viable tool to increase experimental throughput and permit novel experimental possibilities in any biomedical research lab.

Puncture Mechanics of Cnidarian Cnidocysts: a Natural Actuator

Journal of Biological Engineering. 2009  |  Pubmed ID: 19785761

Cnidocysts isolated from cnidarian organisms are attractive as a drug-delivery platform due to their fast, efficient delivery of toxins. The cnidocyst could be utilized as the means to deliver therapeutics in a wearable drug-delivery patch. Cnidocysts have been previously shown to discharge upon stimulation via electrical, mechanical, and chemical pathways. Cnidocysts isolated from the Portuguese Man O' War jellyfish (Physalia physalis) are attractive for this purpose because they possess relatively long threads, are capable of puncturing through hard fish scales, and are stable for years.

Device for the Control of Oxygen Concentration in Multiwell Cell Culture Plates

Conference Proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference. 2009  |  Pubmed ID: 19963531

Oxygen is a key modulator of many cellular pathways but current devices permitting in vitro oxygen modulation fail to meet the needs of many researchers. In this study, a microfabricated insert for multiwell formats has been developed to control the gas concentration of each well independent of the global incubator's condition. The platform consists of a polydimethylsiloxane (PDMS) insert that nests into a standard multiwell plate and serves as a passive network with a gas permeable membrane aimed to deliver gas to adherent cell cultures. Preliminary data demonstrate that the insert is effective in controlling the oxygen concentration at the cell surface inside a well with equilibration times in minutes rather than hours for conventional technologies. A wide variety of oxygen profiles can be attained based on the device design, such as the cyclic profile achieved in this study, and even gradients in local oxygen concentration to mimic those found in vivo for more biomimetic cellular models.

Microfluidic Device for Multimodal Characterization of Pancreatic Islets

Lab on a Chip. Jan, 2009  |  Pubmed ID: 19209341

A microfluidic device to perfuse pancreatic islets while simultaneously characterizing their functionality through fluorescence imaging of the mitochondrial membrane potential and intracellular calcium ([Ca(2+)](i)) in addition to enzyme linked immunosorbent assay (ELISA) quantification of secreted insulin was developed and characterized. This multimodal characterization of islet function will facilitate rapid assessment of tissue quality immediately following isolation from donor pancreas and allow more informed transplantation decisions to be made which may improve transplantation outcomes. The microfluidic perfusion chamber allows flow rates of up to 1 mL min(-1), without any noticeable perturbation or shear of islets. This multimodal quantification was done on both mouse and human islets. The ability of this simple microfluidic device to detect subtle variations in islet responses in different functional assays performed in short time-periods demonstrates that the microfluidic perfusion chamber device can be used as a new gold standard to perform comprehensive islet analysis and obtain a more meaningful predictive value for islet functionality prior to transplantation into recipients, which is currently difficult to predict using a single functional assay.

Statistical Dynamics of Flowing Red Blood Cells by Morphological Image Processing

PLoS Computational Biology. Feb, 2009  |  Pubmed ID: 19214200

Blood is a dense suspension of soft non-Brownian cells of unique importance. Physiological blood flow involves complex interactions of blood cells with each other and with the environment due to the combined effects of varying cell concentration, cell morphology, cell rheology, and confinement. We analyze these interactions using computational morphological image analysis and machine learning algorithms to quantify the non-equilibrium fluctuations of cellular velocities in a minimal, quasi-two-dimensional microfluidic setting that enables high-resolution spatio-temporal measurements of blood cell flow. In particular, we measure the effective hydrodynamic diffusivity of blood cells and analyze its relationship to macroscopic properties such as bulk flow velocity and density. We also use the effective suspension temperature to distinguish the flow of normal red blood cells and pathological sickled red blood cells and suggest that this temperature may help to characterize the propensity for stasis in Virchow's Triad of blood clotting and thrombosis.

Impact of Phlebotomine Sand Flies on U.S. Military Operations at Tallil Air Base, Iraq: 4. Detection and Identification of Leishmania Parasites in Sand Flies

Journal of Medical Entomology. May, 2009  |  Pubmed ID: 19496439

Sand flies collected between April 2003 and November 2004 at Tallil Air Base, Iraq, were evaluated for the presence of Leishmania parasites using a combination of a real-time Leishmania-generic polymerase chain reaction (PCR) assay and sequencing of a 360-bp fragment of the glucose-6-phosphate-isomerase (GPI) gene. A total of 2,505 pools containing 26,574 sand flies were tested using the real-time PCR assay. Leishmania DNA was initially detected in 536 pools; however, after extensive retesting with the real-time PCR assay, a total of 456 pools were considered positive and 80 were considered indeterminate. A total of 532 samples were evaluated for Leishmania GPI by sequencing, to include 439 PCR-positive samples, 80 PCR-indeterminate samples, and 13 PCR-negative samples. Leishmania GPI was detected in 284 samples that were sequenced, to include 281 (64%) of the PCR-positive samples and 3 (4%) of the PCR-indeterminate samples. Of the 284 sequences identified as Leishmania, 261 (91.9%) were L. tarentolae, 18 (6.3%) were L. donovani-complex parasites, 3 (1.1%) were L. tropica, and 2 were similar to both L. major and L. tropica. Minimum field infection rates were 0.09% for L. donovani-complex parasites, 0.02% for L. tropica, and 0.01% for the L. major/tropica-like parasite. Subsequent sequencing of a 600-bp region of the "Hyper" gene of 12 of the L. donovani-complex parasites showed that all 12 parasites were L. infantum. These data suggest that L. infantum was the primary leishmanial threat to U.S. military personnel deployed to Tallil Air Base. The implications of these findings are discussed.

Enhanced Tumor Cell Isolation by a Biomimetic Combination of E-selectin and Anti-EpCAM: Implications for the Effective Separation of Circulating Tumor Cells (CTCs)

Langmuir : the ACS Journal of Surfaces and Colloids. Jun, 2010  |  Pubmed ID: 20155985

The selective detection of circulating tumor cells (CTCs) is of significant clinical importance for the clinical diagnosis and prognosis of cancer metastasis. However, largely because of the extremely low number of CTCs (as low as 1 in 10(9) hematologic cells) in the blood of patients, effective detection and separation of the rare cells remain a tremendous challenge. Cell rolling is known to play a key role in physiological processes such as the recruitment of leukocytes to sites of inflammation and selectin-mediated CTC metastasis. Furthermore, because CTCs typically express the epithelial-cell adhesion molecule (EpCAM) on the surface whereas normal hematologic cells do not, substrates with immobilized antibody against EpCAM may specifically interact with CTCs. In this article, we created biomimetic surfaces functionalized with P- and E-selectin and anti-EpCAM that induce different responses in HL-60 (used as a model of leukocytes in this study) and MCF-7 (a model of CTCs) cells. HL-60 and MCF-7 cells showed different degrees of interaction with P-/E-selectin and anti-EpCAM at a shear stress of 0.32 dyn/cm(2). HL-60 cells exhibited rolling on P-selectin-immobilized substrates at a velocity of 2.26 +/- 0.28 microm/s whereas MCF-7 cells had no interaction with the surface. Both cell lines, however, had interactions with E-selectin, and the rolling velocity of MCF-7 cells (4.24 +/- 0.31 microm/s) was faster than that of HL-60 cells (2.12 +/- 0.15 microm/s). However, only MCF-7 cells interacted with anti-EpCAM-coated surfaces, forming stationary binding under flow. More importantly, the combination of the rolling (E-selectin) and stationary binding (anti-EpCAM) resulted in substantially enhanced separation capacity and capture efficiency (more than 3-fold enhancement), as compared to a surface functionalized solely with anti-EpCAM that has been commonly used for CTC capture. Our results indicate that cell-specific detection and separation may be achieved through mimicking the biological processes of combined dynamic cell rolling and stationary binding, which will likely lead to a CTC detection device with significantly enhanced specificity and sensitivity without a complex fabrication process.

Microfluidic Perifusion and Imaging Device for Multi-parametric Islet Function Assessment

Biomedical Microdevices. Jun, 2010  |  Pubmed ID: 20300858

A microfluidic islet perifusion device was developed for the assessment of dynamic insulin secretion of multiple pancreatic islets and simultaneous fluorescence imaging of calcium influx and mitochondrial potential changes. The fanned out design of the second generation device optimized the efficient mixing and uniform distribution of rapid alternating solutions in the perifusion chamber and allowed for the generation of reproducible glucose gradients. Simultaneous imaging of calcium influx and mitochondrial potential changes in response to glucose stimulation showed high signal-noise ratio and spatial-temporal resolution. These results suggest that this system can be used for detailed study of the endocrine function of pancreatic islets with simultaneous imaging of intracellular ion fluxes and mitochondrial membrane potential changes. This tool can be used for quality assessment of islets preparation before transplantation and for in vitro studies of islet function.

Multiphysics Simulation of a Microfluidic Perfusion Chamber for Brain Slice Physiology

Biomedical Microdevices. Oct, 2010  |  Pubmed ID: 20464499

Understanding and optimizing fluid flows through in vitro microfluidic perfusion systems is essential in mimicking in vivo conditions for biological research. In a previous study a microfluidic brain slice device (microBSD) was developed for microscale electrophysiology investigations. The device consisted of a standard perfusion chamber bonded to a polydimethylsiloxane (PDMS) microchannel substrate. Our objective in this study is to characterize the flows through the microBSD by using multiphysics simulations of injections into a pourous matrix to identify optimal spacing of ports. Three-dimensional computational fluid dynamic (CFD) simulations are performed with CFD-ACE + software to model, simulate, and assess the transport of soluble factors through the perfusion bath, the microchannels, and a material that mimics the porosity, permeability and tortuosity of brain tissue. Additionally, experimental soluble factor transport through a brain slice is predicted by and compared to simulated fluid flow in a volume that represents a porous matrix material. The computational results are validated with fluorescent dye experiments.

Size-based Separation and Collection of Mouse Pancreatic Islets for Functional Analysis

Biomedical Microdevices. Oct, 2010  |  Pubmed ID: 20549367

Islet size has recently been demonstrated to be an important factor in determining human islet transplantation outcomes. In this study, a multi-layered microfluidic device was developed and quantified for size-based separation of a heterogeneous population of mouse islets. The device was fabricated using standard soft lithography and polydimethylsiloxane (PDMS). Size-based separation was first demonstrated via injection of a heterogeneous population of glass beads between 50-300 microm in diameter which were separated into five sub-populations based on their diameter. Next, a heterogeneous population of mouse pancreatic islets, between 50-250 microm in diameter was separated into four sub-populations. Throughout this process the islets remained intact without any signs of damage, as indicated by cell viability staining. Islet glucose-stimulated insulin secretion of each sub-population of islets was also evaluated demonstrating that islets smaller than 150 microm have superior stimulation indexes (SI) compared to islets larger than 150 microm. In this study, we found that islets between 100 microm and 150 microm in diameter had the greatest SI value in a heterogeneous population of islets.

Oxygen Gradients for Open Well Cellular Cultures Via Microfluidic Substrates

Lab on a Chip. Sep, 2010  |  Pubmed ID: 20559583

Controlling oxygen concentration at a microscale level can benefit experimental investigations involving oxidative stress, ischemia, and reactive oxygen species (ROS) mediated cellular pathways. Here, we report the application of microfluidic gradient generation in an open-well culture model, in which a gradient of gas is delivered via diffusion through a gas permeable substrate that separates cells from the gas microchannels below. By using diffusion to localize oxygen delivery, microgradients of oxygen concentrations can be rapidly and controllably applied without exposing cells to mechanical stresses or reducing culture volumes inside microfluidic culture chambers. Furthermore, we demonstrate the modulation of intracellular ROS levels in Madin-Darby Canine Kidney (MDCK) cells by applying these oxygen microgradients. Increases in ROS levels consistent with both oxidative stress and hypoxic exposures were observed in MDCK cells. The measured ROS increases were comparable to 100 microM hydrogen peroxide exposure in a control comparison, which is within the range of standard ROS induction methods. Incubation with 200 microM vitamin C was able to demodulate the ROS response at both hypoxic and hyperoxic exposures. By providing microfluidic controlled gradients, constant ROS exposure, and a shear-free open well design, the devices introduced here greatly improve upon standard oxygen-based culturing methods.

Precise Control over the Oxygen Conditions Within the Boyden Chamber Using a Microfabricated Insert

Lab on a Chip. Sep, 2010  |  Pubmed ID: 20689862

Cell migration is a hallmark of cancer cell metastasis and is highly correlated with hypoxia in tumors. The Boyden chamber is a porous membrane-based migration platform that has seen a great deal of use for both in vitro migration and invasion assays due to its adaptability to common culture vessels and relative ease of use. The hypoxic chamber is a current tool that can be implemented to investigate the cellular response to oxygen paradigms. Unfortunately, this method lacks the spatial and temporal precision to accurately model a number of physiological phenomena. In this article, we present a newly developed microfabricated polydimethylsiloxane (PDMS) device that easily adapts to the Boyden chamber, and provides more control over the oxygenation conditions exposed to cells. The device equilibrates to 1% oxygen in about 20 min, thus demonstrating the capabilities of a system for researchers to establish both short-term continuous and intermittent hypoxia regimes. A Parylene-C thin-film coating was used to prevent ambient air penetration through the bulk PDMS and was found to yield improved equilibration times and end-point concentrations. MDA-MD-231 cells, an invasive breast cancer line, were used as a model cell type to demonstrate the effect of oxygen concentration on cell migration through the Boyden chamber porous membrane. Continuous hypoxia downregulated migration of cells relative to the normoxic control, as did an intermittent hypoxia regime (IH) cycling between 0% and 21% oxygen (0-21% IH). However, cells exposed to 5-21% IH exhibited increased migration compared to the other conditions, as well as relative to the normoxic control. The results presented here show the device can be utilized for experiments implementing the Boyden chamber for in vitro hypoxic studies, allowing experiments to be conducted faster and with more precision than currently possible.

Contributors to the Emerging Investigators Issue

Lab on a Chip. Sep, 2010  |  Pubmed ID: 20717627

Oxygen Sensitive Microwells

Lab on a Chip. Dec, 2010  |  Pubmed ID: 20938500

Oxygen tension is critical in a number of cell pathways but is often overlooked in cell culture. One reason for this is the difficulty in modulating and assessing oxygen tensions without disturbing the culture conditions. Toward this end, a simple method to generate oxygen-sensitive microwells was developed through embossing polystyrene (PS) and platinum(ii) octaethylporphyrin ketone (PtOEPK) thin films. In addition to monitoring the oxygen tension, microwells were employed in order to isolate uniform clusters of cells in microwells. The depth and width of the microwells can be adapted to different experimental parameters easily by altering the thin film processing or embossing stamp geometries. The thin oxygen sensitive microwell substrate is also compatible with high magnification modalities such as confocal imaging. The incorporation of the oxygen sensor into the microwells produces measurements of the oxygen tension near the cell surface. The oxygen sensitive microwells were calibrated and used to monitor oxygen tensions of Madin-Darby Canine Kidney Cells (MDCKs) cultured at high and low densities as a proof of concept. Wells 500 µm in diameter seeded with an average of 330 cells exhibited an oxygen level of 12.6% whereas wells seeded with an average of 20 cells per well exhibited an oxygen level of 19.5%, a 35.7% difference. This platform represents a new tool for culturing cells in microwells in a format amenable to high magnification imaging while monitoring the oxygen state of the culture media.

Application of Microfluidic Technology to Pancreatic Islet Research: First Decade of Endeavor

Bioanalysis. Oct, 2010  |  Pubmed ID: 21083325

β-cells respond to blood glucose by secreting insulin to maintain glucose homeostasis. Perifusion enables manipulation of biological and chemical cues in elucidating the mechanisms of β-cell physiology. Recently, microfluidic devices made of polydimethylsiloxane and Borofloat glass have been developed as miniaturized perifusion setups and demonstrated distinct advantages over conventional techniques in resolving rapid secretory and metabolic waveforms intrinsic to β-cells. In order to enhance sensing and monitoring capabilities, these devices have been integrated with analytical tools to increase assay throughput. The spatio-temporal resolutions of these analyses have been improved through enhanced flow control, valves and compartmentalization. For the first time, this review provides an overview of current devices used in islet studies and analyzes their strengths and experimental suitability. To realize the potential of microfluidic islet applications, it is essential to bridge the gap in design and application between engineers and biologists through the creation of standardized bioassays and user-friendly interfaces.

Direct Measurements on CD24-mediated Rolling of Human Breast Cancer MCF-7 Cells on E-selectin

Analytical Chemistry. Feb, 2011  |  Pubmed ID: 21207944

Tumor cell rolling on the endothelium plays a key role in the initial steps of cancer metastasis, i.e., extravasation of circulating tumor cells (CTCs). Identification of the ligands that induce the rolling of cells is thus critical to understanding how cancers metastasize. We have previously demonstrated that MCF-7 cells, human breast cancer cells, exhibit the rolling response selectively on E-selectin-immobilized surfaces. However, the ligand that induces rolling of MCF-7 cells on E-selectin has not yet been identified, as these cells lack commonly known E-selectin ligands. Here we report, for the first time to our knowledge, a set of quantitative and direct evidence demonstrating that CD24 expressed on MCF-7 cell membranes is responsible for rolling of the cells on E-selectin. The binding kinetics between CD24 and E-selectin was directly measured using surface plasmon resonance (SPR), which revealed that CD24 has a binding affinity against E-selectin (K(D) = 3.4 ± 0.7 nM). The involvement of CD24 in MCF-7 cell rolling was confirmed by the rolling behavior that was completely blocked when cells were treated with anti-CD24. A simulated study by flowing microspheres coated with CD24 onto E-selectin-immobilized surfaces further revealed that the binding is Ca(2+)-dependent. Additionally, we have found that actin filaments are involved in the CD24-mediated cell rolling, as observed by the decreased rolling velocities of the MCF-7 cells upon treatment with cytochalasin D (an inhibitor of actin-filament dynamics) and the stationary binding of CD24-coated microspheres (the lack of actins) on the E-selectin-immobilized slides. Given that CD24 is known to be directly related to enhanced invasiveness of cancer cells, our results imply that CD24-based cell rolling on E-selectin mediates, at least partially, cancer cell extravasation, resulting in metastasis.

Rheologically Biomimetic Cell Suspensions for Decreased Cell Settling in Microfluidic Devices

Biomedical Microdevices. Jun, 2011  |  Pubmed ID: 21409456

Many microfluidic devices operate with cells suspended in buffer solutions. Researchers who work with large cell types in such devices often run into problems with gravitational cell settling in the injection equipment and in the device itself. A method for reducing this problematic settling is discussed in this paper using tumor cell lines as an example. Microfluidic circulating tumor cell (CTC) isolation devices (MCIDs) are benchmarked using buffer solutions spiked with in-vitro tumor cell lines prior to validation with clinical samples (i.e. whole blood). However, buffer solutions have different rheological properties than whole blood. Here we describe the use of alginate in PBS buffer solutions to mimic blood rheology and reduce cell settling during preliminary validation experiments. Because alginate increases the viscosity of a solution, it helps to maintain cells in suspension. We report that vertical equipment configurations are important to further mitigate the effects of cell settling for MDA-MB-468 carcinoma cells. We also report that alginate does not disrupt the specific binding interactions that are the basis of carcinoma cell capture in MCIDs. These results indicate that vertical equipment configurations and the addition of alginates can be used to reduce cell settling in buffer based MCID testing and other applications involving large cells suspended in buffer solution.

Controlling Flow in Microfluidic Channels with a Manually Actuated Pin Valve

Biomedical Microdevices. Aug, 2011  |  Pubmed ID: 21472409

There is a need for a simple method to control fluid flow within microfluidic channels. To meet this need, a simple push pin with a polydimethylsiloxane (PDMS) tip has been integrated into microfluidic networks to be placed within the microchannel to obstruct flow. This new valve design can attain on/off control of fluid flow without an external power source using readily-available, low-cost materials. The valve consists of a 14 gauge (1.6 mm) one inch piece of metal tubing with a PDMS pad at the tip to achieve a fluidic seal when pressed against a microfluidic channel's substrate. The metal tubing or pin is then either manually pushed down to block or pulled up to allow fluid flow. The valve was validated using a pressure transducer and fluorescent dye to determine the breakthrough pressure the valve can withstand over multiple cycles. In the first cycle, the median value for pressure withstood by the valve was 8.8 psi with a range of 17.5-2.7 psi. The pressure the valves were able to withstand during each successive trial was lower suggesting they may be most valuable as a method to control the initial introduction of fluids into a microfluidic device. These valves can achieve flow regulation within microfluidic devices, have a small dead volume, and are simple to fabricate and use, making this technique widely suitable for a range of applications.

Dual Microfluidic Perifusion Networks for Concurrent Islet Perifusion and Optical Imaging

Biomedical Microdevices. Aug, 2011  |  Pubmed ID: 21850483

This study explores a new class of duplex microfluidic device which utilizes a dual perifusion network to simultaneously perform live-cell optical imaging of physiological activities and study insulin release kinetics on two islet populations. This device also incorporates on-chip staggered herringbone mixers (SHMs) to increase mixing efficiency and facilitate the generation of user-defined chemical gradients. Mouse islets are used to simultaneously measure dynamic insulin release, changes in mitochondrial potentials, and calcium influx in response to insulin secretagogues (glucose and tolbutamide), and show a high signal-to-noise ratio and spatiotemporal resolution of all measured parameters for both perifusion chambers. This system has many potential applications for studying β-cell physiology and pathophysiology, as well as for therapeutic drug screening. This dual perifusion device is not limited to islet studies and could easily be applied to other tissues and cells without major modifications.

Dendrimer-mediated Multivalent Binding for the Enhanced Capture of Tumor Cells

Angewandte Chemie (International Ed. in English). Dec, 2011  |  Pubmed ID: 22012872

Systematic Prevention of Bubble Formation and Accumulation for Long-term Culture of Pancreatic Islet Cells in Microfluidic Device

Biomedical Microdevices. Jan, 2012  |  Pubmed ID: 22252566

Reliable long-term cell culture in microfluidic system is limited by air bubble formation and accumulation. In this study, we developed a bubble removal system capable of both trapping and discharging air bubbles in a consistent and reliable manner. Combined with PDMS (Polydimethylsiloxane) hydrophilic surface treatment and vacuum filling, a microfluidic perifusion system equipped with the bubble trap was successfully applied for long-term culture of mouse pancreatic islets with no bubble formation and no flow interruption. In addition to demonstrating normal cell viability and islet morphology, post-cultured islets exhibited normal insulin secretion kinetics, intracellular calcium signaling, and changes in mitochondrial potentials in response to glucose challenge. This design could be easily adapted by other microfluidic systems due to its simple design, ease of fabrication, and portability.

Islet Preconditioning Via Multimodal Microfluidic Modulation of Intermittent Hypoxia

Analytical Chemistry. Feb, 2012  |  Pubmed ID: 22296179

Simultaneous stimulation of ex vivo pancreatic islets with dynamic oxygen and glucose is a critical technique for studying how hypoxia alters glucose-stimulated response, especially in transplant environments. Standard techniques using a hypoxic chamber cannot provide both oxygen and glucose modulations, while monitoring stimulus-secretion coupling factors in real-time. Using novel microfluidic device with integrated glucose and oxygen modulations, we quantified hypoxic impairment of islet response by calcium influx, mitochondrial potentials, and insulin secretion. Glucose-induced calcium response magnitude and phase were suppressed by hypoxia, while mitochondrial hyperpolarization and insulin secretion decreased in coordination. More importantly, hypoxic response was improved by preconditioning islets to intermittent hypoxia (IH, 1 min/1 min 5-21% cycling for 1 h), translating to improved insulin secretion. Moreover, blocking mitochondrial K(ATP) channels removed preconditioning benefits of IH, similar to mechanisms in preconditioned cardiomyocytes. Additionally, the multimodal device can be applied to a variety of dynamic oxygen-metabolic studies in other ex vivo tissues.