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In this study, a new 3D liver model was developed using biomimetic nanofiber scaffolds and co-culture system consisting of hepatocytes and fibroblasts for the maintenance of long-term liver functions. The chitosan nanofiber scaffolds were fabricated by the electrospinning technique. To enhance cellular adhesion and spreading, the surfaces of the chitosan scaffolds were coated with fibronectin (FN) by adsorption and evaluated for various cell types. Cellular phenotype, protein expression, and liver-specific functions were extensively characterized by immunofluorescent and histochemical stainings, albumin enzyme-linked immunosorbent assay and Cytochrome p450 detoxification assays, and scanning electron microscopy. The electrospun chitosan scaffolds exhibited a highly porous and randomly oriented nanofibrous structure. The FN coating on the surface of the chitosan nanofibers significantly enhanced cell attachment and spreading, as expected, as surface modification with this cell adhesion molecule on the chitosan surface is important for focal adhesion formation and integrin binding. Comparison of hepatocyte mono-cultures and co-cultures in 3D culture systems indicated that the hepatocytes in co-cultures formed colonies and maintained their morphologies and functions for prolonged periods of time. The 3D liver tissue model developed in this study will provide useful tools toward the development of engineered liver tissues for drug screening and tissue engineering applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2119-2128, 2017.

Tissue engineering using stem cells is widely used to repair damaged tissues in diverse biological systems; however, this approach has met with less success in regenerating the central nervous system (CNS). In this study we optimized and characterized the surface chemistry of chitosan-based scaffolds for CNS repair. To maintain radial glial cell (RGC) character of primitive neural precursors, fibronectin was adsorbed to chitosan. The chitosan was further modified by covalently linking heparin using genipin, which then served as a linker to immobilize fibroblast growth factor-2 (FGF-2), creating a multifunctional film. Fetal rat neural precursors plated onto this multifunctional film proliferated and remained multipotent for at least 3 days without providing soluble FGF-2. Moreover, they remained less mature and more highly proliferative than cells maintained on fibronectin-coated substrates in culture medium supplemented with soluble FGF-2. To create a vehicle for cell transplantation, a 3% chitosan solution was electrosprayed into a coagulation bath to generate microspheres (range 30-100 µm, mean 64 µm) that were subsequently modified. Radial glial cells seeded onto these multifunctional microspheres proliferated for at least 7 days in culture and the microspheres containing cells were small enough to be injected, using 23 Gauge Hamilton syringes, into the brains of adult rats that had previously sustained cortical contusion injuries. When analysed 3 days later, the transplanted RGCs were positive for the stem cell/progenitor marker Nestin. These results demonstrate that this multifunctional scaffold can be used as a cellular and growth factor delivery vehicle for the use in developing cell transplantation therapies for traumatic brain injuries. Copyright © 2013 John Wiley & Sons, Ltd.

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

Substrate and cell patterning are widely used techniques in cell biology to study cell-to-cell and cell-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This paper describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. Our method builds upon a previous vacuum-assisted method used for micromolding (Jeon et al 1999 Adv. Mater 11 946) and successfully patterned collagen-I, fibronectin and Sal-1 substrates on glass and polystyrene surfaces, filling microchannels with lengths up to 120 mm and covering areas up to 13 × 10 mm(2). Vacuum-patterned substrates were subsequently used to culture mammalian PC12 and fibroblast cells and amphibian neurons. Cells were also patterned directly by injecting cell suspensions into microchannels using vacuum. Fibroblast and neuronal cells patterned using vacuum showed normal growth and minimal cell death indicating no adverse effects of vacuum on cells. Our method fills reversibly sealed PDMS microchannels. This enables the user to remove the PDMS microchannel cast and access the patterned biomaterial or cells for further experimental purposes. Overall, this is a straightforward technique that has broad applicability for cell biology.

Two-dimensional (2D) monolayer cultures are the standard in vitro model for cancer research. However, they fail to recapitulate the three-dimensional (3D) environment and quickly lose their function. In this study, we developed a new 3D multicellular heterospheroid tumor model in a collagen hydrogel culture system that more closely mimics the in vivo tumor microenvironment for anti-cancer drug testing. Three aspects of cancer were chosen to be modeled based on their ability to resist anti-cancer drugs: 3D, multicellularity, and extracellular matrix (ECM) barrier. The hanging drop method and co-culture of liver carcinoma with stromal fibroblasts were used to form controlled and uniform heterospheroids. These heterospheroids were then encapsulated in collagen gel in order to create a 3D model of liver cancer that would act more similarly to in vivo ECM conditions. The 3D heterospheroid tumor model was tested with an anti-cancer drug to determine how each of the above aspects affects drug resistance. The results demonstrate that the 3D heterospheroid model is more resistant to drug over 2D monolayer and homospheroid cultures, indicating stromal fibroblasts and collagen hydrogel culture system provides more resistance to anti-cancer drug. This study will provide useful information toward the development of improved biomimetic tumor models in vitro for cancer research in pre-clinical drug development.

An effective paradigm for transplanting large numbers of neural stem cells after central nervous system (CNS) injury has yet to be established. Biomaterial scaffolds have shown promise in cell transplantation and in regenerative medicine, but improved scaffolds are needed. In this study we designed and optimized multifunctional and biocompatible chitosan-based films and microspheres for the delivery of neural stem cells and growth factors for CNS injuries. The chitosan microspheres were fabricated by coaxial airflow techniques, with the sphere size controlled by varying the syringe needle gauge and the airflow rate. When applying a coaxial airflow at 30 standard cubic feet per hour, ∼300μm diameter spheres were reproducibly generated that were physically stable yet susceptible to enzymatic degradation. Heparin was covalently crosslinked to the chitosan scaffolds using genipin, which bound fibroblast growth factor-2 (FGF-2) with high affinity while retaining its biological activity. At 1μgml(-1) approximately 80% of the FGF-2 bound to the scaffold. A neural stem cell line, GFP+RG3.6 derived from embryonic rat cortex, was used to evaluate cytocompatibility, attachment and survival on the crosslinked chitosan-heparin complex surfaces. The MTT assay and microscopic analysis revealed that the scaffold containing tethered FGF-2 was superior in sustaining survival and growth of neural stem cells compared to standard culture conditions. Altogether, our results demonstrate that this multifunctional scaffold possesses good cytocompatibility and can be used as a growth factor delivery vehicle while supporting neural stem cell attachment and survival.

The in vitro generation of a three-dimensional (3-D) myocardial tissue-like construct employing cells, biomaterials, and biomolecules is a promising strategy in cardiac tissue regeneration, drug testing, and tissue engineering applications. Despite significant progress in this field, current cardiac tissue models are not yet able to stably maintain functional characteristics of cardiomyocytes for long-term culture and therapeutic purposes. The objective of this study was to fabricate bioactive 3-D chitosan nanofiber scaffolds using an electrospinning technique and exploring its potential for long-term cardiac function in the 3-D co-culture model. Chitosan is a natural polysaccharide biomaterial that is biocompatible, biodegradable, non-toxic, and cost effective. Electrospun chitosan was utilized to provide structural scaffolding characterized by scale and architectural resemblance to the extracellular matrix (ECM) in vivo. The chitosan fibers were coated with fibronectin via adsorption in order to enhance cellular adhesion to the fibers and migration into the interfibrous milieu. Ventricular cardiomyocytes were harvested from neonatal rats and studied in various culture conditions (i.e., mono- and co-cultures) for their viability and function. Cellular morphology and functionality were examined using immunofluorescent staining for alpha-sarcomeric actin (SM-actin) and gap junction protein, Connexin-43 (Cx43). Scanning electron microscopy (SEM) and light microscopy were used to investigate cellular morphology, spatial organization, and contractions. Calcium indicator was used to monitor calcium ion flux of beating cardiomyocytes. The results demonstrate that the chitosan nanofibers retained their cylindrical morphology in long-term cell cultures and exhibited good cellular attachment and spreading in the presence of adhesion molecule, fibronectin. Cardiomyocyte mono-cultures resulted in loss of cardiomyocyte polarity and islands of non-coherent contractions. However, the cardiomyocyte-fibroblast co-cultures resulted in polarized cardiomyocyte morphology and retained their morphology and function for long-term culture. The Cx43 expression in the fibroblast co-culture was higher than the cardiomyocytes mono-culture and endothelial cells co-culture. In addition, fibroblast co-cultures demonstrated synchronized contractions involving large tissue-like cellular networks. To our knowledge, this is the first attempt to test chitosan nanofiber scaffolds as a 3-D cardiac co-culture model. Our results demonstrate that chitosan nanofibers can serve as a potential scaffold that can retain cardiac structure and function. These studies will provide useful information to develop a strategy that allows us to generate engineered 3-D cardiac tissue constructs using biocompatible and biodegradable chitosan nanofiber scaffolds for many tissue engineering applications.

The purpose of this study is to develop biodegradable nerve guidance conduits seeded with Schwann cells (SC) using chitosan-based biopolymers for improving nerve regeneration. Chitosan, prepared from alkaline hydrolysis of chitin, is a natural polysaccharide biopolymer, having biocompatibility and biodegradability. In this study, chitosan and chitosan-gelatin membranes were prepared, characterized, and evaluated. The SC isolated from rat sciatic nerve was seeded on chitosan and chitosan-gelatin membranes to investigate their interactions. To enhance the SC attachment and growth on the membranes, we incorporated the poly-L-lysine (PLL) into chitosan and chitosan-gelatin network at various ratios of 1:800, 1:200, 1:50 (chitosan or chitosan-gelatin: PLL). We have also investigated the effect of extracellular matrix (ECM) molecules on the proliferation of SC. We observed that SC proliferation on PLL-coated surface was significantly higher than that of ECM coated and uncoated surfaces. With the increase of PLL ratio into chitosan or chitosan-gelatin network, the attachment and growth of SCs were significantly enhanced. This study indicates that the incorporation of PLL into chitosan and chitosan-gelatin membranes significantly improves the bioactivity of the materials. The Schwann cell-seeded films can be used to construct a three-dimensional nerve conduit for enhancing nerve regeneration.

The objective of this study was to fabricate 3-dimensional (3D) chitosan nanofiber scaffolds using an electrospinning technique and explore its potential for cardiac tissue engineering. Three culture conditions were tested: cardiomyocytes only, cardiomyocytes-fibroblasts cocultures, and cardiomyocyte-endothelial cells cocultures. The cells were seeded on 2-dimensional (2D) chitosan films and 3D chitosan nanofibers. Cellular morphology and functionality were assessed using immunofluorescent staining for alpha-sarcomeric actin (SA) and gap junction protein, connexin-43 (Cx43). In both the 2D and 3D scaffolds, only the cardiomyocyte-fibroblasts cocultures resulted in polarized cardiomyocyte morphology, fibril SA expression and Cx43 expression was higher compared to the other two conditions. In addition, the fibroblasts cocultures demonstrated synchronized contractions involving large tissue-like cellular networks. To our knowledge, this is the first attempt to utilize 3D chitosan nanofibers as cardiomyocyte scaffolds. Our results demonstrate that chitosan nanofibers can serve as a potential scaffold that can retain cardiomyocyte morphology and function.

Bioartificial liver (BAL) devices have been developed to treat patients undergoing acute liver failure. One of the most important parameters to consider in designing these devices is the oxygen consumption rate of the seeded hepatocytes which are known to have oxygen consumption rates 10 times higher than most other cell types. Hepatocytes in various culture configurations have been tested in BAL devices including those formats that involve co-culture of hepatocytes with other cell types. In this study, we investigated, for the first time, oxygen uptake rates (OUR)s of hepatocytes co-cultured with 3T3-J2 fibro- blasts at various hepatocyte to fibroblast seeding ratios. OURs were determined by measuring the rate of oxygen disappearance using a ruthenium-coated optical probe after closing and sealing the culture dish. Albumin and urea production rates were measured to assess hepatocyte func- tion. Lower hepatocyte density co-cultures demonstrated significantly higher OURs (2 to 3.5-fold) and liver- specific functions (1.6-fold for albumin and 4.5-fold for urea pro- duction) on a per cell basis than those seeded at higher densities. Increases in OUR correlated well with increased liver-specific functions. OURs (Vm) were modeled by fitting Michaelis-Menten kinetics and the model predictions closely correlated with the experimental data. This study provides useful information for predicting BAL design parameters that will avoid oxygen limitations, as well as maximize metabolic functions. Biotechnol. Bioeng. 2007;97: 188-199.