A Simple Soft Lithographic Route to Fabrication of Poly(ethylene Glycol) Microstructures for Protein and Cell Patterning

Biomaterials. Feb, 2004  |  Pubmed ID: 14585705

We present a simple, direct soft lithographic method to fabricate poly(ethylene glycol) (PEG) microstructures for protein and cell patterning. This lithographic method involves a molding process in which a uniform PEG film is molded with a patterned polydimethylsiloxane stamp by means of capillary force. The patterned surfaces created by this method provide excellent resistance towards non-specific protein and cell adsorption. The patterned substrates consist of two regions: the molded PEG surface that acts as a resistant layer and the exposed substrate surface that promotes protein or cell adsorption. A notable finding here is that the substrate surface can be directly exposed during the molding process due to the ability to control the wetting properties of the polymer on the stamp, which is a key factor to patterning proteins and cells.

Layer-by-layer Deposition of Hyaluronic Acid and Poly-L-lysine for Patterned Cell Co-cultures

Biomaterials. Aug, 2004  |  Pubmed ID: 15020132

A novel method for patterning cellular co-cultures that uses the layer-by-layer deposition of ionic biopolymers is described. Non-biofouling hyaluronic acid (HA) micropatterns were used to immobilize cells and proteins to glass substrates. Subsequent ionic adsorption of poly-L-lysine (PLL) to HA patterns was used to switch the HA surfaces from cell repulsive to adherent thereby facilitating the adhesion of a second cell type. The utility of this approach to pattern co-cultures of hepatocytes or embryonic stem cells with fibroblasts was demonstrated. In addition, the versatility of this approach to generate patterned co-cultures irrespective of the primary cell seeding and relative adhesion of the seeded cells was demonstrated. Thus, the proposed method may be a useful tool for fabricating controlled cellular co-cultures for cell-cell interaction studies and tissue engineering applications.

A Soft Lithographic Approach to Fabricate Patterned Microfluidic Channels

Analytical Chemistry. Jul, 2004  |  Pubmed ID: 15228340

The control of surface properties and spatial presentation of functional molecules within a microfluidic channel is important for the development of diagnostic assays and microreactors and for performing fundamental studies of cell biology and fluid mechanics. Here, we present a simple technique, applicable to many soft lithographic methods, to fabricate robust microchannels with precise control over the spatial properties of the substrate. In this approach, the patterned regions were protected from oxygen plasma by controlling the dimensions of the poly(dimethylsiloxane) (PDMS) stamp and by leaving the stamp in place during the plasma treatment process. The PDMS stamp was then removed, and the microfluidic mold was irreversibly bonded to the substrate. The approach was used to pattern a nonbiofouling poly(ethylene glycol)-based copolymer or the polysaccharide hyaluronic acid within microfluidic channels. These nonbiofouling patterns were then used to fabricate arrays of fibronectin and bovine serum albumin as well as mammalian cells. In addition, further control over the deposition of multiple proteins onto multiple or individual patterns was achieved using laminar flow. Also, cells that were patterned within channels remained viable and capable of performing intracellular reactions and could be potentially lysed for analysis.

Single Nanocrystal Arrays on Patterned Poly(ethylene Glycol) Copolymer Microstructures Using Selective Wetting and Drying

Langmuir : the ACS Journal of Surfaces and Colloids. Jul, 2004  |  Pubmed ID: 15248685

Single nanocrystal arrays were fabricated on sub-microwells of poly(ethylene glycol) (PEG) copolymer using selective wetting on the hydrophilic regions of the exposed substrate surface and subsequent drying. Templates were produced by molding a thin film of a PEG-based random copolymer on hydrophilic substrates such as glass or silicon dioxide. The polymeric microstructures provide a topographical barrier around the well, which makes it possible to create nanocrystal arrays with controlled geometrical features. The size of the nanocrystal was found to decrease with decreasing well size and also decrease with decreasing topological height. A simple empirical equation was derived to predict the size of the crystal as a function of the pattern size and height, which is in good agreement with the experimental data.

Patterning and Separating Infected Bacteria Using Host-parasite and Virus-antibody Interactions

Biomedical Microdevices. Sep, 2004  |  Pubmed ID: 15377832

Bacteria were selectively deposited on substrates patterned with poly(ethylene glycol) (PEG) microstructures by using host-parasite and virus-antibody interactions. In this scheme viruses were used to attach onto a host bacterium, Escherichia coli (E. coli). The E. coli expressing the virus were selectively adhered to the regions pretreated with an antibody against the virus proteins while E. coli without the virus showed no selectivity. Single or aggregated cell arrays were fabricated depending on the initial pattern size with respect to the size of E. coli. The current approach could be a general route to spatially positioning or controlling adhesion of other biological species that are not accessible by conventional methods and as a tool for separating and isolating specific cell populations based on host-parasite interactions.

Molded Polyethylene Glycol Microstructures for Capturing Cells Within Microfluidic Channels

Lab on a Chip. Oct, 2004  |  Pubmed ID: 15472725

The ability to control the deposition and location of adherent and non-adherent cells within microfluidic devices is beneficial for the development of micro-scale bioanalytical tools and high-throughput screening systems. Here, we introduce a simple technique to fabricate poly(ethylene glycol)(PEG) microstructures within microfluidic channels that can be used to dock cells within pre-defined locations. Microstructures of various shapes were used to capture and shear-protect cells despite medium flow in the channel. Using this approach, PEG microwells were fabricated either with exposed or non-exposed substrates. Proteins and cells adhered within microwells with exposed substrates, while non-exposed substrates prevented protein and cell adhesion (although the cells were captured inside the features). Furthermore, immobilized cells remained viable and were stained for cell surface receptors by sequential flow of antibodies and secondary fluorescent probes. With its unique strengths in utility and control, this approach is potentially beneficial for the development of cell-based analytical devices and microreactors that enable the capture and real-time analysis of cells within microchannels, irrespective of cell anchorage properties.

Nanoparticle-aptamer Bioconjugates: a New Approach for Targeting Prostate Cancer Cells

Cancer Research. Nov, 2004  |  Pubmed ID: 15520166

Nucleic acid ligands (aptamers) are potentially well suited for the therapeutic targeting of drug encapsulated controlled release polymer particles in a cell- or tissue-specific manner. We synthesized a bioconjugate composed of controlled release polymer nanoparticles and aptamers and examined its efficacy for targeted delivery to prostate cancer cells. Specifically, we synthesized poly(lactic acid)-block-polyethylene glycol (PEG) copolymer with a terminal carboxylic acid functional group (PLA-PEG-COOH), and encapsulated rhodamine-labeled dextran (as a model drug) within PLA-PEG-COOH nanoparticles. These nanoparticles have the following desirable characteristics: (a) negative surface charge (-50 +/- 3 mV, mean +/- SD, n = 3), which may minimize nonspecific interaction with the negatively charged nucleic acid aptamers; (b) carboxylic acid groups on the particle surface for potential modification and covalent conjugation to amine-modified aptamers; and (c) presence of PEG on particle surface, which enhances circulating half-life while contributing to decreased uptake in nontargeted cells. Next, we generated nanoparticle-aptamer bioconjugates with RNA aptamers that bind to the prostate-specific membrane antigen, a well-known prostate cancer tumor marker that is overexpressed on prostate acinar epithelial cells. We demonstrated that these bioconjugates can efficiently target and get taken up by the prostate LNCaP epithelial cells, which express the prostate-specific membrane antigen protein (77-fold increase in binding versus control, n = 150 cells per group). In contrast to LNCaP cells, the uptake of these particles is not enhanced in cells that do not express the prostate-specific membrane antigen protein. To our knowledge, this represents the first report of targeted drug delivery with nanoparticle-aptamer bioconjugates.

Fabrication of Gradient Hydrogels Using a Microfluidics/photopolymerization Process

Langmuir : the ACS Journal of Surfaces and Colloids. Jun, 2004  |  Pubmed ID: 15986641

A method of fabricating photo-cross-linked hydrogels with gradients of immobilized molecules and crosslinking densities is introduced. Two macromer/initiator solutions are injected into a unique poly(dimethylsiloxane) channel system that produces a prepolymer gradient that is subsequently polymerized into a water-swollen hydrogel with ultraviolet light exposure. The gradient is controlled by the injection flow rate (optimized to 0.3 microL/min per inlet to produce a linear gradient). The technique is investigated both through fabrication of adhesive ligand gradients that modulate spatial distribution of attached endothelial cells and gradients of cross-linking densities that led to unique hydrogel architectures and spatially dependent swelling.

Microfluidic System for Studying the Interaction of Nanoparticles and Microparticles with Cells

Analytical Chemistry. Sep, 2005  |  Pubmed ID: 16131052

Nanoparticles and microparticles have many potential biomedical applications ranging from imaging to drug delivery. Therefore, in vitro systems that can analyze and optimize the interaction of such particles with cells may be beneficial. Here, we report a microfluidic system that can be used to study these interactions. As a model system, we evaluated the interaction of polymeric nanoparticles and microparticles and similar particles conjugated to aptamers that recognize the transmembrane prostate specific membrane antigen (PSMA), with cells seeded in microchannels. The binding of particles to cells that expressed or did not express the PSMA (LNCaP or PC3, respectively) were evaluated with respect to changes in fluid shear stress, PSMA expression on target cells, and particle size. Nanoparticle-aptamer bioconjugates selectively adhered to LNCaP but not PC3 cells at static and low shear (<1 dyn/cm2) but not higher shear (approximately 4.5 dyn/cm2) conditions. Control nanoparticles and microparticles lacking aptamers and microparticle-aptamer bioconjugates did not adhere to LNCaP cells, even under very low shear conditions (approximately 0.28 dyn/cm2). These results demonstrate that the interaction of particles with cells can be studied under controlled conditions, which may aid in the engineering of desired particle characteristics. The scalability, low cost, reproducibility, and high-throughput capability of this technology is potentially beneficial to examining and optimizing a wide array of cell-particle systems prior to in vivo experiments.

Characterization of Chemisorbed Hyaluronic Acid Directly Immobilized on Solid Substrates

Journal of Biomedical Materials Research. Part B, Applied Biomaterials. Feb, 2005  |  Pubmed ID: 15486967

Hyaluronic acid (HA) has a number of potential biomedical applications in drug delivery and tissue engineering. For these applications, a prerequisite is to understand the characteristic of HA films directly immobilized to solid substrates. Here, we demonstrate that high molecular weight HA can be directly immobilized onto hydrophilic substrates without any chemical manipulation, allowing for the formation of an ultrathin chemisorbed layer. Hyaluronic acid is stabilized on these surfaces through hydrogen bonding between the hydrophilic moieties in HA [such as carboxylic acid (-COOH) or hydroxyl (-OH) groups] with silanol (-SiOH), carboxylic acid or hydroxyl groups on the hydrophilic substrates. Despite the water solubility, the chemisorbed HA layer remained stable on glass or silicon oxide substrates for at least 7 days in phosphate-buffered saline. Furthermore, HA immobilized on silicon and other dioxide surfaces in much higher quantities than other polysaccharides including dextran sulfate, heparin, heparin sulfate, chondroitin sulfate, dermatan sulfate, and alginic acid. This behavior is related to the molecular entanglement and intrinsic stiffness of HA as a result of strong internal and external hydrogen bonding as well as high molecular weight. These results demonstrate that HA can be used to coat surfaces through direct immobilization.

Conformal Coating of Mammalian Cells Immobilized Onto Magnetically Driven Beads

Tissue Engineering. Nov-Dec, 2005  |  Pubmed ID: 16411825

A novel cell bead system, comprising a magnetic core, a spherical annulus of agarose-immobilized cells, all conformally coated within a synthetic polymer, is proposed as a means of immunoisolating mammalian cells in a system that provides a balance between low total implant volume, retrievability, and diffusion limitations. A successful immunoisolation system could be used to transplant cells without eliciting an inappropriate host response. Chinese hamster ovary (CHO) cells were immobilized at the periphery of large (approximately 2 mm) agarose beads containing inert magnetic cores (< or = 1 mm) and coated in a hydroxyethyl methacrylate-methyl methacrylate (HEMA-MMA) copolymer by interfacial precipitation. The beads were coated in liquid gradients containing polyethylene glycol 200 (PEG) or bromooctane. Although many cells were adversely affected by the coating process, the cells that did survive (30-50% of those loaded into the beads) remained viable for a period of at least 2 weeks. This viability was much higher than achieved previously because of a number of factors, such as the aqueous agarose, the hydrophobic bromooctane intermediate layer, and faster coating times that minimize the exposure of the cells to organic solvents. Also, a mathematical model was used to describe oxygen transport within the annular agarose beads. These results provide evidence that the proposed geometry and the fabrication approach may be useful for a variety of applications that involve cell encapsulation.

Chips to Hits: Microarray and Microfluidic Technologies for High-throughput Analysis and Drug Discovery. September 12-15, 2005, MA, USA

Expert Review of Molecular Diagnostics. Nov, 2005  |  Pubmed ID: 16255625

Cell Docking Inside Microwells Within Reversibly Sealed Microfluidic Channels for Fabricating Multiphenotype Cell Arrays

Lab on a Chip. Dec, 2005  |  Pubmed ID: 16286969

We present a soft lithographic method to fabricate multiphenotype cell arrays by capturing cells within an array of reversibly sealed microfluidic channels. The technique uses reversible sealing of elastomeric polydimethylsiloxane (PDMS) molds on surfaces to sequentially deliver various fluids or cells onto specific locations on a substrate. Microwells on the substrate were used to capture and immobilize cells within low shear stress regions inside channels. By using an array of channels it was possible to deposit multiple cell types, such as hepatocytes, fibroblasts, and embryonic stem cells, on the substrates. Upon formation of the cell arrays on the substrate, the PDMS mold could be removed, generating a multiphenotype array of cells. In addition, the orthogonal alignment and subsequent attachment of a secondary array of channels on the patterned substrates could be used to deliver fluids to the patterned cells. The ability to position many cell types on particular regions within a two dimensional substrate could potentially lead to improved high-throughput methods applicable to drug screening and tissue engineering.

Magnetically Responsive Polymeric Microparticles for Oral Delivery of Protein Drugs

Pharmaceutical Research. Mar, 2006  |  Pubmed ID: 16388405

Protein drugs cannot be delivered efficiently through oral routes. To address this challenge, we evaluated the effect of prolonged gastrointestinal transit on the bioavailability of insulin carried by magnetically responsive microparticles in the presence of an external magnetic field.

Interplay of Biomaterials and Micro-scale Technologies for Advancing Biomedical Applications

Journal of Biomaterials Science. Polymer Edition. 2006  |  Pubmed ID: 17176747

Micro-scale technologies have already dramatically changed our society through their use in the microelectronics and telecommunications industries. Today these engineering tools are also useful for many biological applications ranging from drug delivery to DNA sequencing, since they can be used to fabricate small features at a low cost and in a reproducible manner. The discovery and development of new biomaterials aid in the advancement of these micro-scale technologies, which in turn contribute to the engineering and generation of new, custom-designed biomaterials with desired properties. This review aims to present an overview of the merger of micro-scale technologies and biomaterials in two-dimensional (2D) surface patterning, device fabrication and three-dimensional (3D) tissue-engineering applications.

Microscale Technologies for Tissue Engineering and Biology

Proceedings of the National Academy of Sciences of the United States of America. Feb, 2006  |  Pubmed ID: 16477028

Microscale technologies are emerging as powerful tools for tissue engineering and biological studies. In this review, we present an overview of these technologies in various tissue engineering applications, such as for fabricating 3D microfabricated scaffolds, as templates for cell aggregate formation, or for fabricating materials in a spatially regulated manner. In addition, we give examples of the use of microscale technologies for controlling the cellular microenvironment in vitro and for performing high-throughput assays. The use of microfluidics, surface patterning, and patterned cocultures in regulating various aspects of cellular microenvironment is discussed, as well as the application of these technologies in directing cell fate and elucidating the underlying biology. Throughout this review, we will use specific examples where available and will provide trends and future directions in the field.

Direct Confinement of Individual Viruses Within Polyethylene Glycol (PEG) Nanowells

Nano Letters. Jun, 2006  |  Pubmed ID: 16771579

Individual M13 viruses were spatially confined within wells fabricated from nanomolding of a PEG-based random copolymer. The viruses were selectively adhered to the region pretreated with an antibody against the virus, resulting in individual virus arrays. The polymer surface was found to be highly resistant to the attachment of the virus (approximately 0.02 microm-2), approximately 2 orders of magnitude lower than that on a bare silicon surface. The physical height of the template provided an additional barrier to the attachment of the virus due to entropic penalty in bending of a semi-flexible M13 virus. The effects of pattern size and barrier height were investigated, revealing that a certain critical height is needed to ensure successful confinement within the template for a given pattern size.

Micromolding of Photocrosslinkable Hyaluronic Acid for Cell Encapsulation and Entrapment

Journal of Biomedical Materials Research. Part A. Dec, 2006  |  Pubmed ID: 16788972

Micropatterning of hydrogels is potentially useful for a variety of applications, including tissue engineering, fundamental biological studies, diagnostics, and high-throughput screening. Although synthetic polymers have been developed for these applications, natural polymers such as polysaccharides may have advantages for biological samples and cell-based devices because they are natural components of the in vivo microenvironment. In this study, we synthesized and used hyaluronic acid (HA) modified with photoreactive methacrylates to fabricate microstructures as functional components of microfabricated devices. To demonstrate the universality of this approach, two types of microstructures were formed. In the first approach, HA microstructures were fabricated and used as docking templates to enable the subsequent formation of cell microarrays within low shear stress regions of the patterns. Cells within these microwells remained viable, could generate spheroids, and could be retrieved using mechanical disruption. In the second approach, cells were encapsulated directly within the HA hydrogels. Arrays of viable embryonic stem (ES) cells or fibroblasts were encapsulated within HA hydrogels and could later be recovered using enzymatic digestion of the microstructures. These results demonstrate that it is possible to incorporate photocrosslinkable HA, a natural, versatile, degradable, and biocompatible biopolymer, into micro-electromechanical systems.

Micromolding of Photocrosslinkable Chitosan Hydrogel for Spheroid Microarray and Co-cultures

Biomaterials. Oct, 2006  |  Pubmed ID: 16814859

Bioengineering approaches, such as co-cultures of multiple cell types, that aim to mimic the physiological microenvironment may be beneficial for optimizing cell function and for engineering tissues in vitro. This study describes a novel method for preparing a spheroid microarray on microfabricated hydrogels, alone or in co-cultures. Photocrosslinkable chitosan was synthesized and utilized for fabricating hydrogel microstructures through a micromolding process. The chitosan surface was initially cell repellent but became increasingly cell adhesive over time. By using this unique property of chitosan hydrogels, it was possible to generate patterned co-cultures of spheroids and support cells. In this scheme, cells were initially microarrayed within low shear stress regions of microwells. Human hepatoblastoma cells, Hep G2, seeded in these wells formed spheroids with controlled sizes and shapes and stably secreted albumin during the culture period. The change of cell adhesive properties in the chitosan surface facilitated the adhesion and growth of a second cell type, NIH-3T3 fibroblast, and therefore enabled co-cultures of hepatocyte spheroids and fibroblast monolayers. This co-culture system could be a useful platform for studying heterotypic cell-cell interactions, for drug screening, and for developing implantable bioartificial organs.

Micromolding of Shape-controlled, Harvestable Cell-laden Hydrogels

Biomaterials. Nov, 2006  |  Pubmed ID: 16828863

Encapsulation of mammalian cells within hydrogels has great utility for a variety of applications ranging from tissue engineering to cell-based assays. In this work, we present a technique to encapsulate live cells in three-dimensional (3D) microscale hydrogels (microgels) of controlled shapes and sizes in the form of harvestable free standing units. Cells were suspended in methacrylated hyaluronic acid (MeHA) or poly(ethylene glycol) diacrylate (PEGDA) hydrogel precursor solution containing photoinitiator, micromolded using a hydrophilic poly(dimethylsiloxane) (PDMS) stamp, and crosslinked using ultraviolet (UV) radiation. By controlling the features on the PDMS stamp, the size and shape of the molded hydrogels were controlled. Cells within microgels were well distributed and remained viable. These shape-specific microgels could be easily retrieved, cultured and potentially assembled to generate structures with controlled spatial distribution of multiple cell types. Further development of this technique may lead to applications in 3D co-cultures for tissue/organ regeneration and cell-based assays in which it is important to mimic the architectural intricacies of physiological cell-cell interactions.

Co-culture of Human Embryonic Stem Cells with Murine Embryonic Fibroblasts on Microwell-patterned Substrates

Biomaterials. Dec, 2006  |  Pubmed ID: 16901537

Human embryonic stem (hES) cells are generally cultured as cell clusters on top of a feeder layer formed by mitotically inactivated murine embryonic fibroblasts (MEFs) to maintain their undifferentiated state. This co-culture system, which is typically used to expand the population of undifferentiated hES cells, presents several challenges since it is difficult to control cell cluster size. Large cell clusters tend to differentiate at the borders, and clusters with different sizes may lead to heterogeneous differentiation patterns within embryoid bodies. In this work, we develop a new approach to culture hES cells with controlled cluster size and number through merging microfabrication, and biomaterials technologies. Polymeric microwells were fabricated and used to control the size and uniformity of hES cell clusters in co-culture with MEFs. The results show that it is possible to culture hES cells homogeneously while keeping their undifferentiated state as confirmed by the expression of stem cell markers octamer binding protein 4 (Oct-4) and alkaline phosphatase (ALP). In addition, these clusters can be recovered from the microwells to generate nearly homogeneous cell aggregates for differentiation experiments.

Enhanced Angiogenesis Through Controlled Release of Basic Fibroblast Growth Factor from Peptide Amphiphile for Tissue Regeneration

Biomaterials. Dec, 2006  |  Pubmed ID: 16930687

In the present study, we hypothesized that a novel approach to promote vascularization would be to create injectable three-dimensional (3-D) scaffolds with encapsulated growth factor that enhance the sustained release of growth factor and induce the angiogenesis. We demonstrate that a 3-D scaffold can be formed by mixing of peptide-amphiphile (PA) aqueous solution with basic fibroblast growth factor (bFGF) suspension. PA was synthesized by standard solid phase chemistry that ends with the alkylation of the NH(2) terminus of the peptide. A 3-D network of nanofibers was formed by mixing bFGF suspensions with dilute aqueous solutions of PA. Scanning electron microscopy (SEM) observation revealed the formation of fibrous assemblies with an extremely high aspect ratio and high surface areas. In vitro and in vivo release profile of bFGF from 3-D network of nanofibers was investigated while angiogenesis induced by the released bFGF was assessed. When aqueous solution of PA was subcutaneously injected together with bFGF suspension into the back of mice, a transparent 3-D hydrogel was formed at the injected site and induced significant angiogenesis around the injected site, in marked contrast to bFGF injection alone or PA injection alone. The combination of bFGF-induced angiogenesis is a promising procedure to improve tissue regeneration.

Drug Delivery Systems in Urology--getting "smarter"

Urology. Sep, 2006  |  Pubmed ID: 17010721

Fabrication of Non-biofouling Polyethylene Glycol Micro- and Nanochannels by Ultraviolet-assisted Irreversible Sealing

Lab on a Chip. Nov, 2006  |  Pubmed ID: 17066166

We present a simple and widely applicable method to fabricate micro- and nanochannels comprised entirely of crosslinked polyethylene glycol (PEG) by using UV-assisted irreversible sealing to bond partially crosslinked PEG surfaces. The method developed here can be used to form channels as small as approximately 50 nm in diameter without using a sophisticated experimental setup. The manufactured channel is a homogeneous conduit made completely from non-biofouling PEG, exhibits robust sealing with minimal swelling and can be used without additional surface modification chemistries, thus significantly enhancing reliability and durability of microfluidic devices. Furthermore, we demonstrate simple analytical assays using PEG microchannels combined with patterned arrays of supported lipid bilayers (SLBs) to detect ligand (biotin)-receptor (streptavidin) interactions.

A Controlled-release Strategy for the Generation of Cross-linked Hydrogel Microstructures

Journal of the American Chemical Society. Nov, 2006  |  Pubmed ID: 17117838

Microscale hydrogels of controlled sizes and shapes are useful for cell-based screening, in vitro diagnostics, tissue engineering, and drug delivery. However, the rapid cross-linking of many chemically and pH cross-linkable hydrogel materials prevents the application of existing micromolding techniques. In this work we present a method for fabricating micromolded calcium alginate and chitosan structures through controlled release of the gelling agent from a hydrogel mold. Replica molding was employed to generate patterned membranes, whereas microtransfer molding was used to produce microparticles of controlled shapes. To explore the viability of this technique for producing complex tissue engineering micro-architectures, this approach was used to generate cell-laden size- and shape-controlled 3D microgels as well as composite hydrogels with well-defined spatially segregated regions. In addition, shape-controlled microstructures that can exhibit differential release properties were loaded with macromolecules to verify the potential of this approach for drug delivery applications.

Micropatterned Cell Co-cultures Using Layer-by-layer Deposition of Extracellular Matrix Components

Biomaterials. Mar, 2006  |  Pubmed ID: 16242769

Micropatterned cellular co-cultures were fabricated using three major extracellular matrix components: hyaluronic acid (HA), fibronectin (FN) and collagen. To fabricate co-cultures with these components, HA was micropatterned on a glass substrate by capillary force lithography, and the regions of exposed glass were coated with FN to generate cell adhesive islands. Once the first cell type was immobilized on the adhesive islands, the subsequent electrostatic adsorption of collagen to HA patterns switched the non-adherent HA surfaces to adherent, thereby facilitating the adhesion of a second cell type. This technique utilized native extracellular matrix components and therefore affords high biological affinity and no cytotoxicity. This biocompatible co-culture system could potentially provide a new tool to study cell behavior such as cell-cell communication and cell-matrix interactions, as well as tissue-engineering applications.

Cultivation of Human Embryonic Stem Cells Without the Embryoid Body Step Enhances Osteogenesis in Vitro

Stem Cells (Dayton, Ohio). Apr, 2006  |  Pubmed ID: 16253980

Osteogenic cultures of embryonic stem cells (ESCs) are predominately derived from three-dimensional cell spheroids called embryoid bodies (EBs). An alternative method that has been attempted and merits further attention avoids EBs through the immediate separation of ESC colonies into single cells. However, this method has not been well characterized and the effect of omitting the EB step is unknown. Herein, we report that culturing human embryonic stem cells (hESCs) without the EB stage leads to a sevenfold greater number of osteogenic cells and to spontaneous bone nodule formation after 10-12 days. In contrast, when hESCs were differentiated as EBs for 5 days followed by plating of single cells, bone nodules formed after 4 weeks only in the presence of dexamethasone. Furthermore, regardless of the inclusion of EBs, bone matrix formed, including cement line matrix and mineralized collagen, which displayed apatitic mineral (PO4) with calcium-to-phosphorous ratios similar to those of hydroxyapatite and human bone. Together these results demonstrate that culturing hESCs without an EB step can be used to derive large quantities of functional osteogenic cells for bone tissue engineering.

Bone Morphogenetic Protein-4 Enhances Cardiomyocyte Differentiation of Cynomolgus Monkey ESCs in Knockout Serum Replacement Medium

Stem Cells (Dayton, Ohio). Mar, 2007  |  Pubmed ID: 17138962

Despite extensive research in the differentiation of rodent ESCs into cardiomyocytes, there have been few studies of this process in primates. In this study, we examined the role of bone morphogenic protein-4 (BMP-4) to induce cardiomyocyte differentiation of cynomolgus monkey ESCs. To study the role of BMP-4, EBs were formed and cultured in Knockout Serum Replacement (KSR) medium containing BMP-4 for 8 days and subsequently seeded in gelatin-coated dishes for 20 days. It was found that ESCs differentiated into cardiomyocytes upon stimulation with BMP-4 in KSR medium, which resulted in a large fraction of beating EBs ( approximately 16%) and the upregulation of cardiac-specific proteins in a dose and time-dependent manner. In contrast, the addition of BMP-4 in FBS-containing medium resulted in a lower fraction of beating EBs ( approximately 6%). BMP-4 acted principally between mesendodermal and mesoderm progenitors and subsequently enhanced their expression. Ultrastructural observation revealed that beating EBs contained mature cardiomyocytes with sarcomeric structures. In addition, immunostaining, reverse transcription-polymerase chain reaction, and Western blotting for cardiac markers confirmed the increased differentiation of cardiomyocytes in these cultures. Moreover, electrophysiological studies demonstrated that the differentiated cardiomyocytes were electrically activated. These findings may be useful in developing effective culture conditions to differentiate cynomolgus monkey ESCs into cardiomyocytes for studying developmental biology and for regenerative medicine.

Microfluidic Patterning for Fabrication of Contractile Cardiac Organoids

Biomedical Microdevices. Apr, 2007  |  Pubmed ID: 17146728

The development of in vitro methods of engineering three-dimensional cardiac tissues can be useful for tissue replacement, diagnostics and drug discovery. Here, we introduce the use of patterned hyaluronic acid (HA) substrates generated using microfluidic patterning as a method of fabricating 3D cardiac organoids. HA micropatterns served as inductive templates for organoid assembly. Upon seeding, cardiomyocytes elongated and aligned along the pattern direction attaching preferentially to the glass substrate and the interface between HA patterns and glass substrate. After 3 days in culture, the linearly aligned myocytes detached from the surface and formed contractile cardiac organoids. The procedure can be utilized to simply, rapidly and inexpensively create in vitro cardiac tissue models.

Generation of Static and Dynamic Patterned Co-cultures Using Microfabricated Parylene-C Stencils

Lab on a Chip. Oct, 2007  |  Pubmed ID: 17896010

Many biological processes, such as stem cell differentiation, wound healing and development, involve dynamic interactions between cells and their microenvironment. The ability to control these dynamic processes in vitro would be potentially useful to fabricate tissue engineering constructs, study biological processes, and direct stem cell differentiation. In this paper, we used a parylene-C microstencil to develop two methods of creating patterned co-cultures using either static or dynamic conditions. In the static case, embryonic stem (ES) cells were co-cultured with fibroblasts or hepatocytes by using the reversible sealing of the stencil on the substrate. In the dynamic case, ES cells were co-cultured with NIH-3T3 fibroblasts and AML12 hepatocytes sequentially by engineering the surface properties of the stencil. In this approach, the top surface of the parylene-C stencil was initially treated with hyaluronic acid (HA) to reduce non-specific cell adhesion. The stencil was then sealed on a substrate and seeded with ES cells which adhered to the underlying substrate through the holes in the membrane. To switch the surface properties of the parylene-C stencils to cell adhesive, collagen was deposited on the parylene-C surfaces. Subsequently, a second cell type was seeded on the parylene-C stencils to form a patterned co-culture. This group of cells was removed by peeling off the parylene-C stencils, which enabled the patterning of a third cell type. Although the static patterned co-culture approach has been demonstrated previously with a variety of methods, layer-by-layer modification of microfabricated parylene-C stencils enables dynamic patterning of multiple cell types in sequence. Thus, this method is a promising approach to engineering the complexity of cell-cell interactions in tissue culture in a spatially and temporally regulated manner.

Cell and Protein Compatibility of Parylene-C Surfaces

Langmuir : the ACS Journal of Surfaces and Colloids. Nov, 2007  |  Pubmed ID: 17915896

Parylene-C, which is traditionally used to coat implantable devices, has emerged as a promising material to generate miniaturized devices due to its unique mechanical properties and inertness. In this paper we compared the surface properties and cell and protein compatibility of parylene-C relative to other commonly used BioMEMS materials. We evaluated the surface hydrophobicity and roughness of parylene-C and compared these results to those of tissue culture-treated polystyrene, poly(dimethylsiloxane) (PDMS), and glass. We also treated parylene-C and PDMS with air plasma, and coated the surfaces with fibronectin to demonstrate that biochemical treatments modify the surface properties of parylene-C. Although plasma treatment caused both parylene-C and PDMS to become hydrophilic, only parylene-C substrates retained their hydrophilic properties over time. Furthermore, parylene-C substrates display a higher degree of nanoscale surface roughness (>20 nm) than the other substrates. We also examined the level of BSA and IgG protein adsorption on various surfaces and found that surface plasma treatment decreased the degree of protein adsorption on both PDMS and parylene-C substrates. After testing the degree of cell adhesion and spreading of two mammalian cell types, NIH-3T3 fibroblasts and AML-12 hepatocytes, we found that the adhesion of both cell types to surface-treated parylene-C variants were comparable to standard tissue culture substrates, such as polystyrene. Overall, these results indicate that parylene-C, along with its surface-treated variants, could potentially be a useful material for fabricating cell-based microdevices.

Covalent Immobilization of P-selectin Enhances Cell Rolling

Langmuir : the ACS Journal of Surfaces and Colloids. Nov, 2007  |  Pubmed ID: 17949112

Cell rolling is an important physiological and pathological process that is used to recruit specific cells in the bloodstream to a target tissue. This process may be exploited for biomedical applications to capture and separate specific cell types. One of the most commonly studied proteins that regulate cell rolling is P-selectin. By coating surfaces with this protein, biofunctional surfaces that induce cell rolling can be prepared. Although most immobilization methods have relied on physisorption, chemical immobilization has obvious advantages, including longer functional stability and better control over ligand density and orientation. Here we describe chemical methods to immobilize P-selectin covalently on glass substrates. The chemistry was categorized on the basis of the functional groups on modified glass substrates: amine, aldehyde, and epoxy. The prepared surfaces were first tested in a flow chamber by flowing microspheres functionalized with a cell surface carbohydrate (sialyl Lewis(x)) that binds to P-selectin. Adhesion bonds between P-selectin and sialyl Lewis(x) dissociate readily under shear forces, leading to cell rolling. P-selectin immobilized on the epoxy glass surfaces exhibited enhanced long-term stability of the function and better homogeneity as compared to that for surfaces prepared by other methods and physisorbed controls. The microsphere rolling results were confirmed in vitro with isolated human neutrophils. This work is essential for the future development of devices for isolating specific cell types based on cell rolling, which may be useful for hematologic cancers and certain metastatic cancer cells that are responsive to immobilized selectins.

Bone Regeneration Through Controlled Release of Bone Morphogenetic Protein-2 from 3-D Tissue Engineered Nano-scaffold

Journal of Controlled Release : Official Journal of the Controlled Release Society. Feb, 2007  |  Pubmed ID: 17207881

The objective of the present study was to enhance ectopic bone formation through the controlled release of bone morphogenetic protein-2 (BMP-2) from an injectable three dimensional (3-D) tissue engineered nano-scaffold. We demonstrate that a 3-D scaffold can be formed by mixing of peptide-amphiphile (PA) aqueous solution with BMP-2 suspension. A 3-D network of nanofibers was formed by mixing BMP-2 suspensions with dilute aqueous solutions of PA. Scanning electron microscopy (SEM) observation revealed the formation of fibrous assemblies with an extremely high aspect ratio and high surface areas. In vivo release profile of BMP-2 from 3-D network of nanofibers was investigated. In addition, ectopic bone formation induced by the released BMP-2 was assessed in a rat model using histological and biochemical examinations. It was demonstrated that the injection of an aqueous solution of PA together with BMP-2 into the back subcutis of rats, resulted in the formation of a transparent 3-D hydrogel at the injected site and induced significant homogeneous ectopic bone formation around the injected site, in marked contrast to BMP-2 injection alone or PA injection alone. The combination of BMP-2-induced bone formation is a promising procedure to improve tissue regeneration.

A Cell-laden Microfluidic Hydrogel

Lab on a Chip. Jun, 2007  |  Pubmed ID: 17538718

The encapsulation of mammalian cells within the bulk material of microfluidic channels may be beneficial for applications ranging from tissue engineering to cell-based diagnostic assays. In this work, we present a technique for fabricating microfluidic channels from cell-laden agarose hydrogels. Using standard soft lithographic techniques, molten agarose was molded against a SU-8 patterned silicon wafer. To generate sealed and water-tight microfluidic channels, the surface of the molded agarose was heated at 71 degrees C for 3 s and sealed to another surface-heated slab of agarose. Channels of different dimensions were generated and it was shown that agarose, though highly porous, is a suitable material for performing microfluidics. Cells embedded within the microfluidic molds were well distributed and media pumped through the channels allowed the exchange of nutrients and waste products. While most cells were found to be viable upon initial device fabrication, only those cells near the microfluidic channels remained viable after 3 days, demonstrating the importance of a perfused network of microchannels for delivering nutrients and oxygen to maintain cell viability in large hydrogels. Further development of this technique may lead to the generation of biomimetic synthetic vasculature for tissue engineering, diagnostics, and drug screening applications.

Controlling Size, Shape and Homogeneity of Embryoid Bodies Using Poly(ethylene Glycol) Microwells

Lab on a Chip. Jun, 2007  |  Pubmed ID: 17538722

Directed differentiation of embryonic stem (ES) cells is useful for creating models of human disease and could potentially generate a wide array of functional cell types for therapeutic applications. Methods to differentiate ES cells often involve the formation of cell aggregates called embryoid bodies (EBs), which recapitulate early stages of embryonic development. EBs are typically made from suspension cultures, resulting in heterogeneous structures with a wide range of sizes and shapes, which may influence differentiation. Here, we use microfabricated cell-repellant poly(ethylene glycol) (PEG) wells as templates to initiate the formation of homogenous EBs. ES cell aggregates were formed with controlled sizes and shapes defined by the geometry of the microwells. EBs generated in this manner remained viable and maintained their size and shape within the microwells relative to their suspension counterparts. Intact EBs could be easily retrieved from the microwells with high viability (>95%). These results suggest that the microwell technique could be a useful approach for in vitro studies involving ES cells and, more specifically, for initiating the differentiation of EBs of greater uniformity based on controlled microenvironments.

Microengineered Hydrogels for Tissue Engineering

Biomaterials. Dec, 2007  |  Pubmed ID: 17707502

Hydrogels have been extensively used in various biomedical applications such as drug delivery and biosensing. More recently the ability to engineer the size and shape of biologically relevant hydrogels has generated new opportunities in addressing challenges in tissue engineering such as vascularization, tissue architecture and cell seeding. Here, we discuss the use of microengineered hydrogels for tissue engineering applications. We will initially provide an overview of the various approaches that can be used to synthesize hydrogels with controlled features and will subsequently discuss the emerging applications of these hydrogels.

Reusable, Reversibly Sealable Parylene Membranes for Cell and Protein Patterning

Journal of Biomedical Materials Research. Part A. May, 2008  |  Pubmed ID: 17729252

The patterned deposition of cells and biomolecules on surfaces is a potentially useful tool for in vitro diagnostics, high-throughput screening, and tissue engineering. Here, we describe an inexpensive and potentially widely applicable micropatterning technique that uses reversible sealing of microfabricated parylene-C stencils on surfaces to enable surface patterning. Using these stencils it is possible to generate micropatterns and copatterns of proteins and cells, including NIH-3T3 fibroblasts, hepatocytes and embryonic stem cells. After patterning, the stencils can be removed from the surface, plasma treated to remove adsorbed proteins, and reused. A variety of hydrophobic surfaces including PDMS, polystyrene and acrylated glass were patterned using this approach. Furthermore, we demonstrated the reusability and mechanical integrity of the parylene membrane for at least 10 consecutive patterning processes. These parylene-C stencils are potentially scalable commercially and easily accessible for many biological and biomedical applications.

Quantitative Analysis of Cell Adhesion on Aligned Micro- and Nanofibers

Journal of Biomedical Materials Research. Part A. Feb, 2008  |  Pubmed ID: 17607759

In this study, we quantitatively analyzed the affinity of cell adhesion to aligned nanofibers composed of composites of poly(glycolic acid) (PGA) and collagen. Electrospun composite fibers were fabricated at various PGA/collagen weight mixing ratio (7, 18, 40, 67, and 86%) to generate fibers that ranged in diameter from 10 mum to 500 nm. Scanning electron microscopy (SEM) observation revealed that the PGA/collagen fibers were long and uniformly aligned, irrespective of the PGA/collagen weight mixing ratio. In addition, it was observed that a significantly higher number of NIH3T3 fibroblasts adhered to nanofibers with smaller diameters in comparison to fibers with larger diameters. The highest affinity of cell adhesion was observed in the PGA/collagen fibers with diameter of 500 nm and PGA/collagen weight mixing ratio of 40%. Furthermore, the adherent cells were more elongated on fibers with smaller diameters. Thus, based on the results here, PGA/collagen composite fibers are suitable for tissue culture studies and provide an attractive material for tissue engineering applications.

DNA Nanoparticles Encapsulated in 3D Tissue-engineered Scaffolds Enhance Osteogenic Differentiation of Mesenchymal Stem Cells

Journal of Biomedical Materials Research. Part A. Apr, 2008  |  Pubmed ID: 17688252

In this study, we enhanced the expression of a plasmid DNA in mesenchymal stem cells (MSC) by the combination of three-dimensional (3D) tissue-engineered scaffold and nonviral gene carrier. To function as an enhanced delivery of plasmid DNA, acetic anhydride was reacted with polyethylenimine (PEI) to acetylate 80% of the primary and 20% of the secondary amines (PEI-Ac(80)). This acetylated PEI has been demonstrated to show enhanced gene-delivery efficiency over unmodified PEI. Collagen sponges reinforced by incorporating of poly(glycolic acid) (PGA) fibers were used as the scaffold material. DNA nanoparticles formed through simple mixing of plasmid DNA encoding bone morphogenetic protein-2 (BMP-2) and PEI-Ac(80) solutions were encapsulated within these scaffolds. MSC were seeded into each scaffold and cultured for several weeks. Within these scaffolds, the level of BMP-2 expression by transfected MSC was significantly enhanced compared to MSC transfected by DNA nanoparticles in solution (in 2D tissue culture plates). Homogeneous bone formation was histologically observed throughout the sponges seeded with transfected MSC by using DNA nanoparticles after subcutaneous implantation into the back of rats. The level of alkaline phosphatase activity and osteocalcin content at the implanted sites of sponges seeded with transfected MSC by using DNA nanoparticles were significantly higher when compared with those seeded with other agents.

Method of Bottom-Up Directed Assembly of Cell-Laden Microgels

Cellular and Molecular Bioengineering. 2008  |  Pubmed ID: 19953195

The paper describes a protocol to fabricate cell-laden microgel assemblies with pre-defined micro-architecture and complexity by a bottom-up approach, which can be used for tissue engineering applications. The assembly process was driven by the hydrophobic effect in the water/oil interface. By agitating hydrophilic microgels in hydrophobic medium, the shape-controlled microgel units assemble in an organized manner to locally minimize the interaction free energy (the surface area exposed to the oil). The assembly process was shown to be controlled by several parameters, such as external energy input, surface tension, and microgel dimensions. This assembly approach was used to build multi-component cell-laden constructs by assembling microgel building blocks and performing a secondary cross-linking reaction. This bottom-up approach for the directed assembly of cell-laden microgels offers a scalable method to fabricate 3D tissue constructs with biomimetic structure.

A Microwell Array System for Stem Cell Culture

Biomaterials. Feb, 2008  |  Pubmed ID: 18001830

Directed embryonic stem (ES) cell differentiation is a potentially powerful approach for generating a renewable source of cells for regenerative medicine. Typical in vitro ES cell differentiation protocols involve the formation of ES cell aggregate intermediates called embryoid bodies (EBs). Recently, we demonstrated the use of poly(ethylene glycol) (PEG) microwells as templates for directing the formation of these aggregates, offering control over parameters such as size, shape, and homogeneity. Despite these promising results, the previously developed technology was limited as it was difficult to reproducibly obtain cultures of homogeneous EBs with high efficiency and retrievability. In this study, we improve the platform by optimizing a number of features: material composition of the microwells, cell seeding procedures, and aggregate retrieval methods. Adopting these modifications, we demonstrate an improved degree of homogeneity of the resulting aggregate populations and establish a robust protocol for eliciting high EB formation efficiencies. The optimized microwell array system is a potentially versatile tool for ES cell differentiation studies and high-throughput stem cell experimentation.

The Use of Charge-coupled Polymeric Microparticles and Micromagnets for Modulating the Bioavailability of Orally Delivered Macromolecules

Biomaterials. Mar, 2008  |  Pubmed ID: 18082254

Protein drugs have low bioavailability after oral administration, which is due in part to fast transit of the drugs or drug delivery vehicles through the gastrointestinal tract. Increasing the time that the drugs spend in the intestine after dosing would allow for greater absorption and increased bioavailability. We developed a formulation strategy that can be used to prolong intestinal retention of drug delivery vehicles without substantial alterations to current polymeric encapsulation strategies. A model drug, insulin, was encapsulated in negatively charged poly(lactic-co-glycolic acid) (PLGA) microparticles, and the microparticles were subsequently mixed with positively charged micromagnets, whose size will prevent them from being absorbed. Stable complexes formed through electrostatic interaction. The complexes were effectively immobilized in vitro in a model of the mouse small intestine by application of an external magnetic field. Mice that were gavaged with radio-labeled complexes and fitted with a magnetic belt retained 32.5% of the (125)I-insulin in the small intestine compared with 5.4% for the control group 6h after administration (p=0.005). Furthermore, mice similarly gavaged with complexes encapsulating insulin (120 Units/kg) exhibited long-term glucose reduction in the groups with magnetic belts. The corresponding bioavailability of insulin was 5.11% compared with 0.87% for the control group (p=0.007).

Microfluidics for Drug Discovery and Development: from Target Selection to Product Lifecycle Management

Drug Discovery Today. Jan, 2008  |  Pubmed ID: 18190858

Microfluidic technologies' ability to miniaturize assays and increase experimental throughput have generated significant interest in the drug discovery and development domain. These characteristics make microfluidic systems a potentially valuable tool for many drug discovery and development applications. Here, we review the recent advances of microfluidic devices for drug discovery and development and highlight their applications in different stages of the process, including target selection, lead identification, preclinical tests, clinical trials, chemical synthesis, formulations studies and product management.

Microcirculation Within Grooved Substrates Regulates Cell Positioning and Cell Docking Inside Microfluidic Channels

Lab on a Chip. May, 2008  |  Pubmed ID: 18432345

Immobilization of cells inside microfluidic devices is a promising approach for enabling studies related to drug screening and cell biology. Despite extensive studies in using grooved substrates for immobilizing cells inside channels, a systematic study of the effects of various parameters that influence cell docking and retention within grooved substrates has not been performed. We demonstrate using computational simulations that the fluid dynamic environment within microgrooves significantly varies with groove width, generating microcirculation areas in smaller microgrooves. Wall shear stress simulation predicted that shear stresses were in the opposite direction in smaller grooves (25 and 50 microm wide) in comparison to those in wider grooves (75 and 100 microm wide). To validate the simulations, cells were seeded within microfluidic devices, where microgrooves of different widths were aligned perpendicularly to the direction of the flow. Experimental results showed that, as predicted, the inversion of the local direction of shear stress within the smaller grooves resulted in alignment of cells on two opposite sides of the grooves under the same flow conditions. Also, the amplitude of shear stress within microgrooved channels significantly influenced cell retainment in the channels. Therefore, our studies suggest that microscale shear stresses greatly influence cellular docking, immobilization, and retention in fluidic systems and should be considered for the design of cell-based microdevices.

Microfabricated Multilayer Parylene-C Stencils for the Generation of Patterned Dynamic Co-cultures

Journal of Biomedical Materials Research. Part A. Jul, 2008  |  Pubmed ID: 18442109

Co-culturing different cell types can be useful to engineer a more in vivo-like microenvironment for cells in culture. Recent approaches to generating cellular co-cultures have used microfabrication technologies to regulate the degree of cell-cell contact between different cell types. However, these approaches are often limited to the co-culture of only two cell types in static cultures. The dynamic aspect of cell-cell interaction, however, is a key regulator of many biological processes such as early development, stem cell differentiation, and tissue regeneration. In this study, we describe a micropatterning technique based on microfabricated multilayer parylene-C stencils and demonstrate the potential of parylene-C technology for co-patterning of proteins and cells with the ability to generate a series of at least five temporally controlled patterned co-cultures. We generated dynamic co-cultures of murine embryonic stem cells in culture with various secondary cell types that could be sequentially introduced and removed from the co-cultures. Our studies suggested that dynamic co-cultures generated by using parylene-C stencils may be applicable in studies investigating cellular interactions in controlled microenvironments such as studies of ES cell differentiation, wound healing and development.

Microfluidic Chip-based Fabrication of PLGA Microfiber Scaffolds for Tissue Engineering

Langmuir : the ACS Journal of Surfaces and Colloids. Jun, 2008  |  Pubmed ID: 18512874

In this paper, we have developed a method to produce poly(lactic- co-glycolic acid) (PLGA) microfibers within a microfluidic chip for the generation of 3D tissue engineering scaffolds. The synthesis of PLGA fibers was achieved by using a polydimethylsiloxane (PDMS)-based microfluidic spinning device in which linear streams of PLGA dissolved in dimethyl sulfoxide (DMSO) were precipitated in a glycerol-containing water solution. By changing the flow rate of PLGA solution from 1 to 50 microL/min with a sheath flow rate of 250 or 1000 microL/min, fibers were formed with diameters that ranged from 20 to 230 microm. The PLGA fibers were comprised of a dense outer surface and a highly porous interior. To evaluate the applicability of PLGA microfibers generated in this process as a cell culture scaffold, L929 fibroblasts were seeded on the PLGA fibers either as-fabricated or coated with fibronectin. L929 fibroblasts showed no significant difference in proliferation on both PLGA microfibers after 5 days of culture. As a test for application as nerve guide, neural progenitor cells were cultured and the neural axons elongated along the PLGA microfibers. Thus our experiments suggest that microfluidic chip-based PLGA microfiber fabrication may be useful for 3D cell culture tissue engineering applications.

WITHDRAWN: Microfabrication-based Engineering of the Embryonic and Adult Stem Cell Niches

Biomaterials. Jun, 2008  |  Pubmed ID: 18533250

This article has been withdrawn consistent with Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). The Publisher apologizes for any inconvenience this may cause.

Stop-flow Lithography to Generate Cell-laden Microgel Particles

Lab on a Chip. Jul, 2008  |  Pubmed ID: 18584079

Encapsulating cells within hydrogels is important for generating three-dimensional (3D) tissue constructs for drug delivery and tissue engineering. This paper describes, for the first time, the fabrication of large numbers of cell-laden microgel particles using a continuous microfluidic process called stop-flow lithography (SFL). Prepolymer solution containing cells was flowed through a microfluidic device and arrays of individual particles were repeatedly defined using pulses of UV light through a transparency mask. Unlike photolithography, SFL can be used to synthesize microgel particles continuously while maintaining control over particle size, shape and anisotropy. Therefore, SFL may become a useful tool for generating cell-laden microgels for various biomedical applications.

Directed Assembly of Cell-laden Microgels for Fabrication of 3D Tissue Constructs

Proceedings of the National Academy of Sciences of the United States of America. Jul, 2008  |  Pubmed ID: 18599452

We present a bottom-up approach to direct the assembly of cell-laden microgels to generate tissue constructs with tunable microarchitecture and complexity. This assembly process is driven by the tendency of multiphase liquid-liquid systems to minimize the surface area and the resulting surface free energy between the phases. We demonstrate that shape-controlled microgels spontaneously assemble within multiphase reactor systems into predetermined geometric configurations. Furthermore, we characterize the parameters that influence the assembly process, such as external energy input, surface tension, and microgel dimensions. Finally, we show that multicomponent cell-laden constructs could be generated by assembling microgel building blocks and performing a secondary cross-linking reaction. This bottom-up approach for the directed assembly of cell-laden microgels provides a powerful and highly scalable approach to form biomimetic 3D tissue constructs and opens a paradigm for directing the assembly of mesoscale materials.

Mechanically Robust and Bioadhesive Collagen and Photocrosslinkable Hyaluronic Acid Semi-interpenetrating Networks

Tissue Engineering. Part A. Jul, 2009  |  Pubmed ID: 19105604

In this work, we present a class of hydrogels that leverage the favorable properties of the photo-cross-linkable hyaluronic acid (HA) and semi-interpenetrating collagen components. The mechanical properties of the semi-interpenetrating-network (semi-IPN) hydrogels far surpass those achievable with collagen gels or collagen gel-based semi-IPNs. Furthermore, the inclusion of the semi-interpenetrating collagen chains provides a synergistic mechanical improvement over unmodified HA hydrogels. Collagen-HA semi-IPNs supported fibroblast adhesion and proliferation and were shown to be suitable for cell encapsulation at high levels of cell viability. To demonstrate the utility of the semi-IPNs as a microscale tissue engineering material, cell-laden microstructures and microchannels were fabricated using soft lithographic techniques. Given their enhanced mechanical and biomimetic properties, we anticipate that these materials will be of value in tissue engineering and three-dimensional cell culture applications.

Cell Docking in Double Grooves in a Microfluidic Channel

Small (Weinheim an Der Bergstrasse, Germany). May, 2009  |  Pubmed ID: 19242937

Microstructures that generate shear-protected regions in microchannels can rapidly immobilize cells for cell-based biosensing and drug screening. Here, a two-step fabrication method is used to generate double microgrooves with various depth ratios to achieve controlled double-level cell patterning while still providing shear protection. Six microgroove geometries are fabricated with different groove widths and depth ratios. Two modes of cell docking are observed: cells docked upstream in sufficiently deep and narrow grooves, and downstream in shallow, wide grooves. Computational flow simulations link the groove geometry and bottom shear stress to the experimental cell docking patterns. Analysis of the experimental cell retention in the double grooves demonstrates its linear dependence on inlet flow speed, with slope inversely proportional to the sheltering provided by the groove geometry. Thus, double-grooved microstructures in microfluidic channels provide shear-protected regions for cell docking and immobilization and appear promising for cell-based biosensing and drug discovery.

Rapid Generation of Spatially and Temporally Controllable Long-range Concentration Gradients in a Microfluidic Device

Lab on a Chip. Mar, 2009  |  Pubmed ID: 19255657

The ability to rapidly generate concentration gradients of diffusible molecules has important applications in many chemical and biological studies. Here we established spatially and temporally controllable concentration gradients of molecules (i.e. proteins or toxins) in a portable microfluidic device in an easy and rapid manner. The formation of the concentration gradients was initiated by a passive-pump-induced forward flow and further optimized during an evaporation-induced backward flow. The centimeter-long gradients along the microfluidic channel were shown to be spatially and temporally controlled by the backward flow. The gradient profile was stabilized by stopping the flow. Computational simulations of this dynamic process illustrated the combined effects of convection and diffusion on the gradient generation, and fit well with the experimental data. To demonstrate the applications of this methodology, a stabilized concentration gradient of a cardiac toxin, alpha-cypermethrin, along the microchannel was used to test the response of HL-1 cardiac cells in the micro-device, which correlated with toxicity data obtained from multi-well plates. The approach presented here may be useful for many biological and chemical processes that require rapid generation of long-range gradients in a portable microfluidic device.

Electrochemical Desorption of Self-assembled Monolayers for Engineering Cellular Tissues

Biomaterials. Jul, 2009  |  Pubmed ID: 19362363

Adherent cells, cell sheets, and spheroids were harvested noninvasively from a culture surface by means of electrochemical desorption of a self-assembled monolayer (SAM) of alkanethiol. The SAM surface was made adhesive by the covalent bonding of Arg-Gly-Asp (RGD)-peptides to the alkanethiol molecules. The application of a negative electrical potential caused the reductive desorption of the SAM, resulting in the detachment of the cells. Using this approach greater than 90% of adherent cells detached within 5 min. Furthermore, this approach was used to obtain two-dimensional (2D) cell sheets. The detached cell sheets consisted of viable cells, which could be easily attached to other cell sheets in succession to form a multilayered cell sheet. Moreover, spheroids of hepatocytes of a uniform diameter were formed in an array of cylindrical cavities at a density of 280 spheroids/cm(2) and were harvested by applying a negative electrical potential. This cell manipulation technology could potentially be a useful tool for the fabrication and assembly of building blocks such as cell sheets and spheroids for regenerative medicine and tissue engineering applications.

Progress in Tissue Engineering

Scientific American. May, 2009  |  Pubmed ID: 19438051

Integrating Microfluidics and Lensless Imaging for Point-of-care Testing

Biosensors & Bioelectronics. Jul, 2009  |  Pubmed ID: 19467854

We demonstrate an integrated platform that merges a microfluidic chip with lensless imaging to target CD4(+) T-lymphocyte counts for HIV point-of-care testing at resource-limited settings. The chips were designed and fabricated simply with a laser cutter without using expensive cleanroom equipment. To capture CD4(+) T-lymphocytes from blood, anti-CD4 antibody was immobilized on only one side of the microfluidic chip. These captured cells were detected through an optically clear chip using a charge coupled device (CCD) sensor by lensless shadow imaging techniques. Gray scale image of the captured cells in a 24 mm x 4 mm x 50 microm microfluidic chip was obtained by the lensless imaging platform. The automatic cell counting software enumerated the captured cells in 3s. Captured cells were also imaged with a fluorescence microscope and manually counted to characterize functionality of the integrated platform. The integrated platform achieved 70.2+/-6.5% capture efficiency, 88.8+/-5.4% capture specificity for CD4(+) T-lymphocytes, 96+/-1.6% CCD efficiency, and 83.5+/-2.4% overall platform performance (n=9 devices) compared to the gold standard, i.e. flow cytometry count. The integrated system gives a CD4 count from blood within 10 min. The integrated platform points a promising direction for point-of-care testing (POCT) to rapidly capture, image and count subpopulations of cells from blood samples in an automated matter.

Microwell-mediated Control of Embryoid Body Size Regulates Embryonic Stem Cell Fate Via Differential Expression of WNT5a and WNT11

Proceedings of the National Academy of Sciences of the United States of America. Oct, 2009  |  Pubmed ID: 19805103

Recently, various approaches for controlling the embryonic stem (ES) cell microenvironment have been developed for regulating cellular fate decisions. It has been reported that the lineage specific differentiation could be affected by the size of ES cell colonies and embryoid bodies (EBs). However, much of the underlying biology has not been well elucidated. In this study, we used microengineered hydrogel microwells to direct ES cell differentiation and determined the role of WNT signaling pathway in directing the differentiation. This was accomplished by forming ES cell aggregates within microwells to form different size EBs. We determined that cardiogenesis was enhanced in larger EBs (450 microm in diameter), and in contrast, endothelial cell differentiation was increased in smaller EBs (150 microm in diameter). Furthermore, we demonstrated that the EB-size mediated differentiation was driven by differential expression of WNTs, particularly noncanonical WNT pathway, according to EB size. The higher expression of WNT5a in smaller EBs enhanced endothelial cell differentiation. In contrast, the increased expression of WNT11 enhanced cardiogenesis. This was further validated by WNT5a-siRNA transfection assay and the addition of recombinant WNT5a. Our data suggest that EB size could be an important parameter in ES cell fate specification via differential gene expression of members of the noncanonical WNT pathway. Given the size-dependent response of EBs to differentiate to endothelial and cardiac lineages, hydrogel microwell arrays could be useful for directing stem cell fates and studying ES cell differentiation in a controlled manner.

Arraycount, an Algorithm for Automatic Cell Counting in Microwell Arrays

BioTechniques. Sep, 2009  |  Pubmed ID: 19852758

Microscale technologies have emerged as a powerful tool for studying and manipulating biological systems and miniaturizing experiments. However, the lack of software complementing these techniques has made it difficult to apply them for many high-throughput experiments. This work establishes Arraycount, an approach to automatically count cells in microwell arrays. The procedure consists of fluorescent microscope imaging of cells that are seeded in microwells of a microarray system and then analyzing images via computer to recognize the array and count cells inside each microwell. To start counting, green and red fluorescent images (representing live and dead cells, respectively) are extracted from the original image and processed separately. A template-matching algorithm is proposed in which pre-defined well and cell templates are matched against the red and green images to locate microwells and cells. Subsequently, local maxima in the correlation maps are determined and local maxima maps are thresholded. At the end, the software records the cell counts for each detected microwell on the original image in high-throughput. The automated counting was shown to be accurate compared with manual counting, with a difference of approximately 1-2 cells per microwell: based on cell concentration, the absolute difference between manual and automatic counting measurements was 2.5-13%.

Micro- and Nanoscale Control of the Cardiac Stem Cell Niche for Tissue Fabrication

Tissue Engineering. Part B, Reviews. Dec, 2009  |  Pubmed ID: 19552604

Advances in stem cell (SC) biology have greatly enhanced our understanding of SC self-renewal and differentiation. Both embryonic and adult SCs can be differentiated into a great variety of tissue cell types, including cardiac myocytes. In vivo studies and clinical trials, however, have demonstrated major limitations in reconstituting the myocardium in failing hearts. These limitations include precise control of SC proliferation, survival and phenotype both prior and subsequent to transplantation and avoidance of serious adverse effects such as tumorigenesis and arrhythmias. Micro- and nanoscale techniques to recreate SC niches, the natural environment for the maintenance and regulation of SCs, have enabled the elucidation of novel SC behaviors and offer great promise in the fabrication of cardiac tissue constructs. The ability to precisely manipulate the interface between biopolymeric scaffolds and SCs at in vivo scale resolutions is unique to micro- and nanoscale approaches and may help overcome limitations of conventional biological scaffolds and methods for cell delivery. We now know that micro- and nanoscale manipulation of scaffold composition, mechanical properties, and three-dimensional architecture have profound influences on SC fate and will likely prove important in developing the next generation of "transplantable SC niches" for regeneration of heart and other tissues. In this review, we examine two key aspects of micro- and nanofabricated SC-based cardiac tissue constructs: the role of scaffold composition and the role of scaffold architecture and detail how recent work in these areas brings us closer to clinical solutions for cardiovascular regeneration.

Engineered 3D Tissue Models for Cell-laden Microfluidic Channels

Analytical and Bioanalytical Chemistry. Sep, 2009  |  Pubmed ID: 19629459

Delivery of nutrients and oxygen within three-dimensional (3D) tissue constructs is important to maintain cell viability. We built 3D cell-laden hydrogels to validate a new tissue perfusion model that takes into account nutrition consumption. The model system was analyzed by simulating theoretical nutrient diffusion into cell-laden hydrogels. We carried out a parametric study considering different microchannel sizes and inter-channel separation in the hydrogel. We hypothesized that nutrient consumption needs to be taken into account when optimizing the perfusion channel size and separation. We validated the hypothesis by experiments. We fabricated circular microchannels (r = 400 microm) in 3D cell-laden hydrogel constructs (R = 7.5 mm, volume = 5 ml). These channels were positioned either individually or in parallel within hydrogels to increase nutrient and oxygen transport as a way to improve cell viability. We quantified the spatial distribution of viable cells within 3D hydrogel scaffolds without channels and with single- and dual-perfusion microfluidic channels. We investigated quantitatively the cell viability as a function of radial distance from the channels using experimental data and mathematical modeling of diffusion profiles. Our simulations show that a large-channel radius as well as a large channel to channel distance diffuse nutrients farther through a 3D hydrogel. This is important since our results reveal that there is a close correlation between nutrient profiles and cell viability across the hydrogel.

Future Approaches to Organ Regeneration: Microscale Environments, Stem Cell Engineering, and Self-assembly of Living Tissues

Studies in Health Technology and Informatics. 2009  |  Pubmed ID: 19745484

A promising means to address the limited supply of donor tissue is through the generation of artificial organs consisting of cells and materials. Progress towards this goal is limited by three main obstacles namely the generation of a sufficient number of cells specific to the organ, the arrangement of these cells in a functional tissue architecture and the delivery of nutrients and removal of waste from the tissue mass. This chapter describes the emerging approaches that may be achieved by the control of stem cell differentiation, control of the local tissue environment on the microscale, and the generation of complex structures containing multiple cell types.

Rapid Formation of Acrylated Microstructures by Microwave-Induced Thermal Crosslinking

Macromolecular Rapid Communications. Jun, 2009  |  Pubmed ID: 20011617

We present a rapid and highly efficient method to form microstructure of poly(ethylene glycol) (PEG)-based acrylates by microwave-induced thermal crosslinking. PEG-based polymeric microstructures such as polymer microarrays and microwells were fabricated on 3-(trimethoxysilyl)propyl methacrylate (TMSPMA)-coated glass slides that were placed on top of a silicon wafer. In comparison to ultraviolet (UV) irradiation curing, microwave-induced thermal crosslinking could be completed within 10 s, without thermal degradation or oxygen inhibition in the presence of ambient oxygen. Furthermore, the activation of surviving free radical impurities by microwave-induced heating enabled crosslinking even without an exogenous radical initiator (e.g., 2,2'-azoisobutyronitrile (AIBN)). This approach can be beneficial for fabricating various PEG-based microstructures for high-throughput screening assays, cell-based biosensors, and biomedical microdevices.

Microscale Electroporation: Challenges and Perspectives for Clinical Applications

Integrative Biology : Quantitative Biosciences from Nano to Macro. Mar, 2009  |  Pubmed ID: 20023735

Microscale engineering plays a significant role in developing tools for biological applications by miniaturizing devices and providing controllable microenvironments for in vitro cell research. Miniaturized devices offer numerous benefits in comparison to their macroscale counterparts, such as lower use of expensive reagents, biomimetic environments, and the ability to manipulate single cells. Microscale electroporation is one of the main beneficiaries of microscale engineering as it provides spatial and temporal control of various electrical parameters. Microscale electroporation devices can be used to reduce limitations associated with the conventional electroporation approaches such as variations in the local pH, electric field distortion, sample contamination, and the difficulties in transfecting and maintaining the viability of desired cell types. Here, we present an overview of recent advances of the microscale electroporation methods and their applications in biology, as well as current challenges for its use for clinical applications. We categorize microscale electroporation into microchannel and microcapillary electroporation. Microchannel-based electroporation can be used for transfecting cells within microchannels under dynamic flow conditions in a controlled and high-throughput fashion. In contrast, microcapillary-based electroporation can be used for transfecting cells within controlled reaction chambers under static flow conditions. Using these categories we examine the use of microscale electroporation for clinical applications related to HIV-1, stem cells, cancer and other diseases and discuss the challenges in further advancing this technology for use in clinical medicine and biology.

Integration Column: Microwell Arrays for Mammalian Cell Culture

Integrative Biology : Quantitative Biosciences from Nano to Macro. Dec, 2009  |  Pubmed ID: 20027371

Microwell arrays have emerged as robust and versatile alternatives to conventional mammalian cell culture substrates. Using standard microfabrication processes, biomaterials surfaces can be topographically patterned to comprise high-density arrays of micron-sized cavities with desirable geometry. Hundreds to thousands of individual cells or cell colonies with controlled size and shape can be trapped in these cavities by simple gravitational sedimentation. Efficient long-term cell confinement allows for parallel analyses and manipulation of cell fate during in vitro culture. These live-cell arrays have already found applications in cell biology, for example to probe the effect of cell colony size on embryonic stem cell differentiation, to dissect the heterogeneity in single cell proliferation kinetics of neural or hematopoietic stem/progenitor cell populations, or to elucidate the role of cell shape on cell function. Here, we highlight the key applications of these platforms, hopefully inspiring biologists to apply these systems for their own studies.

Modular Tissue Engineering: Engineering Biological Tissues from the Bottom Up

Soft Matter. 2009  |  Pubmed ID: 20179781

Tissue engineering creates biological tissues that aim to improve the function of diseased or damaged tissues. To enhance the function of engineered tissues there is a need to generate structures that mimic the intricate architecture and complexity of native organs and tissues. With the desire to create more complex tissues with features such as developed and functional microvasculature, cell binding motifs and tissue specific morphology, tissue engineering techniques are beginning to focus on building modular microtissues with repeated functional units. The emerging field known as modular tissue engineering focuses on fabricating tissue building blocks with specific microarchitectural features and using these modular units to engineer biological tissues from the bottom up. In this review we will examine the promise and shortcomings of "bottom-up" approaches to creating engineered biological tissues. Specifically, we will survey the current techniques for controlling cell aggregation, proliferation and extracellular matrix deposition, as well as approaches to generating shape-controlled tissue modules. We will then highlight techniques utilized to create macroscale engineered biological tissues from modular microscale units.

Stochastic Model of Self-assembly of Cell-laden Hydrogels

Physical Review. E, Statistical, Nonlinear, and Soft Matter Physics. Dec, 2009  |  Pubmed ID: 20365184

Recent progress in bottom-up tissue engineering has demonstrated that three-dimensional tissue constructs with predefined architectures may be obtained by assembling shape-controlled hydrogels in multiphase reactor systems. Driven by the hydrophobic force between gel unit and liquid media, highly ordered hydrogel clusters can be formed. Many complex factors occurring at microscale (i.e., gel unit collisions, hydrophobic forces, and gel unit movement) are involved in the self-assembly process. In this paper a two-dimensional off-lattice Monte Carlo model with Lennard-Jones-type potential describing unit-unit interactions is introduced for studying this process. Simulations are shown to agree well with the experimental results for hydrogel assembly in mineral oil. The simulation method is demonstrated for rectangular hydrogel units of different aspect ratios as well as extended to the case of more complex hydrogel unit geometries.

UV-assisted Capillary Force Lithography for Engineering Biomimetic Multiscale Hierarchical Structures: From Lotus Leaf to Gecko Foot Hairs

Nanoscale. Dec, 2009  |  Pubmed ID: 20648269

This feature article provides an overview of the recently developed two-step UV-assisted capillary force lithography and its application to fabricating well-defined micro/nanoscale hierarchical structures. This method utilizes an oxygen inhibition effect in the course of UV irradiation curing and a two-step moulding process, to form multiscale hierarchical or suspended nanobridge structures in a rapid and reproducible manner. After a brief description of the fabrication principles, several examples of the two-step UV-assisted moulding technique are presented. In addition, emerging applications of the multiscale hierarchical structures are briefly described.

Hydrogels in Regenerative Medicine

Advanced Materials (Deerfield Beach, Fla.). Sep, 2009  |  Pubmed ID: 20882499

Hydrogels, due to their unique biocompatibility, flexible methods of synthesis, range of constituents, and desirable physical characteristics, have been the material of choice for many applications in regenerative medicine. They can serve as scaffolds that provide structural integrity to tissue constructs, control drug and protein delivery to tissues and cultures, and serve as adhesives or barriers between tissue and material surfaces. In this work, the properties of hydrogels that are important for tissue engineering applications and the inherent material design constraints and challenges are discussed. Recent research involving several different hydrogels polymerized from a variety of synthetic and natural monomers using typical and novel synthetic methods are highlighted. Finally, special attention is given to the microfabrication techniques that are currently resulting in important advances in the field.

Patterned Differentiation of Individual Embryoid Bodies in Spatially Organized 3D Hybrid Microgels

Advanced Materials (Deerfield Beach, Fla.). Dec, 2010  |  Pubmed ID: 20941801

Contributors to the Emerging Investigators Issue

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

Fabrication of Three-dimensional Porous Cell-laden Hydrogel for Tissue Engineering

Biofabrication. Sep, 2010  |  Pubmed ID: 20823504

For tissue engineering applications, scaffolds should be porous to enable rapid nutrient and oxygen transfer while providing a three-dimensional (3D) microenvironment for the encapsulated cells. This dual characteristic can be achieved by fabrication of porous hydrogels that contain encapsulated cells. In this work, we developed a simple method that allows cell encapsulation and pore generation inside alginate hydrogels simultaneously. Gelatin beads of 150-300 microm diameter were used as a sacrificial porogen for generating pores within cell-laden hydrogels. Gelation of gelatin at low temperature (4 degrees C) was used to form beads without chemical crosslinking and their subsequent dissolution after cell encapsulation led to generation of pores within cell-laden hydrogels. The pore size and porosity of the scaffolds were controlled by the gelatin bead size and their volume ratio, respectively. Fabricated hydrogels were characterized for their internal microarchitecture, mechanical properties and permeability. Hydrogels exhibited a high degree of porosity with increasing gelatin bead content in contrast to nonporous alginate hydrogel. Furthermore, permeability increased by two to three orders while compressive modulus decreased with increasing porosity of the scaffolds. Application of these scaffolds for tissue engineering was tested by encapsulation of hepatocarcinoma cell line (HepG2). All the scaffolds showed similar cell viability; however, cell proliferation was enhanced under porous conditions. Furthermore, porous alginate hydrogels resulted in formation of larger spheroids and higher albumin secretion compared to nonporous conditions. These data suggest that porous alginate hydrogels may have provided a better environment for cell proliferation and albumin production. This may be due to the enhanced mass transfer of nutrients, oxygen and waste removal, which is potentially beneficial for tissue engineering and regenerative medicine applications.

Constrained Watershed Method to Infer Morphology of Mammalian Cells in Microscopic Images

Cytometry. Part A : the Journal of the International Society for Analytical Cytology. Dec, 2010  |  Pubmed ID: 20872884

Precise information about the size, shape, temporal dynamics, and spatial distribution of cells is beneficial for the understanding of cell behavior and may play a key role in drug development, regenerative medicine, and disease research. The traditional method of manual observation and measurement of cells from microscopic images is tedious, expensive, and time consuming. Thus, automated methods are in high demand, especially given the increasing quantity of cell data being collected. In this article, an automated method to measure cell morphology from microscopic images is proposed to outline the boundaries of individual hematopoietic stem cells (HSCs). The proposed method outlines the cell regions using a constrained watershed method which is derived as an inverse problem. The experimental results generated by applying the proposed method to different HSC image sequences showed robust performance to detect and segment individual and dividing cells. The performance of the proposed method for individual cell segmentation for single frame high-resolution images was more than 97%, and decreased slightly to 90% for low-resolution multiframe stitched images.

Directed Assembly of Cell-laden Hydrogels for Engineering Functional Tissues

Organogenesis. Oct-Dec, 2010  |  Pubmed ID: 21220962

Tissue engineering aims to develop functionalized tissues for organ replacement or restoration. Biodegradable scaffolds have been used in tissue engineering to support cell growth and maintain mechanical and biological properties of tissue constructs. Ideally cells on these scaffolds adhere, proliferate, and deposit matrix at a rate that is consistent with scaffold degradation. However, the cellular rearrangement within these scaffolds often does not recapitulate the architecture of the native tissues. Directed assembly of tissue-like structures is an attractive alternative to scaffold-based approach for tissue engineering which potentially can build tissue constructs with biomimetic architecture and function. In directed assembly, shape-controlled microstructures are fabricated in which organized structures of different cell types can be used as tissue building blocks. To fabricate tissue building blocks, hydrogels are commonly used as biomaterials for cell encapsulation to mimic the matrix in vivo. The hydrogel-based tissue building blocks can be arranged in pre-defined architectures by various directed tissue assembly techniques. In this paper, recent advances in directed assembly-based tissue engineering are summarized as an emerging alternative to meet challenges associated with scaffold-based tissue engineering and future directions are addressed.

Microengineering Hydrogels for Stem Cell Bioengineering and Tissue Regeneration

JALA (Charlottesville, Va.). Dec, 2010  |  Pubmed ID: 21344063

The integration of microfabrication technologies with advanced biomaterials has led to the development of powerful tools to control the cellular microenvironment and the microarchitecture of engineered tissue constructs. Here we review this area, with a focus on the work accomplished in our laboratory. In particular, we discuss techniques to develop hydrogel microstructures for controlling cell aggregate formation to regulate stem cell behavior as well as a bottom-up and a top-down microengineering approach to creating biomimic tissue-like structures.

Benchtop Fabrication of PDMS Microstructures by an Unconventional Photolithographic Method

Biofabrication. Dec, 2010  |  Pubmed ID: 21076185

Poly(dimethylsiloxane) (PDMS) microstructures have been widely used in bio-microelectromechanical systems (bio-MEMS) for various types of analytical, diagnostic and therapeutic applications. However, PDMS-based soft lithographic techniques still use conventional microfabrication processes to generate a master mold, which requires access to clean room facilities and costly equipment. With the increasing use of these systems in various fields, the development of benchtop systems for fabricating microdevices is emerging as an important challenge in their widespread use. Here we demonstrate a simple, low-cost and rapid method to fabricate PDMS microstructures by using micropatterned poly(ethylene glycol) diacrylate (PEGDA) master molds. In this method, PEGDA microstructures were patterned on a glass substrate by photolithography under ambient conditions and by using simple tools. The resulting PEGDA structures were subsequently used to generate PDMS microstructures by standard molding in a reproducible and repeatable manner. The thickness of the PEGDA microstructures was controllable from 15 to 300 µm by using commonly available spacer materials. We also demonstrate the use of this method to fabricate microfluidic channels capable of generating concentration gradients. In addition, we fabricated PEGDA microstructures by photolithography from the light generated from commonly available laminar cell culture hood. These data suggest that this approach could be beneficial for fabricating low-cost PDMS-based microdevices in resource limited settings.

Preventing Cardiac Remodeling: the Combination of Cell-based Therapy and Cardiac Support Therapy Preserves Left Ventricular Function in Rodent Model of Myocardial Ischemia

The Journal of Thoracic and Cardiovascular Surgery. Dec, 2010  |  Pubmed ID: 21078426

Cellular and mechanical treatment to prevent heart failure each holds therapeutic promise but together have not been reported yet. The goal of the present study was to determine whether combining a cardiac support device with cell-based therapy could prevent adverse left ventricular remodeling, more than either therapy alone.

Fabrication and Characterization of Tough Elastomeric Fibrous Scaffolds for Tissue Engineering Applications

Conference Proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference. 2010  |  Pubmed ID: 21096824

Development of biodegradable tough elastomeric scaffolds are important for engineering tissues such as myocardium and heart valves that experience dynamic environments in vivo. Biomaterial scaffolds should ideally provide appropriate physical, chemical and mechanical cues to the seeded cells to closely mimic the native ECM. Collagen fibers form an important component of native myocardium as well as heart valve leaflets and provide necessary tensile properties to these tissues. Amongst various polymers, collagen mimicking biodegradable elastomer, Poly-(glycerol-sebacate) (PGS) has shown great promise in microfabricated scaffolds for cardiac tissue engineering. However, its use is limited by its solubility and the ability to cast nano-/microfibrous structures. For its superior mechanical properties, thermal or UV crosslinking of the pre-polymer is required under high temperatures and vacuum limiting fabrication of fibers. In this work, we fabricated electrospun PGS fibers were fabricated by simply blending it with biodegradable polycaprolactone (PCL) polymer without any post-processing. It was hypothesized that microfibrous PGS-PCL scaffolds would provide appropriate physical (fibrous structure) and chemical (balanced hydrophilicity and hydrophobicity) to the cells in addition to the mechanical properties.

BIOMIMETIC GRADIENT HYDROGELS FOR TISSUE ENGINEERING

The Canadian Journal of Chemical Engineering. Dec, 2010  |  Pubmed ID: 21874065

During tissue morphogenesis and homeostasis, cells experience various signals in their environments, including gradients of physical and chemical cues. Spatial and temporal gradients regulate various cell behaviours such as proliferation, migration, and differentiation during development, inflammation, wound healing, and cancer. One of the goals of functional tissue engineering is to create microenvironments that mimic the cellular and tissue complexity found in vivo by incorporating physical, chemical, temporal, and spatial gradients within engineered three-dimensional (3D) scaffolds. Hydrogels are ideal materials for 3D tissue scaffolds that mimic the extracellular matrix (ECM). Various techniques from material science, microscale engineering, and microfluidics are used to synthesise biomimetic hydrogels with encapsulated cells and tailored microenvironments. In particular, a host of methods exist to incorporate micrometer to centimetre scale chemical and physical gradients within hydrogels to mimic the cellular cues found in vivo. In this review, we draw on specific biological examples to motivate hydrogel gradients as tools for studying cell-material interactions. We provide a brief overview of techniques to generate gradient hydrogels and showcase their use to study particular cell behaviours in two-dimensional (2D) and 3D environments. We conclude by summarizing the current and future trends in gradient hydrogels and cell-material interactions in context with the long-term goals of tissue engineering.

Modified Gellan Gum Hydrogels with Tunable Physical and Mechanical Properties

Biomaterials. Oct, 2010  |  Pubmed ID: 20663552

Gellan Gum (GG) has been recently proposed for tissue engineering applications. GG hydrogels are produced by physical crosslinking methods induced by temperature variation or by the presence of divalent cations. However, physical crosslinking methods may yield hydrogels that become weaker in physiological conditions due to the exchange of divalent cations by monovalent ones. Hence, this work presents a new class of GG hydrogels crosslinkable by both physical and chemical mechanisms. Methacrylate groups were incorporated in the GG chain, leading to the production of a methacrylated Gellan Gum (MeGG) hydrogel with highly tunable physical and mechanical properties. The chemical modification was confirmed by proton nuclear magnetic resonance (1H NMR) and Fourier transform infrared spectroscopy (FTIR-ATR). The mechanical properties of the developed hydrogel networks, with Young's modulus values between 0.15 and 148 kPa, showed to be tuned by the different crosslinking mechanisms used. The in vitro swelling kinetics and hydrolytic degradation rate were dependent on the crosslinking mechanisms used to form the hydrogels. Three-dimensional (3D) encapsulation of NIH-3T3 fibroblast cells in MeGG networks demonstrated in vitro biocompatibility confirmed by high cell survival. Given the highly tunable mechanical and degradation properties of MeGG, it may be applicable for a wide range of tissue engineering approaches.

Stimuli-responsive Microwells for Formation and Retrieval of Cell Aggregates

Lab on a Chip. Sep, 2010  |  Pubmed ID: 20664846

Generating cell aggregates is beneficial for various applications ranging from biotechnology to regenerative therapies. Previously, poly(ethylene glycol) (PEG) microwells have been demonstrated as a potentially useful method for generating controlled-size cell aggregates. In addition to controlling cell aggregate size and homogeneity, the ability to confine cell aggregates on glass adhesive substrates and subsequently retrieve aggregates from microwells for further experimentation and analysis could be beneficial for various applications. However, it is often difficult to retrieve cell aggregates from these microwells without the use of digestive enzymes. This study describes the stable formation of cell aggregates in responsive microwells with adhesive substrates and their further retrieval in a temperature dependent manner by exploiting the stimuli responsiveness of these microwells. The responsive polymer structure of the arrays can be used to thermally regulate the microwell diameters causing a mechanical force on the aggregates, subsequently facilitating the retrieval of cell aggregates from the microwells with high efficiency compared to PEG arrays. This approach can be potentially integrated into high-throughput systems and may become a versatile tool for various applications that require aggregate formation and experimentation, such as tissue engineering, drug discovery, and stem cell biology.

Cell-laden Microengineered Gelatin Methacrylate Hydrogels

Biomaterials. Jul, 2010  |  Pubmed ID: 20417964

The cellular microenvironment plays an integral role in improving the function of microengineered tissues. Control of the microarchitecture in engineered tissues can be achieved through photopatterning of cell-laden hydrogels. However, despite high pattern fidelity of photopolymerizable hydrogels, many such materials are not cell-responsive and have limited biodegradability. Here, we demonstrate gelatin methacrylate (GelMA) as an inexpensive, cell-responsive hydrogel platform for creating cell-laden microtissues and microfluidic devices. Cells readily bound to, proliferated, elongated, and migrated both when seeded on micropatterned GelMA substrates as well as when encapsulated in microfabricated GelMA hydrogels. The hydration and mechanical properties of GelMA were demonstrated to be tunable for various applications through modification of the methacrylation degree and gel concentration. The pattern fidelity and resolution of GelMA were high and it could be patterned to create perfusable microfluidic channels. Furthermore, GelMA micropatterns could be used to create cellular micropatterns for in vitro cell studies or 3D microtissue fabrication. These data suggest that GelMA hydrogels could be useful for creating complex, cell-responsive microtissues, such as endothelialized microvasculature, or for other applications that require cell-responsive microengineered hydrogels.

Micro-masonry: Construction of 3D Structures by Microscale Self-assembly

Advanced Materials (Deerfield Beach, Fla.). Jun, 2010  |  Pubmed ID: 20440697

Preparation of Arrays of Cell Spheroids and Spheroid-monolayer Cocultures Within a Microfluidic Device

Journal of Bioscience and Bioengineering. Nov, 2010  |  Pubmed ID: 20591731

This study describes a novel method for generation of an array of three-dimensional (3D) multicellular spheroids within a microchannel in patterned cultures containing one or multiple cell types. This method uses a unique property of a cross-linked albumin coated surface in which the surface can be switched from non-adhesive to cell adhesive upon electrostatic adsorption of a polycation. Introduction of a solution containing albumin and a cross-linking agent into a microchannel with an array of microwells caused the entire surface, with the exception of the interior of the microwells, to become coated with the cross-linked albumin layer. Cells that were seeded within the microchannel did not adhere to the surface of the microchannel and became entrapped in the microwells. HepG2 cells seeded in the microwells formed 3D spheroids with controlled sizes and shapes depending upon the dimensions of the microwells. When the albumin coated surface was subsequently exposed to an aqueous solution containing poly(ethyleneimine) (PEI), adhesion of secondary cells, fibroblasts, occurred in the regions surrounding the arrayed spheroids. This coculture system can be coupled with spatially controlled fluids such as gradients and focused flow generators for various biological and tissue engineering applications.

Directed 3D Cell Alignment and Elongation in Microengineered Hydrogels

Biomaterials. Sep, 2010  |  Pubmed ID: 20638973

Organized cellular alignment is critical to controlling tissue microarchitecture and biological function. Although a multitude of techniques have been described to control cellular alignment in 2D, recapitulating the cellular alignment of highly organized native tissues in 3D engineered tissues remains a challenge. While cellular alignment in engineered tissues can be induced through the use of external physical stimuli, there are few simple techniques for microscale control of cell behavior that are largely cell-driven. In this study we present a simple and direct method to control the alignment and elongation of fibroblasts, myoblasts, endothelial cells and cardiac stem cells encapsulated in microengineered 3D gelatin methacrylate (GelMA) hydrogels, demonstrating that cells with the intrinsic potential to form aligned tissues in vivo will self-organize into functional tissues in vitro if confined in the appropriate 3D microarchitecture. The presented system may be used as an in vitro model for investigating cell and tissue morphogenesis in 3D, as well as for creating tissue constructs with microscale control of 3D cellular alignment and elongation, that could have great potential for the engineering of functional tissues with aligned cells and anisotropic function.

Controlled-size Embryoid Body Formation in Concave Microwell Arrays

Biomaterials. May, 2010  |  Pubmed ID: 20206991

Embryonic stem (ES) cells hold great potential as a renewable cell source for regenerative medicine and cell-based therapy. Despite the potential of ES cells, conventional stem cell culture methods do not enable the control of the microenvironment. A number of microscale engineering approaches have been recently developed to control the extracellular microenvironment and to direct embryonic stem cell fate. Here, we used engineered concave microwell arrays to regulate the size and shape of embryoid bodies (EBs)-cell aggregate intermediates derived from ES cells. Murine ES cells were aggregated within concave microwells, and their aggregate sizes were controlled by varying the microwell widths (200, 500, and 1000 mum). Differentiation of murine ES cells into three germ layers was assessed by analyzing gene expression. We found that ES cell-derived cardiogenesis and neurogenesis were strongly regulated by the EB size, showing that larger concave microwell arrays induced more neuronal and cardiomyocyte differentiation than did smaller microwell arrays. Therefore, this engineered concave microwell array could be a potentially useful tool for controlling ES cell behavior.

Rapid Generation of Biologically Relevant Hydrogels Containing Long-range Chemical Gradients

Advanced Functional Materials. 2010  |  Pubmed ID: 20216924

Many biological processes are regulated by gradients of bioactive chemicals. Thus, the generation of materials with embedded chemical gradients may be beneficial for understanding biological phenomena and generating tissue-mimetic constructs. Here we describe a simple and versatile method to rapidly generate materials containing centimeter-long gradients of chemical properties in a microfluidic channel. The formation of chemical gradient was initiated by a passive-pump-induced forward flow and further developed during an evaporation-induced backward flow. The gradient was spatially controlled by the backward flow time and the hydrogel material containing the gradient was synthesized via photopolymerization. Gradients of a cell-adhesion ligand, Arg-Gly-Asp-Ser (RGDS), was incorporated in the poly(ethylene glycol)-diacrylate (PEG-DA) hydrogels to test the response of endothelial cells. The cells attached and spread along the hydrogel material in a manner consistent with the RGDS gradient profile. A hydrogel containing PEG-DA concentration gradient and constant RGDS concentration was also generated. The morphology of cells cultured on such hydrogel changed from round in the lower PEG-DA concentration regions to well-spread in the higher PEG-DA concentration regions. This approach is expected to be a valuable tool to investigate the cell-material interactions in a simple and high-throughput manner and to design graded biomimetic materials for tissue engineering applications.

A Computational and Experimental Study Inside Microfluidic Systems: the Role of Shear Stress and Flow Recirculation in Cell Docking

Biomedical Microdevices. Aug, 2010  |  Pubmed ID: 20300857

In this paper, microfluidic devices containing microwells that enabled cell docking were investigated. We theoretically assessed the effect of geometry on recirculation areas and wall shear stress patterns within microwells and studied the relationship between the computational predictions and experimental cell docking. We used microchannels with 150 microm diameter microwells that had either 20 or 80 microm thickness. Flow within 80 microm deep microwells was subject to extensive recirculation areas and low shear stresses (<0.5 mPa) near the well base; whilst these were only presented within a 10 microm peripheral ring in 20 microm thick microwells. We also experimentally demonstrated that cell docking was significantly higher (p < 0.01) in 80 microm thick microwells as compared to 20 microm thick microwells. Finally, a computational tool which correlated physical and geometrical parameters of microwells with their fluid dynamic environment was developed and was also experimentally confirmed.

Interface-directed Self-assembly of Cell-laden Microgels

Small (Weinheim an Der Bergstrasse, Germany). Apr, 2010  |  Pubmed ID: 20358531

Cell-laden hydrogels show great promise for creating engineered tissues. However, a major shortcoming with these systems has been the inability to fabricate structures with controlled micrometer-scale features on a biologically relevant length scale. In this Full Paper, a rapid method is demonstrated for creating centimeter-scale, cell-laden hydrogels through the assembly of shape-controlled microgels or a liquid-air interface. Cell-laden microgels of specific shapes are randomly placed on the surface of a high-density, hydrophobic solution, induced to aggregate and then crosslinked into macroscale tissue-like structures. The resulting assemblies are cell-laden hydrogel sheets consisting of tightly packed, ordered microgel units. In addition, a hierarchical approach creates complex multigel building blocks, which are then assembled into tissues with precise spatial control over the cell distribution. The results demonstrate that forces at an air-liquid interface can be used to self-assemble spatially controllable, cocultured tissue-like structures.

Convection-driven Generation of Long-range Material Gradients

Biomaterials. Mar, 2010  |  Pubmed ID: 20035990

Natural materials exhibit anisotropy with variations in soluble factors, cell distribution, and matrix properties. The ability to recreate the heterogeneity of the natural materials is a major challenge for investigating cell-material interactions and for developing biomimetic materials. Here we present a generic fluidic approach using convection and alternating flow to rapidly generate multi-centimeter gradients of biomolecules, polymers, beads and cells and cross-gradients of two species in a microchannel. Accompanying theoretical estimates and simulations of gradient growth provide design criteria over a range of material properties. A poly(ethylene-glycol) hydrogel gradient, a porous collagen gradient and a composite material with a hyaluronic acid/gelatin cross-gradient were generated with continuous variations in material properties and in their ability to regulate cellular response. This simple yet generic fluidic platform should prove useful for creating anisotropic biomimetic materials and high-throughput platforms for investigating cell-microenvironment interactions.

Bioinspired Materials for Controlling Stem Cell Fate

Accounts of Chemical Research. Mar, 2010  |  Pubmed ID: 20043634

Although researchers currently have limited ability to mimic the natural stem cell microenvironment, recent work at the interface of stem biology and biomaterials science has demonstrated that control over stem cell behavior with artificial microenvironments is quite advanced. Embryonic and adult stem cells are potentially useful platforms for tissue regeneration, cell-based therapeutics, and disease-in-a-dish models for drug screening. The major challenge in this field is to reliably control stem cell behavior outside the body. Common biological control schemes often ignore physicochemical parameters that materials scientists and engineers commonly manipulate, such as substrate topography and mechanical and rheological properties. However, with appropriate attention to these parameters, researchers have designed novel synthetic microenvironments to control stem cell behavior in rather unnatural ways. In this Account, we review synthetic microenvironments that aim to overcome the limitations of natural niches rather than to mimic them. A biomimetic stem cell control strategy is often limited by an incomplete understanding of the complex signaling pathways that drive stem cell behavior from early embryogenesis to late adulthood. The stem cell extracellular environment presents a miscellany of competing biological signals that keep the cell in a state of unstable equilibrium. Using synthetic polymers, researchers have designed synthetic microenvironments with an uncluttered array of cell signals, both specific and nonspecific, that are motivated by rather than modeled after biology. These have proven useful in maintaining cell potency, studying asymmetric cell division, and controlling cellular differentiation. We discuss recent research that highlights important biomaterials properties for controlling stem cell behavior, as well as advanced processes for selecting those materials, such as combinatorial and high-throughput screening. Much of this work has utilized micro- and nanoscale fabrication tools for controlling material properties and generating diversity in both two and three dimensions. Because of their ease of synthesis and similarity to biological soft matter, hydrogels have become a biomaterial of choice for generating 3D microenvironments. In presenting these efforts within the framework of synthetic biology, we anticipate that future researchers may exploit synthetic polymers to create microenvironments that control stem cell behavior in clinically relevant ways.

Microporous Cell-laden Hydrogels for Engineered Tissue Constructs

Biotechnology and Bioengineering. May, 2010  |  Pubmed ID: 20091766

In this article, we describe an approach to generate microporous cell-laden hydrogels for fabricating biomimetic tissue engineered constructs. Micropores at different length scales were fabricated in cell-laden hydrogels by micromolding fluidic channels and leaching sucrose crystals. Microengineered channels were created within cell-laden hydrogel precursors containing agarose solution mixed with sucrose crystals. The rapid cooling of the agarose solution was used to gel the solution and form micropores in place of the sucrose crystals. The sucrose leaching process generated homogeneously distributed micropores within the gels, while enabling the direct immobilization of cells within the gels. We also characterized the physical, mechanical, and biological properties (i.e., microporosity, diffusivity, and cell viability) of cell-laden agarose gels as a function of engineered porosity. The microporosity was controlled from 0% to 40% and the diffusivity of molecules in the porous agarose gels increased as compared to controls. Furthermore, the viability of human hepatic carcinoma cells that were cultured in microporous agarose gels corresponded to the diffusion profile generated away from the microchannels. Based on their enhanced diffusive properties, microporous cell-laden hydrogels containing a microengineered fluidic channel can be a useful tool for generating tissue structures for regenerative medicine and drug discovery applications.

Controlling the Porosity and Microarchitecture of Hydrogels for Tissue Engineering

Tissue Engineering. Part B, Reviews. Aug, 2010  |  Pubmed ID: 20121414

Tissue engineering holds great promise for regeneration and repair of diseased tissues, making the development of tissue engineering scaffolds a topic of great interest in biomedical research. Because of their biocompatibility and similarities to native extracellular matrix, hydrogels have emerged as leading candidates for engineered tissue scaffolds. However, precise control of hydrogel properties, such as porosity, remains a challenge. Traditional techniques for creating bulk porosity in polymers have demonstrated success in hydrogels for tissue engineering; however, often the conditions are incompatible with direct cell encapsulation. Emerging technologies have demonstrated the ability to control porosity and the microarchitectural features in hydrogels, creating engineered tissues with structure and function similar to native tissues. In this review, we explore the various technologies for controlling the porosity and microarchitecture within hydrogels, and demonstrate successful applications of combining these techniques.

Surface-directed Assembly of Cell-laden Microgels

Biotechnology and Bioengineering. Feb, 2010  |  Pubmed ID: 19777588

Cell-laden microscale hydrogels (microgels) can be used as tissue building blocks and assembled to create 3D tissue constructs with well-defined microarchitecture. In this article, we present a bottom-up approach to achieve microgel assembly on a patterned surface. Driven by surface tension, the hydrophilic microgels can be assembled into well-defined shapes on a glass surface patterned with hydrophobic and hydrophilic regions. We found that the cuboidic microgels ( approximately 100-200 microm in width) could self-assemble into defined shapes with high fidelity to the surface patterns. The microgel assembly process was improved by increasing the hydrophilicity of the microgels and reducing the surface tension of the surrounding solution. The assembled microgels were stabilized by a secondary crosslinking step. Assembled microgels containing cells stained with different dyes were fabricated to demonstrate the application of this approach for engineering microscale tissue constructs containing multiple cell types. This bottom-up approach enables rapid fabrication of cell-laden microgel assemblies with pre-defined geometrical and biological features, which is easily scalable and can be potentially used in microscale tissue engineering applications.

Cell Confinement in Patterned Nanoliter Droplets in a Microwell Array by Wiping

Journal of Biomedical Materials Research. Part A. May, 2010  |  Pubmed ID: 19585570

Cell patterning is useful for a variety of biological applications such as tissue engineering and drug discovery. In particular, the ability to localize cells within distinct fluids is beneficial for a variety of applications ranging from microencapsulation to high-throughput analysis. However, despite much progress, cell immobilization and maintenance within patterned microscale droplets remains a challenge. In particular, no method currently exists to rapidly seed cells into microwell arrays in a controllable and reliable manner. In this study, we present a simple wiping technique to localize cells within arrays of polymeric microwells. This robust method produces cell seeding densities that vary consistently with microwell geometry and cell concentration. Moreover, we develop a simple theoretical model to accurately predict cell seeding density and seeding efficiency in terms of the design parameters of the microwell array and the cell density. This short-term cell patterning approach is an enabling tool to develop new high-throughput screening technologies that utilize microwell arrays containing cells for screening applications.

Layer by Layer Three-dimensional Tissue Epitaxy by Cell-laden Hydrogel Droplets

Tissue Engineering. Part C, Methods. Feb, 2010  |  Pubmed ID: 19586367

The ability to bioengineer three-dimensional (3D) tissues is a potentially powerful approach to treat diverse diseases such as cancer, loss of tissue function, or organ failure. Traditional tissue engineering methods, however, face challenges in fabricating 3D tissue constructs that resemble the native tissue microvasculature and microarchitectures. We have developed a bioprinter that can be used to print 3D patches of smooth muscle cells (5 mm x 5 mm x 81 microm) encapsulated within collagen. Current inkjet printing systems suffer from loss of cell viability and clogging. To overcome these limitations, we developed a system that uses mechanical valves to print high viscosity hydrogel precursors containing cells. The bioprinting platform that we developed enables (i) printing of multilayered 3D cell-laden hydrogel structures (16.2 microm thick per layer) with controlled spatial resolution (proximal axis: 18.0 +/- 7.0 microm and distal axis: 0.5 +/- 4.9 microm), (ii) high-throughput droplet generation (1 s per layer, 160 droplets/s), (iii) cell seeding uniformity (26 +/- 2 cells/mm(2) at 1 million cells/mL, 122 +/- 20 cells/mm(2) at 5 million cells/mL, and 216 +/- 38 cells/mm(2) at 10 million cells/mL), and (iv) long-term viability in culture (>90%, 14 days). This platform to print 3D tissue constructs may be beneficial for regenerative medicine applications by enabling the fabrication of printed replacement tissues.

Nano/Microfluidics for Diagnosis of Infectious Diseases in Developing Countries

Advanced Drug Delivery Reviews. Mar, 2010  |  Pubmed ID: 19954755

Nano/Microfluidic technologies are emerging as powerful enabling tools for diagnosis and monitoring of infectious diseases in both developed and developing countries. Miniaturized nano/microfluidic platforms that precisely manipulate small fluid volumes can be used to enable medical diagnosis in a more rapid and accurate manner. In particular, these nano/microfluidic diagnostic technologies are potentially applicable to global health applications, since they are disposable, inexpensive, portable, and easy-to-use for detection of infectious diseases. In this paper, we review recent advances in nano/microfluidic technologies for clinical point-of-care applications at resource-limited settings in developing countries.

A Hollow Sphere Soft Lithography Approach for Long-term Hanging Drop Methods

Tissue Engineering. Part C, Methods. Apr, 2010  |  Pubmed ID: 19505251

In conventional hanging drop (HD) methods, embryonic stem cell aggregates or embryoid bodies (EBs) are often maintained in small inverted droplets. Gravity limits the volumes of these droplets to less than 50 microL, and hence such cell cultures can only be sustained for a few days without frequent media changes. Here we present a new approach to performing long-term HD methods (10-15 days) that can provide larger media reservoirs in a HD format to maintain more consistent culture media conditions. To implement this approach, we fabricated hollow sphere (HS) structures by injecting liquid drops into noncured poly(dimethylsiloxane) mixtures. These structures served as cell culture chambers with large media volumes (500 microL in each sphere) where EBs could grow without media depletion. The results showed that the sizes of the EBs cultured in the HS structures in a long-term HD format were approximately twice those of conventional HD methods after 10 days in culture. Further, HS cultures showed multilineage differentiation, similar to EBs cultured in the HD method. Due to its ease of fabrication and enhanced features, this approach may be of potential benefit as a stem cell culture method for regenerative medicine.

Cell-adhesive and Mechanically Tunable Glucose-based Biodegradable Hydrogels

Acta Biomaterialia. Jan, 2011  |  Pubmed ID: 20647064

The development of materials with biomimetic mechanical and biological properties is of great interest for regenerative medicine applications. In particular, hydrogels are a promising class of biomaterials due to their high water content, which mimics that of natural tissues. We have synthesized a hydrophilic biodegradable polymer, designated poly(glucose malate)methacrylate (PGMma), which is composed of glucose and malic acid, commonly found in the human metabolic system. This polymer is made photocrosslinkable by the incorporation of methacrylate groups. The resulting properties of the hydrogels can be tuned by altering the reacting ratio of the starting materials, the degree of methacrylation, and the polymer concentration of the resultant hydrogel. Hydrogels exhibited compressive moduli ranging from 1.8 ± 0.4 kPa to 172.7 ± 36 kPa with compressive strain at failure from 37.5 ± 0.9% to 61.2 ± 1.1%, and hydration by mass ranging from 18.7 ± 0.5% to 114.1 ± 1.3%. PGMma hydrogels also showed a broad range of degradation rates and were cell-adhesive, enabling the spreading of adherent cells. Overall, this work introduces a class of cell-adhesive, mechanically tunable and biodegradable glucose-based hydrogels that may be useful for various tissue engineering and cell culture applications.

Hybrid PGS-PCL Microfibrous Scaffolds with Improved Mechanical and Biological Properties

Journal of Tissue Engineering and Regenerative Medicine. Apr, 2011  |  Pubmed ID: 20669260

Poly(glycerol sebacate) (PGS) is a biodegradable elastomer that has generated great interest as a scaffold material due to its desirable mechanical properties. However, the use of PGS in tissue engineering is limited by difficulties in casting micro- and nanofibrous structures, due to high temperatures and vacuum required for its curing and limited solubility of the cured polymer. In this paper, we developed microfibrous scaffolds made from blends of PGS and poly(ε-caprolactone) (PCL) using a standard electrospinning set-up. At a given PGS:PCL ratio, higher voltage resulted in significantly smaller fibre diameters (reduced from ∼4 µm to 2.8 µm; p < 0.05). Further increase in voltage resulted in the fusion of fibres. Similarly, higher PGS concentrations in the polymer blend resulted in significantly increased fibre diameter (p < 0.01). We further compared the mechanical properties of electrospun PGS:PCL scaffolds with those made from PCL. Scaffolds with higher PGS concentrations showed higher elastic modulus (EM), ultimate tensile strength (UTS) and ultimate elongation (UE) (p < 0.01) without the need for thermal curing or photocrosslinking. Biological evaluation of these scaffolds showed significantly improved HUVEC attachment and proliferation compared to PCL-only scaffolds (p < 0.05). Thus, we have demonstrated that simple blends of PGS prepolymer with PCL can be used to fabricate microfibrous scaffolds with mechanical properties in the range of a human aortic valve leaflet.

Engineering Systems for the Generation of Patterned Co-cultures for Controlling Cell-cell Interactions

Biochimica Et Biophysica Acta. Mar, 2011  |  Pubmed ID: 20655984

Inside the body, cells lie in direct contact or in close proximity to other cell types in a tightly controlled architecture that often regulates the resulting tissue function. Therefore, tissue engineering constructs that aim to reproduce the architecture and the geometry of tissues will benefit from methods of controlling cell-cell interactions with microscale resolution. SCOPE OF THE REVIEW: We discuss the use of microfabrication technologies for generating patterned co-cultures. In addition, we categorize patterned co-culture systems by cell type and discuss the implications of regulating cell-cell interactions in the resulting biological function of the tissues.

Digitally Tunable Physicochemical Coding of Material Composition and Topography in Continuous Microfibres

Nature Materials. Nov, 2011  |  Pubmed ID: 21892177

Heterotypic functional materials with compositional and topographical properties that vary spatiotemporally on the micro- or nanoscale are common in nature. However, fabricating such complex materials in the laboratory remains challenging. Here we describe a method to continuously create microfibres with tunable morphological, structural and chemical features using a microfluidic system consisting of a digital, programmable flow control that mimics the silk-spinning process of spiders. With this method we fabricated hydrogel microfibres coded with varying chemical composition and topography along the fibre, including gas micro-bubbles as well as nanoporous spindle-knots and joints that enabled directional water collection. We also explored the potential use of the coded microfibres for tissue engineering applications by creating multifunctional microfibres with a spatially controlled co-culture of encapsulated cells.

Enhancing Cell Penetration and Proliferation in Chitosan Hydrogels for Tissue Engineering Applications

Biomaterials. Dec, 2011  |  Pubmed ID: 21925727

The aim of this study was to develop a process to create highly porous three-dimensional (3D) chitosan hydrogels suitable for tissue engineering applications. Chitosan was crosslinked by glutaraldehyde (0.5 vol %) under high pressure CO(2) at 60 bar and 4 °C for a period of 90 min. A gradient-depressurisation strategy was developed, which was efficient in increasing pore size and the overall porosity of resultant hydrogels. The average pore diameter increased two fold (59 μm) compared with the sample that was depressurised after complete crosslinking and hydrogel formation (32 μm). It was feasible to achieve a pore diameter of 140 μm and the porosity of hydrogels to 87% by addition of Acacia gum (AG) as a surfactant to the media. The enhancement in porosity resulted in an increased swelling ratio and decreased mechanical strength. On hydrogels with large pores (>90 μm) and high porosities (>85%), fibroblasts were able to penetrate up to 400 μm into the hydrogels with reasonable viabilities (~80%) upon static seeding. MTS assays showed that fibroblasts proliferated over 14 days. Furthermore, aligned microchannels were produced within porous hydrogels to further promote cell proliferation. The developed process can be easily used to generate homogenous pores of controlled sizes in 3D chitosan hydrogels and may be of use for a broad range of tissue engineering applications.

Application of Microtechnologies for the Vascularization of Engineered Tissues

Vascular Cell. 2011  |  Pubmed ID: 22040627

ABSTRACT: Recent advances in medicine and healthcare allow people to live longer, increasing the need for the number of organ transplants. However, the number of organ donors has not been able to meet the demand, resulting in an organ shortage. The field of tissue engineering has emerged to produce organs to overcome this limitation. While tissue engineering of connective tissues such as skin and blood vessels have currently reached clinical studies, more complex organs are still far away from commercial availability due to pending challenges with in vitro engineering of 3D tissues. One of the major limitations of engineering large tissue structures is cell death resulting from the inability of nutrients to diffuse across large distances inside a scaffold. This task, carried out by the vasculature inside the body, has largely been described as one of the foremost important challenges in engineering 3D tissues since it remains one of the key steps for both in vitro production of tissue engineered construct and the in vivo integration of a transplanted tissue. This short review highlights the important challenges for vascularization and control of the microcirculatory system within engineered tissues, with particular emphasis on the use of microfabrication approaches.

Fabrication of Porous Chitosan Scaffolds for Soft Tissue Engineering Using Dense Gas CO2

Acta Biomaterialia. Apr, 2011  |  Pubmed ID: 21130905

The aim of this study was to investigate the feasibility of fabricating porous crosslinked chitosan hydrogels in an aqueous phase using dense gas CO(2) as a foaming agent. Highly porous chitosan hydrogels were formed by using glutaraldehyde and genipin as crosslinkers. The method developed here eliminates the formation of a skin layer, and does not require the use of surfactants or other toxic reagents to generate porosity. The chitosan hydrogel scaffolds had an average pore diameter of 30-40 μm. The operating pressure had a negligible effect on the pore characteristics of chitosan hydrogels. Temperature, reaction period, type of biopolymer and crosslinker had a significant impact on the pore size and characteristics of the hydrogel produced by dense gas CO(2). Scanning electron microscopy and histological analysis confirmed that the resulting porous structures allowed fibroblasts seeded on these scaffolds to proliferate into the three-dimensional (3-D) structure of these chitosan hydrogels. Live/dead staining and MTS analysis demonstrated that fibroblast cells proliferated over 7 days. The fabricated hydrogels exhibited comparable mechanical strength and swelling ratio and are potentially useful for soft tissue engineering applications such as skin and cartilage regeneration.

Engineering Approaches Toward Deconstructing and Controlling the Stem Cell Environment

Annals of Biomedical Engineering. Nov, 2011  |  Pubmed ID: 22101755

Stem cell-based therapeutics have become a vital component in tissue engineering and regenerative medicine. The microenvironment within which stem cells reside, i.e., the niche, plays a crucial role in regulating stem cell self-renewal and differentiation. However, current biological techniques lack the means to recapitulate the complexity of this microenvironment. Nano- and microengineered materials offer innovative methods to (1) deconstruct the stem cell niche to understand the effects of individual elements; (2) construct complex tissue-like structures resembling the niche to better predict and control cellular processes; and (3) transplant stem cells or activate endogenous stem cell populations for regeneration of aged or diseased tissues. In this article, we highlight some of the latest advances in this field and discuss future applications and directions of the use of nano- and microtechnologies for stem cell engineering.

Hydrogels and Microtechnologies for Engineering the Cellular Microenvironment

Wiley Interdisciplinary Reviews. Nanomedicine and Nanobiotechnology. Dec, 2011  |  Pubmed ID: 22144036

Hydrogels represent a class of materials suitable for numerous biomedical applications such as tissue engineering and drug delivery. Hydrogels are by definition capable of absorbing large amount of fluid, making them adequate for cell seeding and encapsulation as well as for implantation because of their biocompatibility and excellent diffusion properties. They also possess other desirable properties for fundamental research as they have the ability to mimic the basic three-dimensional (3D) biological, chemical, and mechanical properties of native tissues. Furthermore, their biological interactions with cells can be modified through the numerous side groups of the polymeric chains. Thus, the biological, chemical, and mechanical properties, as well as the degradation kinetics of hydrogels can be tailored depending on the application. In addition, their fabrication process can be combined with microtechnologies to enable precise control of cell-scale features such as surface topography and the presence of adhesion motifs on the hydrogel material. This ability to control the microscale structure of hydrogels has been used to engineer tissue models and to study cell behavior mechanisms in vitro. New approaches such as bottom-up and directed assembly of microscale hydrogels (microgels) are currently emerging as powerful methods to enable the fabrication of 3D constructs replicating the microenvironment found in vivo. WIREs Nanomed Nanobiotechnol 2011. doi: 10.1002/wnan.171 For further resources related to this article, please visit the WIREs website.

Microfabrication Technologies for Oral Drug Delivery

Advanced Drug Delivery Reviews. Dec, 2011  |  Pubmed ID: 22166590

Micro-/nanoscale technologies such as lithographic techniques and microfluidics offer promising avenues to revolutionalize the fields of tissue engineering, drug discovery, diagnostics and personalized medicine. Microfabrication techniques are being explored for drug delivery applications due to their ability to combine several features such as precise shape and size into a single drug delivery vehicle. They also offer to create unique asymmetrical features incorporated into single or multiple reservoir systems maximizing contact area with the intestinal lining. Combined with intelligent materials, such microfabricated platforms can be designed to be bioadhesive and stimuli-responsive. Apart from drug delivery devices, microfabrication technologies offer exciting opportunities to create biomimetic gastrointestinal tract models incorporating physiological cell types, flow patterns and brush-border like structures. Here we review the recent developments in this field with a focus on the applications of microfabrication in the development of oral drug delivery devices and biomimetic gastrointestinal tract models that can be used to evaluate the drug delivery efficacy.

Delving into BioMEMS

IEEE Pulse. Nov, 2011  |  Pubmed ID: 22250335

Generating Nonlinear Concentration Gradients in Microfluidic Devices for Cell Studies

Analytical Chemistry. Mar, 2011  |  Pubmed ID: 21344866

We describe a microfluidic device for generating nonlinear (exponential and sigmoidal) concentration gradients, coupled with a microwell array for cell storage and analysis. The device has two inputs for coflowing multiple aqueous solutions, a main coflow channel and an asymmetrical grid of fluidic channels that allows the two solutions to combine at intersection points without fully mixing. Due to this asymmetry and diffusion of the two species in the coflow channel, varying amounts of the two solutions enter each fluidic path. This induces exponential and sigmoidal concentration gradients at low and high flow rates, respectively, making the microfluidic device versatile. A key feature of this design is that it is space-saving, as it does not require multiplexing or a separate array of mixing channels. Furthermore, the gradient structure can be utilized in concert with cell experiments, to expose cells captured in microwells to various concentrations of soluble factors. We demonstrate the utility of this design to assess the viability of fibroblast cells in response to a range of hydrogen peroxide (H(2)O(2)) concentrations.

Surface-tension-driven Gradient Generation in a Fluid Stripe for Bench-top and Microwell Applications

Small (Weinheim an Der Bergstrasse, Germany). Apr, 2011  |  Pubmed ID: 21374805

A simple and inexpensive method is presented employing passive mechanisms to generate centimeters-long gradients of molecules and particles in under a second with only a coated glass slide and a micropipette. A drop of solution is pipetted onto a fluid stripe held in place on a glass slide by a hydrophobic boundary. The resulting difference in curvature pressure drives the flow and creates a concentration gradient by convection. Experiments and theoretical models characterize the flows and gradient profiles and their dependence on the fluid volumes, properties, and stripe geometry. A bench-top rapid prototyping method is outlined to allow the user to design and fabricate the coated slides using only tape and hydrophobic spray. The rapid prototyping method is compatible with microwell arrays, allowing soluble gradients to be applied to cells in shear-protected microwells. The method's simplicity makes it accessible to virtually any researcher or student and its use of passive mechanisms makes it ideal for field use and compatible with point-of-care and global health initiatives.

Cell-laden Microengineered Pullulan Methacrylate Hydrogels Promote Cell Proliferation and 3D Cluster Formation

Soft Matter. Jan, 2011  |  Pubmed ID: 21415929

The ability to encapsulate cells in three-dimensional (3D) environments is potentially of benefit for tissue engineering and regenerative medicine. In this paper, we introduce pullulan methacrylate (PulMA) as a promising hydrogel platform for creating cell-laden microscale tissues. The hydration and mechanical properties of PulMA were demonstrated to be tunable through modulation of the degree of methacrylation and gel concentration. Cells encapsulated in PulMA exhibited excellent viability. Interestingly, while cells did not elongate in PulMA hydrogels, cells proliferated and organized into clusters, the size of which could be controlled by the hydrogel composition. By mixing with gelatin methacrylate (GelMA), the biological properties of PulMA could be enhanced as demonstrated by cells readily attaching to, proliferating, and elongating within the PulMA/GelMA composite hydrogels. These data suggest that PulMA hydrogels could be useful for creating complex, cell-responsive microtissues, especially for applications that require controlled cell clustering and proliferation.

Responsive Microgrooves for the Formation of Harvestable Tissue Constructs

Langmuir : the ACS Journal of Surfaces and Colloids. May, 2011  |  Pubmed ID: 21449596

Given its biocompatibility, elasticity, and gas permeability, poly(dimethylsiloxane) (PDMS) is widely used to fabricate microgrooves and microfluidic devices for three-dimensional (3D) cell culture studies. However, conformal coating of complex PDMS devices prepared by standard microfabrication techniques with desired chemical functionality is challenging. This study describes the conformal coating of PDMS microgrooves with poly(N-isopropylacrylamide) (PNIPAAm) by using initiated chemical vapor deposition (iCVD). These microgrooves guided the formation of tissue constructs from NIH-3T3 fibroblasts that could be retrieved by the temperature-dependent swelling property and hydrophilicity change of the PNIPAAm. The thickness of swollen PNIPAAm films at 24 °C was approximately 3 times greater than at 37 °C. Furthermore, PNIPAAm-coated microgroove surfaces exhibit increased hydrophilicity at 24 °C (contact angle θ = 30° ± 2) compared to 37 °C (θ = 50° ± 1). Thus PNIPAAm film on the microgrooves exhibits responsive swelling with higher hydrophilicity at room temperature, which could be used to retrieve tissue constructs. The resulting tissue constructs were the same size as the grooves and could be used as modules in tissue fabrication. Given its ability to form and retrieve cell aggregates and its integration with standard microfabrication, PNIPAAm-coated PDMS templates may become useful for 3D cell culture applications in tissue engineering and drug discovery.

Nanoscale Tissue Engineering: Spatial Control over Cell-materials Interactions

Nanotechnology. May, 2011  |  Pubmed ID: 21451238

Cells interact with the surrounding environment by making tens to hundreds of thousands of nanoscale interactions with extracellular signals and features. The goal of nanoscale tissue engineering is to harness these interactions through nanoscale biomaterials engineering in order to study and direct cellular behavior. Here, we review two- and three-dimensional (2- and 3D) nanoscale tissue engineering technologies, and provide a holistic overview of the field. Techniques that can control the average spacing and clustering of cell adhesion ligands are well established and have been highly successful in describing cell adhesion and migration in 2D. Extension of these engineering tools to 3D biomaterials has created many new hydrogel and nanofiber scaffold technologies that are being used to design in vitro experiments with more physiologically relevant conditions. Researchers are beginning to study complex cell functions in 3D. However, there is a need for biomaterials systems that provide fine control over the nanoscale presentation of bioactive ligands in 3D. Additionally, there is a need for 2- and 3D techniques that can control the nanoscale presentation of multiple bioactive ligands and that can control the temporal changes in the cellular microenvironment.

Drug-eluting Microarrays for Cell-based Screening of Chemical-induced Apoptosis

Analytical Chemistry. Jun, 2011  |  Pubmed ID: 21476591

Traditional high-throughput screening (HTS) is carried out in centralized facilities that require extensive robotic liquid and plate handling equipment. This model of HTS is restrictive as such facilities are not accessible to many researchers. We have designed a simple microarray platform for cell-based screening that can be carried out at the benchtop. The device creates a microarray of 2100 individual cell-based assays in a standard microscope slide format. A microarray of chemical-laden hydrogels addresses a matching array of cell-laden microwells thus creating a microarray of sealed microscale cell cultures each with unique conditions. We demonstrate the utility of the device by screening the extent of apoptosis and necrosis in MCF-7 breast cancer cells in response to exposure to a small library of chemical compounds. From a set of screens we produced a rank order of chemicals that preferentially induce apoptosis over necrosis in MCF-7 cells. Treatment with doxorubicin induced high levels of apoptosis in comparison with staurosporine, ethanol, and hydrogen peroxide, whereas treatment with 100 μM ethanol induced minimal apoptosis with high levels of necrosis. We anticipate broad application of the device for various research and discovery applications as it is easy to use, scalable, and can be fabricated and operated with minimal peripheral equipment.

A Cell-based Biosensor for Real-time Detection of Cardiotoxicity Using Lensfree Imaging

Lab on a Chip. May, 2011  |  Pubmed ID: 21483937

A portable and cost-effective real-time cardiotoxicity biosensor was developed using a CMOS imaging module extracted from a commercially available webcam. The detection system consists of a CMOS imaging module, a white LED and a pinhole. Real-time image processing was conducted by comparing reference and live frame images. To evaluate the engineered system, the effects of two different drugs, isoprenaline and doxorubicin, on the beating rate and beat-to-beat variations of ESC-derived cardiomyocytes were measured. The detection system was used to conclude that the beat-to-beat variability increased under treatment with both isoprenaline and doxorubicin. However, the beating rates increased upon the addition of isoprenaline but decreased for cultures supplemented with doxorubicin. Moreover, the response time for both the beating rates and the beat-to-beat variability of ESC-derived cardiomyocytes under treatment of isoprenaline was shorter than for doxorubicin, although the amount of isoprenaline used in the measurement was three orders of magnitude lower than that of doxorubicin. Given its ability to perform real-time cell monitoring in a simple and inexpensive manner, the proposed system may be useful for a range of cell-based biosensing applications.

Surface Functionalization of Hyaluronic Acid Hydrogels by Polyelectrolyte Multilayer Films

Biomaterials. Aug, 2011  |  Pubmed ID: 21571364

Hyaluronic acid (HA), an anionic polysaccharide, is one of the major components of the natural extracellular matrix (ECM). Although HA has been widely used for tissue engineering applications, it does not support cell attachment and spreading and needs chemical modification to support cellular adhesion. Here, we present a simple approach to functionalize photocrosslinked HA hydrogels by deposition of poly(l-lysine) (PLL) and HA multilayer films made by the layer-by-layer (LbL) technique. PLL/HA multilayer film formation was assessed by using fluorescence microscopy, contact angle measurements, cationic dye loading and confocal microscopy. The effect of polyelectrolyte multilayer film (PEM) formation on the physicochemical and mechanical properties of hydrogels revealed polyelectrolyte diffusion inside the hydrogel pores, increased hydrophobicity of the surface, reduced equilibrium swelling, and reduced compressive moduli of the modified hydrogels. Furthermore, NIH-3T3 fibroblasts seeded on the surface showed improved cell attachment and spreading on the multilayer functionalized hydrogels. Thus, modification of HA hydrogel surfaces with multilayer films affected their physicochemical properties and improved cell adhesion and spreading on these surfaces. This new hydrogel/PEM composite system may offer possibilities for various biomedical and tissue engineering applications, including growth factor delivery and co-culture systems.

Microfabricated Polyester Conical Microwells for Cell Culture Applications

Lab on a Chip. Jul, 2011  |  Pubmed ID: 21614380

Over the past few years there has been a great deal of interest in reducing experimental systems to a lab-on-a-chip scale. There has been particular interest in conducting high-throughput screening studies using microscale devices, for example in stem cell research. Microwells have emerged as the structure of choice for such tests. Most manufacturing approaches for microwell fabrication are based on photolithography, soft lithography, and etching. However, some of these approaches require extensive equipment, lengthy fabrication process, and modifications to the existing microwell patterns are costly. Here we show a convenient, fast, and low-cost method for fabricating microwells for cell culture applications by laser ablation of a polyester film coated with silicone glue. Microwell diameter was controlled by adjusting the laser power and speed, and the well depth by stacking several layers of film. By using this setup, a device containing hundreds of microwells can be fabricated in a few minutes to analyze cell behavior. Murine embryonic stem cells and human hepatoblastoma cells were seeded in polyester microwells of different sizes and showed that after 9 days in culture cell aggregates were formed without a noticeable deleterious effect of the polyester film and glue. These results show that the polyester microwell platform may be useful for cell culture applications. The ease of fabrication adds to the appeal of this device as minimal technological skill and equipment is required.

Microscale Technologies and Modular Approaches for Tissue Engineering: Moving Toward the Fabrication of Complex Functional Structures

ACS Nano. Jun, 2011  |  Pubmed ID: 21627163

Micro- and nanoscale technologies have emerged as powerful tools in the fabrication of engineered tissues and organs. Here we focus on the application of these techniques to improve engineered tissue architecture and function using modular and directed self-assembly and highlight the emergence of this new class of materials for biomedical applications.

Creation of Bony Microenvironment with CaP and Cell-derived ECM to Enhance Human Bone-marrow MSC Behavior and Delivery of BMP-2

Biomaterials. Sep, 2011  |  Pubmed ID: 21632105

Extracellular matrix (ECM) comprises a rich meshwork of proteins and proteoglycans, which not only contains biological cues for cell behavior, but is also a reservoir for binding growth factors and controlling their release. Here we aimed to create a suitable bony microenvironment with cell-derived ECM and biodegradable β-tricalcium phosphate (β-TCP). More specifically, we investigated whether the ECM produced by bone marrow-derived mesenchymal stem cells (hBMSC) on a β-TCP scaffold can bind bone morphogenetic protein-2 (BMP-2) and control its release in a sustained manner, and further examined the effect of ECM and the BMP-2 released from ECM on cell behaviors. The ECM was obtained through culturing the hBMSC on a β-TCP porous scaffold and performing decellularization and sterilization. SEM, XPS, FTIR, and immunofluorescent staining results indicated the presence of ECM on the β-TCP and the amount of ECM increased with the incubation time. BMP-2 was loaded onto the β-TCP with and without ECM by immersing the scaffolds in the BMP-2 solution. The loading and release kinetics of the BMP-2 on the β-TCP/ECM were significantly slower than those on the β-TCP. The β-TCP/ECM exhibited a sustained release profile of the BMP-2, which was also affected by the amount of ECM. This is probably because the β-TCP/ECM has different binding mechanisms with BMP-2. The β-TCP/ECM promoted cell proliferation. Furthermore, the BMP-2-loaded β-TCP/ECM stimulated reorganization of the actin cytoskeleton, increased expression of alkaline phosphatase and calcium deposition by the cells compared to those without BMP-2 loading and the β-TCP with BMP-2 loading.

Anisotropic Material Synthesis by Capillary Flow in a Fluid Stripe

Biomaterials. Sep, 2011  |  Pubmed ID: 21684595

We present a simple bench-top technique to produce centimeter long concentration gradients in biomaterials incorporating soluble, material, and particle gradients. By patterning hydrophilic regions on a substrate, a stripe of prepolymer solution is held in place on a glass slide by a hydrophobic boundary. Adding a droplet to one end of this "pre-wet" stripe causes a rapid capillary flow that spreads the droplet along the stripe to generate a gradient in the relative concentrations of the droplet and pre-wet solutions. The gradient length and shape are controlled by the pre-wet and droplet volumes, stripe thickness, fluid viscosity and surface tension. Gradient biomaterials are produced by crosslinking gradients of prepolymer solutions. Demonstrated examples include a concentration gradient of cells encapsulated in three dimensions (3D) within a homogeneous biopolymer and a constant concentration of cells encapsulated in 3D within a biomaterial gradient exhibiting a gradient in cell spreading. The technique employs coated glass slides that may be purchased or custom made from tape and hydrophobic spray. The approach is accessible to virtually any researcher or student and should dramatically reduce the time required to synthesize a wide range of gradient biomaterials. Moreover, since the technique employs passive mechanisms it is ideal for remote or resource poor settings.

Controlling the Fibroblastic Differentiation of Mesenchymal Stem Cells Via the Combination of Fibrous Scaffolds and Connective Tissue Growth Factor

Tissue Engineering. Part A. Nov, 2011  |  Pubmed ID: 21689062

Controlled differentiation of multi-potent mesenchymal stem cells (MSCs) into vocal fold-specific, fibroblast-like cells in vitro is an attractive strategy for vocal fold repair and regeneration. The goal of the current study was to define experimental parameters that can be used to control the initial fibroblastic differentiation of MSCs in vitro. To this end, connective tissue growth factor (CTGF) and micro-structured, fibrous scaffolds based on poly(glycerol sebacate) (PGS) and poly(ɛ-caprolactone) (PCL) were used to create a three-dimensional, connective tissue-like microenvironment. MSCs readily attached to and elongated along the microfibers, adopting a spindle-shaped morphology during the initial 3 days of preculture in an MSC maintenance medium. The cell-laden scaffolds were subsequently cultivated in a conditioned medium containing CTGF and ascorbic acids for up to 21 days. Cell morphology, proliferation, and differentiation were analyzed collectively by quantitative PCR analyses, and biochemical and immunocytochemical assays. F-actin staining showed that MSCs maintained their fibroblastic morphology during the 3 weeks of culture. The addition of CTGF to the constructs resulted in an enhanced cell proliferation, elevated expression of fibroblast-specific protein-1, and decreased expression of mesenchymal surface epitopes without markedly triggering chondrogenesis, osteogenesis, adipogenesis, or apoptosis. At the mRNA level, CTGF supplement resulted in a decreased expression of collagen I and tissue inhibitor of metalloproteinase 1, but an increased expression of decorin and hyaluronic acid synthesase 3. At the protein level, collagen I, collagen III, sulfated glycosaminoglycan, and elastin productivity was higher in the conditioned PGS-PCL culture than in the normal culture. These findings collectively demonstrate that the fibrous mesh, when combined with defined biochemical cues, is capable of fostering MSC fibroblastic differentiation in vitro.

EMT-inducing Biomaterials for Heart Valve Engineering: Taking Cues from Developmental Biology

Journal of Cardiovascular Translational Research. Oct, 2011  |  Pubmed ID: 21751069

Although artificial prostheses for diseased heart valves have been around for several decades, viable heart valve replacements have yet to be developed due to their complicated nature. The majority of research in heart valve replacement technology seeks to improve decellularization techniques for porcine valves or bovine pericardium as an effort to improve current clinically used valves. The drawback of clinically used valves is that they are nonviable and thus do not grow or remodel once implanted inside patients. This is particularly detrimental for pediatric patients, who will likely need several reoperations over the course of their lifetimes to implant larger valves as the patient grows. Due to this limitation, additional biomaterials, both synthetic and natural in origin, are also being investigated as novel scaffolds for tissue-engineered heart valves, specifically for the pediatric population. Here, we provide a brief overview of valves in clinical use as well as of the materials being investigated as novel tissue-engineered heart valve scaffolds. Additionally, we focus on natural-based biomaterials for promoting cell behavior that is indicative of the developmental biology process that occurs in the formation of heart valves in utero, such as epithelial-to-mesenchymal transition or transformation. By engineering materials that promote native developmental biology cues and signaling, while also providing mechanical integrity once implanted, a viable tissue-engineered heart valve may one day be realized. A viable tissue-engineered heart valve, capable of growing and remodeling actively inside a patient, could reduce risks and complications associated with current valve replacement options and improve overall quality of life in the thousands of patients who received such valves each year, particularly for children.

Responsive Micromolds for Sequential Patterning of Hydrogel Microstructures

Journal of the American Chemical Society. Aug, 2011  |  Pubmed ID: 21766872

Microscale hydrogels have been shown to be beneficial for various applications such as tissue engineering and drug delivery. A key aspect in these applications is the spatial organization of biological entities or chemical compounds within hydrogel microstructures. For this purpose, sequentially patterned microgels can be used to spatially organize either living materials to mimic biological complexity or multiple chemicals to design functional microparticles for drug delivery. Photolithographic methods are the most common way to pattern microscale hydrogels but are limited to photocrosslinkable polymers. So far, conventional micromolding approaches use static molds to fabricate structures, limiting the resulting shapes that can be generated. Herein, we describe a dynamic micromolding technique to fabricate sequentially patterned hydrogel microstructures by exploiting the thermoresponsiveness of poly(N-isopropylacrylamide)-based micromolds. These responsive micromolds exhibited shape changes under temperature variations, facilitating the sequential molding of microgels at two different temperatures. We fabricated multicompartmental striped, cylindrical, and cubic microgels that encapsulated fluorescent polymer microspheres or different cell types. These responsive micromolds can be used to immobilize living materials or chemicals into sequentially patterned hydrogel microstructures which may potentially be useful for a range of applications at the interface of chemistry, materials science and engineering, and biology.

Research Highlights. Cell Beads for Building Macroscopic Tissues

Lab on a Chip. Aug, 2011  |  Pubmed ID: 21773643

Boston Research Takes Center Stage. Interview by Pamela Reynolds

IEEE Pulse. Jul-Aug, 2011  |  Pubmed ID: 21791397

Preface to Special Topic: Microfluidics in Cell Biology and Tissue Engineering

Biomicrofluidics. Jun, 2011  |  Pubmed ID: 21799707

In this special issue of Biomicrofluidics, a wide variety of applications of microfluidics to tissue engineering and cell biology are presented. The articles illustrate the benefits of using microfluidics for controlling the cellular environment in a precise yet high rate manner using minimum reagents. The topic is very timely and takes a stab at portraying a glimpse of what is to come in this exciting and emerging field of research.

A Microfluidic-based Neurotoxin Concentration Gradient for the Generation of an in Vitro Model of Parkinson's Disease

Biomicrofluidics. Jun, 2011  |  Pubmed ID: 21799720

In this study, we developed a miniaturized microfluidic-based high-throughput cell toxicity assay to create an in vitro model of Parkinson's disease (PD). In particular, we generated concentration gradients of 6-hydroxydopamine (6-OHDA) to trigger a process of neuronal apoptosis in pheochromocytoma PC12 neuronal cell line. PC12 cells were cultured in a microfluidic channel, and a concentration gradient of 6-OHDA was generated in the channel by using a back and forth movement of the fluid flow. Cellular apoptosis was then analyzed along the channel. The results indicate that at low concentrations of 6-OHDA along the gradient (i.e., approximately less than 260 μM), the neuronal death in the channel was mainly induced by apoptosis, while at higher concentrations, 6-OHDA induced neuronal death mainly through necrosis. Thus, this concentration appears to be useful for creating an in vitro model of PD by inducing the highest level of apoptosis in PC12 cells. As microfluidic systems are advantageous in a range of properties such as throughput and lower use of reagents, they may provide a useful approach for generating in vitro models of disease for drug discovery applications.

SAM-based Cell Transfer to Photopatterned Hydrogels for Microengineering Vascular-like Structures

Biomaterials. Oct, 2011  |  Pubmed ID: 21802723

A major challenge in tissue engineering is to reproduce the native 3D microvascular architecture fundamental for in vivo functions. Current approaches still lack a network of perfusable vessels with native 3D structural organization. Here we present a new method combining self-assembled monolayer (SAM)-based cell transfer and gelatin methacrylate hydrogel photopatterning techniques for microengineering vascular structures. Human umbilical vein cell (HUVEC) transfer from oligopeptide SAM-coated surfaces to the hydrogel revealed two SAM desorption mechanisms: photoinduced and electrochemically triggered. The former, occurs concomitantly to hydrogel photocrosslinking, and resulted in efficient (>97%) monolayer transfer. The latter, prompted by additional potential application, preserved cell morphology and maintained high transfer efficiency of VE-cadherin positive monolayers over longer culture periods. This approach was also applied to transfer HUVECs to 3D geometrically defined vascular-like structures in hydrogels, which were then maintained in perfusion culture for 15 days. As a step toward more complex constructs, a cell-laden hydrogel layer was photopatterned around the endothelialized channel to mimic the vascular smooth muscle structure of distal arterioles. This study shows that the coupling of the SAM-based cell transfer and hydrogel photocrosslinking could potentially open up new avenues in engineering more complex, vascularized tissue constructs for regenerative medicine and tissue engineering applications.

An Integrated Microfluidic Device for Two-dimensional Combinatorial Dilution

Lab on a Chip. Oct, 2011  |  Pubmed ID: 21837312

High-throughput preparation of multi-component solutions is an integral process in biology, chemistry and materials science for screening, diagnostics and analysis. Compact microfluidic systems enable such processing with low reagent volumes and rapid testing. Here we present a microfluidic device that incorporates two gradient generators, a tree-like generator and a new microfluidic active injection system, interfaced by intermediate solution reservoirs to generate diluted combinations of input solutions within an 8 × 8 or 10 × 10 array of isolated test chambers. Three input solutions were fed into the device, two to the tree-like gradient generator and one to pre-fill the test chamber array. The relative concentrations of these three input solutions in the test chambers completely characterized device behaviour and were controlled by the number of injection cycles and the flow rate. Device behaviour was modelled by computational fluid dynamics simulations and an approximate analytic formula. The device may be used for two-dimensional (2D) combinatorial dilution by adding two solutions in different relative concentrations to each of its three inputs. By appropriate choice of the two-component input solutions, test chamber concentrations that span any triangle in 2D concentration space may be obtained. In particular, explicit inputs are given for a coarse screening of a large region in concentration space followed by a more refined screening of a smaller region, including alternate inputs that span the same concentration region but with different distributions. The ability to probe arbitrary subspaces of concentration space and to control the distribution of discrete test points within those subspaces makes the device of potential benefit for high-throughput cell biology studies and drug screening.

Engineering of Pathways, Cells and Tissues

Current Opinion in Biotechnology. Oct, 2011  |  Pubmed ID: 21862311

Controlling the Porosity of Fibrous Scaffolds by Modulating the Fiber Diameter and Packing Density

Journal of Biomedical Materials Research. Part A. Jan, 2011  |  Pubmed ID: 21225711

Porosity has been shown to be a key determinant of the success of tissue engineered scaffolds. A high degree of porosity and an appropriate pore size are necessary to provide adequate space for cell spreading and migration as well as to allow for proper exchange of nutrients and waste between the scaffold and the surrounding environment. Electrospun scaffolds offer an attractive approach for mimicking the natural extracellular matrix (ECM) for tissue engineering applications. The efficacy of electrospinning is likely to depend on the interaction between cells and the geometric features and physicochemical composition of the scaffold. A major problem in electrospinning is the tendency of fibers to accumulate densely, resulting in poor porosity and small pore size. The porosity and pore sizes in the electrospun scaffolds are mainly dependent on the fiber diameter and their packing density. Here we report a method of modulating porosity in three dimensional (3D) scaffolds by simultaneously tuning the fiber diameter and the fiber packing density. Nonwoven poly(ε-caprolactone) mats were formed by electrospinning under various conditions to generate sparse or highly dense micro- and nanofibrous scaffolds and characterized for their physicochemical and biological properties. We found that microfibers with low packing density resulted in improved cell viability, proliferation and infiltration compared to tightly packed scaffolds. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A: , 2011.

Gradient Biomaterials for Soft-to-hard Interface Tissue Engineering

Acta Biomaterialia. Apr, 2011  |  Pubmed ID: 21232635

Interface tissue engineering (ITE) is a rapidly developing field that aims to fabricate biological tissue alternates with the goal of repairing or regenerating the functions of diseased or damaged zones at the interface of different tissue types (also called "interface tissues"). Notable examples of the interface tissues in the human body include ligament-to-bone, tendon-to-bone and cartilage-to-bone. Engineering interface tissues is a complex process, which requires a combination of specialized biomaterials with spatially organized material composition, cell types and signaling molecules. Therefore, the use of conventional biomaterials (monophasic or composites) for ITE has certain limitations to help stimulate the tissue integration or recreating the structural organization at the junction of different tissue types. The advancement of micro- and nanotechnologies enable us to develop systems with gradients in biomaterials properties that encourage the differentiation of multiple cell phenotypes and subsequent tissue development. In this review we discuss recent developments in the fabrication of gradient biomaterials for controlling cellular behavior such as migration, differentiation and heterotypic interactions. Moreover, we give an overview of potential uses of gradient biomaterials in engineering interface tissues such as soft tissues (e.g. cartilage) to hard tissues (e.g. bone), with illustrated experimental examples. We also address fundamentals of interface tissue organization, various gradient biomaterials used in ITE, micro- and nanotechnologies employed for the fabrication of those gradients, and certain challenges that must be met in order for ITE to reach its full potential.

Controlling the Porosity of Fibrous Scaffolds by Modulating the Fiber Diameter and Packing Density

Journal of Biomedical Materials Research. Part A. Mar, 2011  |  Pubmed ID: 21254388

Porosity has been shown to be a key determinant of the success of tissue engineered scaffolds. A high degree of porosity and an appropriate pore size are necessary to provide adequate space for cell spreading and migration as well as to allow for proper exchange of nutrients and waste between the scaffold and the surrounding environment. Electrospun scaffolds offer an attractive approach for mimicking the natural extracellular matrix (ECM) for tissue engineering applications. The efficacy of electrospinning is likely to depend on the interaction between cells and the geometric features and physicochemical composition of the scaffold. A major problem in electrospinning is the tendency of fibers to accumulate densely, resulting in poor porosity and small pore size. The porosity and pore sizes in the electrospun scaffolds are mainly dependent on the fiber diameter and their packing density. Here we report a method of modulating porosity in three dimensional (3D) scaffolds by simultaneously tuning the fiber diameter and the fiber packing density. Nonwoven poly(ε-caprolactone) mats were formed by electrospinning under various conditions to generate sparse or highly dense micro- and nanofibrous scaffolds and characterized for their physicochemical and biological properties. We found that microfibers with low packing density resulted in improved cell viability, proliferation and infiltration compared to tightly packed scaffolds.

Synthesis and Characterization of Photocrosslinkable Gelatin and Silk Fibroin Interpenetrating Polymer Network Hydrogels

Acta Biomaterialia. Jun, 2011  |  Pubmed ID: 21295165

To effectively repair or replace damaged tissues, it is necessary to design scaffolds with tunable structural and biomechanical properties that closely mimic the host tissue. In this paper, we describe a newly synthesized photocrosslinkable interpenetrating polymer network (IPN) hydrogel based on gelatin methacrylate (GelMA) and silk fibroin (SF) formed by sequential polymerization, which possesses tunable structural and biological properties. Experimental results revealed that IPNs, where both the GelMA and SF were independently crosslinked in interpenetrating networks, demonstrated a lower swelling ratio, higher compressive modulus and lower degradation rate as compared to the GelMA and semi-IPN hydrogels, where only GelMA was crosslinked. These differences were likely caused by a higher degree of overall crosslinking due to the presence of crystallized SF in the IPN hydrogels. NIH-3T3 fibroblasts readily attached to, spread and proliferated on the surface of IPN hydrogels, as demonstrated by F-actin staining and analysis of mitochondrial activity (MTT). In addition, photolithography combined with lyophilization techniques was used to fabricate three-dimensional micropatterned and porous microscaffolds from GelMA-SF IPN hydrogels, furthering their versatility for use in various microscale tissue engineering applications. Overall, this study introduces a class of photocrosslinkable, mechanically robust and tunable IPN hydrogels that could be useful for various tissue engineering and regenerative medicine applications.

Deep Wells Integrated with Microfluidic Valves for Stable Docking and Storage of Cells

Biotechnology Journal. Feb, 2011  |  Pubmed ID: 21298801

In this paper, we describe a microfluidic mechanism that combines microfluidic valves and deep wells for cell localization and storage. Cells are first introduced into the device via externally controlled flow. Activating on-chip valves was used to interrupt the flow and to sediment the cells floating above the wells. Thus, valves could be used to localize the cells in the desired locations. We quantified the effect of valves in the cell storage process by comparing the total number of cells stored with and without valve activation. We hypothesized that in deep wells external flows generate low shear stress regions that enable stable, long-term docking of cells. To assess this hypothesis we conducted numerical calculations to understand the influence of well depth on the forces acting on cells. We verified those predictions experimentally by comparing the fraction of stored cells as a function of the well depth and input flow rate upon activation of the valves. As expected, upon reintroduction of the flow the cells in the deep wells were not moved whereas those in shallow wells were washed away. Taken together, our paper demonstrates that deep wells and valves can be combined to enable a broad range of cell studies.

Synthesis and Characterization of Tunable Poly(ethylene Glycol): Gelatin Methacrylate Composite Hydrogels

Tissue Engineering. Part A. Jul, 2011  |  Pubmed ID: 21306293

Poly(ethylene glycol) (PEG) hydrogels are popular for cell culture and tissue-engineering applications because they are nontoxic and exhibit favorable hydration and nutrient transport properties. However, cells cannot adhere to, remodel, proliferate within, or degrade PEG hydrogels. Methacrylated gelatin (GelMA), derived from denatured collagen, yields an enzymatically degradable, photocrosslinkable hydrogel that cells can degrade, adhere to and spread within. To combine the desirable features of each of these materials we synthesized PEG-GelMA composite hydrogels, hypothesizing that copolymerization would enable adjustable cell binding, mechanical, and degradation properties. The addition of GelMA to PEG resulted in a composite hydrogel that exhibited tunable mechanical and biological profiles. Adding GelMA (5%-15% w/v) to PEG (5% and 10% w/v) proportionally increased fibroblast surface binding and spreading as compared to PEG hydrogels (p<0.05). Encapsulated fibroblasts were also able to form 3D cellular networks 7 days after photoencapsulation only within composite hydrogels as compared to PEG alone. Additionally, PEG-GelMA hydrogels displayed tunable enzymatic degradation and stiffness profiles. PEG-GelMA composite hydrogels show great promise as tunable, cell-responsive hydrogels for 3D cell culture and regenerative medicine applications.

Directed Assembly of Cell-laden Microgels for Building Porous Three-dimensional Tissue Constructs

Journal of Biomedical Materials Research. Part A. Feb, 2011  |  Pubmed ID: 21319297

The organization of cells within a well-defined microenvironment is important in generating the resulting tissue function. However, the cellular organization within biodegradable scaffolds often does not resemble those of native tissues. In this study, we present directed assembly of microgels to organize cells for building porous 3D tissue constructs. Cell-laden microgels were generated by molding photocrosslinkable polyethylene glycol diacrylate within a poly(dimethyl siloxane) stencil. The resulting microgels were subsequently packed as individual layers (1 mm in height) on a glass substrate by removing the excess prepolymer solution around the microgels. These clusters were crosslinked and stacked on one another to fabricate thick 3D constructs that were greater than 1 cm in width and 3 mm in thickness. To generate pores within the engineered structures, sodium alginate microgels were integrated in the engineered constructs and used as a sacrificial template. These pores may be potentially useful for fabricating a vascular network to supply oxygen and nutrients to the engineered tissue constructs. This simple and versatile building approach may be a useful tool for various 3D tissue culture and engineering applications. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2011.

Sequential Assembly of Cell-laden Hydrogel Constructs to Engineer Vascular-like Microchannels

Biotechnology and Bioengineering. Jul, 2011  |  Pubmed ID: 21337336

Microscale technologies, such as microfluidic systems, provide powerful tools for building biomimetic vascular-like structures for tissue engineering or in vitro tissue models. Recently, modular approaches have emerged as attractive approaches in tissue engineering to achieve precisely controlled architectures by using microengineered components. Here, we sequentially assembled microengineered hydrogels (microgels) into hydrogel constructs with an embedded network of microchannels. Arrays of microgels with predefined internal microchannels were fabricated by photolithography and assembled into 3D tubular construct with multi-level interconnected lumens. In the current setting, the sequential assembly of microgels occurred in a biphasic reactor and was initiated by swiping a needle to generate physical forces and fluidic shear. We optimized the conditions for assembly and successfully perfused fluids through the interconnected constructs. The sequential assembly process does not significantly influence cell viability within the microgels indicating its promise as a biofabrication method. Finally, in an attempt to build a biomimetic 3D vasculature, we incorporated endothelial cells and smooth muscle cells into an assembled construct with a concentric microgel design. The sequential assembly is simple, rapid, cost-effective, and could be used for fabricating tissue constructs with biomimetic vasculature and other complex architectures.

Microfluidic Synthesis of Composite Cross-gradient Materials for Investigating Cell-biomaterial Interactions

Biotechnology and Bioengineering. Jan, 2011  |  Pubmed ID: 20721897

Combinatorial material synthesis is a powerful approach for creating composite material libraries for the high-throughput screening of cell-material interactions. Although current combinatorial screening platforms have been tremendously successful in identifying target (termed "hit") materials from composite material libraries, new material synthesis approaches are needed to further optimize the concentrations and blending ratios of the component materials. Here we employed a microfluidic platform to rapidly synthesize composite materials containing cross-gradients of gelatin and chitosan for investigating cell-biomaterial interactions. The microfluidic synthesis of the cross-gradient was optimized experimentally and theoretically to produce quantitatively controllable variations in the concentrations and blending ratios of the two components. The anisotropic chemical compositions of the gelatin/chitosan cross-gradients were characterized by Fourier transform infrared spectrometry and X-ray photoelectron spectrometry. The three-dimensional (3D) porous gelatin/chitosan cross-gradient materials were shown to regulate the cellular morphology and proliferation of smooth muscle cells (SMCs) in a gradient-dependent manner. We envision that our microfluidic cross-gradient platform may accelerate the material development processes involved in a wide range of biomedical applications.

A Sandwiched Microarray Platform for Benchtop Cell-based High Throughput Screening

Biomaterials. Jan, 2011  |  Pubmed ID: 20965560

The emergence of combinatorial chemistries and the increased discovery of natural compounds have led to the production of expansive libraries of drug candidates and vast numbers of compounds with potentially interesting biological activities. Despite broad interest in high throughput screening (HTS) across varied fields of biological research, there has not been an increase in accessible HTS technologies. Here, we present a simple microarray sandwich system suitable for screening chemical libraries in cell-based assays at the benchtop. The microarray platform delivers chemical compounds to isolated cell cultures by 'sandwiching' chemical-laden arrayed posts with cell-seeded microwells. In this way, an array of sealed cell-based assays was generated without cross-contamination between neighbouring assays. After chemical exposure, cell viability was analyzed by fluorescence detection of cell viability assays on a per microwell basis using a standard microarray scanner. We demonstrate the efficacy of the system by generating four hits from toxicology screens towards MCF-7 human breast cancer cells. Three of the hits were identified in a combinatorial screen of a library of natural compounds in combination with verapamil, a P-glycoprotein inhibitor. A fourth hit, 9-methoxy-camptothecin, was identified by screening the natural compound library in the absence of verapamil. The method developed here miniaturizes existing HTS systems and enables the screening of a wide array of individual or combinatorial libraries in a reproducible and scalable manner. We anticipate broad application of such a system as it is amenable to combinatorial drug screening in a simple, robust and portable platform.

Methods for Embryoid Body Formation: the Microwell Approach

Methods in Molecular Biology (Clifton, N.J.). 2011  |  Pubmed ID: 21042991

Embyroid body (EB) formation is a key step in many embryonic stem cell (ESC) differentiation protocols. The EB mimics the structure of the developing embryo, thereby providing a means of obtaining any cell lineage. Traditionally, the two methods of EB formation are suspension and hanging drop. The suspension method allows ESCs to self-aggregate into EBs in a nonadherent dish. The hanging drop method suspends ESCs on the lid of a dish and EBs form through aggregation at the bottom of the drops. Recently, alternative methods of EB formation have been developed that allow for highly accurate control of EB size and shape, resulting in reproducibly produced homogeneous EBs. This control is potentially useful for directed differentiation, as recent studies have shown that EB size may be a useful determinant of the resulting differentiated cell types. One particular approach to generate homogeneous EBs utilizes nonadhesive microwell structures. The methodology associated with this technique, along with the traditional approaches of suspension and hanging drop, is the focus of this chapter.

The Mechanical Properties and Cytotoxicity of Cell-laden Double-network Hydrogels Based on Photocrosslinkable Gelatin and Gellan Gum Biomacromolecules

Biomaterials. Apr, 2012  |  Pubmed ID: 22265786

A major goal in the application of hydrogels for tissue engineering scaffolds, especially for load-bearing tissues such as cartilage, is to develop hydrogels with high mechanical strength. In this study, a double-network (DN) strategy was used to engineer strong hydrogels that can encapsulate cells. We improved upon previously studied double-network (DN) hydrogels by using a processing condition compatible with cell survival. The DN hydrogels were created by a two-step photocrosslinking using gellan gum methacrylate (GGMA) for the rigid and brittle first network, and gelatin methacrylamide (GelMA) for the soft and ductile second network. We controlled the degree of methacrylation of each polymer so that they obtain relevant mechanical properties as each network. The DN was formed by photocrosslinking the GGMA, diffusing GelMA into the first network, and photocrosslinking the GelMA to form the second network. The formation of the DN was examined by diffusion tests of the large GelMA molecules into the GGMA network, the resulting enhancement in the mechanical properties, and the difference in mechanical properties between GGMA/GelMA single networks (SN) and DNs. The resulting DN hydrogels exhibited the compressive failure stress of up to 6.9 MPa, which approaches the strength of cartilage. It was found that there is an optimal range of the crosslink density of the second network for high strength of DN hydrogels. DN hydrogels with a higher mass ratio of GelMA to GGMA exhibited higher strength, which shows promise in developing even stronger DN hydrogels in the future. Three dimensional (3D) encapsulation of NIH-3T3 fibroblasts and the following viability test showed the cell-compatibility of the DN formation process. Given the high strength and the ability to encapsulate cells, the DN hydrogels made from photocrosslinkable macromolecules could be useful for the regeneration of load-bearing tissues.

Research Highlights

Lab on a Chip. Feb, 2012  |  Pubmed ID: 22298296

Research Highlights

Lab on a Chip. Feb, 2012  |  Pubmed ID: 22334379

Multi-gradient Hydrogels Produced Layer by Layer with Capillary Flow and Crosslinking in Open Microchannels

Lab on a Chip. Feb, 2012  |  Pubmed ID: 22167009

This technical note describes a new bench-top method for producing anisotropic hydrogels composed of gradient layers of soluble factors, particles, polymer concentrations or material properties. Each gradient layer was produced by a previous gradient method in which a droplet of one precursor solution was added to a thin layer of a second solution. The ensuing rapid capillary flow along the open channel generated a gradient precursor solution, which was then crosslinked to form a gradient gel. Repeating these steps allowed a layered gel to be iteratively constructed with as many gradient layers as desired. This technique renders the synthesis of multi-layered gradient gels accessible to virtually any researcher and should help simplify the production of more biologically relevant cellular microenvironments.

Controlled Release of Drugs from Gradient Hydrogels for High-throughput Analysis of Cell-drug Interactions

Analytical Chemistry. Feb, 2012  |  Pubmed ID: 22220576

In this paper, we report a method to fabricate microengineered hydrogels that contain a concentration gradient of a drug for high-throughput analysis of cell-drug interactions. A microfluidic gradient generator was used to create a concentration gradient of okadaic acid (OA) as a model drug within poly(ethylene glycol) diacrylate hydrogels. These hydrogels were then incubated with MC3T3-E1 cell seeded glass slides to investigate the cell viability through the spatially controlled release of OA. The drug was released from the hydrogel in a gradient manner and induced a gradient of the cell viability. The drug concentration gradient containing hydrogels developed in this study have the potential to be used for drug discovery and diagnostics applications due to their ability to simultaneously test the effects of different concentrations of various chemicals.

Hydrogel Surfaces to Promote Attachment and Spreading of Endothelial Progenitor Cells

Journal of Tissue Engineering and Regenerative Medicine. Jan, 2012  |  Pubmed ID: 22223475

Endothelialization of artificial vascular grafts is a challenging process in cardiovascular tissue engineering. Functionalized biomaterials could be promising candidates to promote endothelialization in repair of cardiovascular injuries. The purpose of this study was to synthesize hyaluronic acid (HA) and heparin-based hydrogels that could promote adhesion and spreading of endothelial progenitor cells (EPCs). We report that the addition of heparin into HA-based hydrogels provides an attractive surface for EPCs promoting spreading and the formation of an endothelial monolayer on the hydrogel surface. To increase EPC adhesion and spreading, we covalently immobilized CD34 antibody (Ab) on HA-heparin hydrogels, using standard EDC/NHS amine-coupling strategies. We found that EPC adhesion and spreading on CD34 Ab-immobilized HA-heparin hydrogels was significantly higher than their non-modified analogues. Once adhered, EPCs spread and formed an endothelial layer on both non-modified and CD34 Ab-modified HA-heparin hydrogels after 3 days of culture. We did not observe significant adhesion and spreading when heparin was not included in the control hydrogels. In addition to EPCs, we also used human umbilical cord vein endothelial cells (HUVECs), which adhered and spread on HA-heparin hydrogels. Macrophages exhibited significantly less adhesion compared to EPCs on the same hydrogels. This composite material could possibly be used to develop surface coatings for artificial cardiovascular implants, due to its specificity for EPC and endothelial cells on an otherwise non-thrombogenic surface. Copyright © 2012 John Wiley & Sons, Ltd.

Osteoblastic/Cementoblastic and Neural Differentiation of Dental Stem Cells and Their Applications to Tissue Engineering and Regenerative Medicine

Tissue Engineering. Part B, Reviews. Jan, 2012  |  Pubmed ID: 22224548

Recently, dental stem and progenitor cells have been harvested from periodontal tissues such as dental pulp, periodontal ligament, follicle and papilla. These cells have received extensive attention in the field of tissue engineering and regenerative medicine due to their accessibility and multilineage differentiation capacity. These dental stem and progenitor cells are known to be derived from ectomesenchymal origin formed during tooth development. A great deal of research has been accomplished for directing osteoblastic/cementoblastic differentiation and neural differentiation from dental stem cells. To differentiate dental stem cells for use in tissue engineering and regenerative medicine, there needs to be efficient in vitro differentiation towards the osteoblastic/cementoblastic and neural lineage with well-defined and proficient protocols. This would reduce the likelihood of spontaneous differentiation into divergent lineages and increase the available cell source. This review focuses on the multilineage differentiation capacity, especially into osteoblastic/cementoblastic lineage and neural lineages, of dental stem cells such as dental pulp stem cells (DPSC), dental follicle stem cells (DFSC), periodontal ligament stem cells (PDLSC), and dental papilla stem cells (DPPSC). It also covers various experimental strategies that could be used to direct lineage specific differentiation, and their potential applications in tissue engineering and regenerative medicine.

Research Highlights

Lab on a Chip. Feb, 2012  |  Pubmed ID: 22249860

Designer Hydrophilic Regions Regulate Droplet Shape for Controlled Surface Patterning and 3D Microgel Synthesis

Small (Weinheim an Der Bergstrasse, Germany). Feb, 2012  |  Pubmed ID: 22162397

A simple technique is presented for controlling the shapes of micro- and nanodrops by patterning surfaces with special hydrophilic regions surrounded by hydrophobic boundaries. Finite element method simulations link the shape of the hydrophilic regions to that of the droplets. Shaped droplets are used to controllably pattern planar surfaces and microwell arrays with microparticles and cells at the micro- and macroscales. Droplets containing suspended sedimenting particles, initially at uniform concentration, deposit more particles under deeper regions than under shallow regions. The resulting surface concentration is thus proportional to the local fluid depth and agrees well with the measured and simulated droplet profiles. A second application is also highlighted in which shaped droplets of prepolymer solution are crosslinked to synthesize microgels with tailored 3D geometry.

Microfluidic Fabrication of Microengineered Hydrogels and Their Application in Tissue Engineering

Lab on a Chip. Jan, 2012  |  Pubmed ID: 22105780

Microfluidic technologies are emerging as an enabling tool for various applications in tissue engineering and cell biology. One emerging use of microfluidic systems is the generation of shape-controlled hydrogels (i.e., microfibers, microparticles, and hydrogel building blocks) for various biological applications. Furthermore, the microfluidic fabrication of cell-laden hydrogels is of great benefit for creating artificial scaffolds. In this paper, we review the current development of microfluidic-based fabrication techniques for the creation of fibers, particles, and cell-laden hydrogels. We also highlight their emerging applications in tissue engineering and regenerative medicine.

Carbon Nanotube Reinforced Hybrid Microgels As Scaffold Materials for Cell Encapsulation

ACS Nano. Jan, 2012  |  Pubmed ID: 22117858

Hydrogels that mimic biological extracellular matrix (ECM) can provide cells with mechanical support and signaling cues to regulate their behavior. However, despite the ability of hydrogels to generate artificial ECM that can modulate cellular behavior, they often lack the mechanical strength needed for many tissue constructs. Here, we present reinforced CNT-gelatin methacrylate (GelMA) hybrid as a biocompatible, cell-responsive hydrogel platform for creating cell-laden three-dimensional (3D) constructs. The addition of carbon nanotubes (CNTs) successfully reinforced GelMA hydrogels without decreasing their porosity or inhibiting cell growth. The CNT-GelMA hybrids were also photopatternable allowing for easy fabrication of microscale structures without harsh processes. NIH-3T3 cells and human mesenchymal stem cells (hMSCs) readily spread and proliferated after encapsulation in CNT-GelMA hybrid microgels. By controlling the amount of CNTs incorporated into the GelMA hydrogel system, we demonstrated that the mechanical properties of the hybrid material can be tuned making it suitable for various tissue engineering applications. Furthermore, due to the high pattern fidelity and resolution of CNT incorporated GelMA, it can be used for in vitro cell studies or fabricating complex 3D biomimetic tissue-like structures.

Biomimetic Tissues on a Chip for Drug Discovery

Drug Discovery Today. Feb, 2012  |  Pubmed ID: 22094245

Developing biologically relevant models of human tissues and organs is an important enabling step for disease modeling and drug discovery. Recent advances in tissue engineering, biomaterials and microfluidics have led to the development of microscale functional units of such models also referred to as 'organs on a chip'. In this review, we provide an overview of key enabling technologies and highlight the wealth of recent work regarding on-chip tissue models. In addition, we discuss the current challenges and future directions of organ-on-chip development.