Cultivating liver cells on printed arrays of hepatocyte growth factor

Department of Biomedical Engineering, University of California, Davis, 451 East Health Sciences Dr. #2519, Davis, CA 95616, USA.
Biomaterials (Impact Factor: 8.56). 05/2009; 30(22):3733-41. DOI: 10.1016/j.biomaterials.2009.03.039
Source: PubMed


Growth factors are commonly present in soluble form during in vitro cell cultivation experiments in order to provide signals for cellular proliferation or differentiation. In contrast to these traditional experiments, we investigated solid-phase presentation of a hepatocyte growth factor (HGF), a protein important in liver development and regeneration, on microarrays of extracellular matrix (ECM) proteins. In our experiments, HGF was mixed in solution with ECM proteins (collagen (I), (IV) or laminin) and robotically printed onto silane-modified glass slides. Primary rat hepatocytes were seeded onto HGF/ECM protein microarrays and formed cellular clusters that corresponded in size to the dimensions of individual protein spots (500 microm diameter). Analysis of liver-specific products, albumin and alpha1-antitrypsin, revealed several fold higher levels of expression of these proteins in hepatocytes cultured on HGF/ECM microarrays compared to cells cultivated on ECM proteins alone. In addition, cultivation of hepatocytes on HGF/ECM protein spots led to spontaneous reorganization of cellular clusters from a monolayer into three-dimensional spheroids. We also investigated the effects of surface-tethered HGF on hepatocytes co-cultivated with stromal cells and observed a significantly higher level of albumin in co-cultures where hepatocytes were stimulated by HGF/ECM spots compared to co-cultures created on ECM protein islands without the growth factor. In summary, our study suggests that incorporation of HGF into ECM protein microarrays has a profound and long-lasting effect on the morphology and phenotype of primary hepatocytes. In the future, the number of growth factors printed on ECM microarrays will be expanded to enable multiplexed and combinatorial screening of inducers of cellular differentiation or proliferation.

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Available from: Caroline Jones, Sep 16, 2014
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    • "In another experiment, Lee et al showed that the function and behavior of cultivated cells on ECM micro-patterns could be analyzed through different ways such as gene expression levels (12). In their experiment, HepG2 cells were cultivated on collagen type I micropatterns and then the cells were extracted by laser catapulting for further gene expression analysis (12, 18). These studies reveal micropatterning as a very strong tool for both in vitro studies and tissue engineering. "
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    ABSTRACT: Micropatterning is becoming a powerful tool for studying cells in vitro. This method not only uses very small amount of material but also mimic the microenvironment structure present in living tissues better than flask culturing techniques. In previous studies using micropatterning of extracellular matrix proteins on glass surfaces, the rate of protein detachment from the surface was so high that the proteins and the cultivated cells detached after 3 three days of cell seeding. Here we optimized the glass surface modification method to fulfill the requirement of most in vitro studies. In our study we showed that the optimum time for glass surface modification reaction is 1.5 hr, and the cells could be tracked in vitro for over 15 days after cell seeding which is enough for the most in vitro studies. As a model, we cultivated HEK 293T and HepG2 cells on the collagen micro-patterns and showed that they have normal growth and morphology on these micropatterns. The HEK cells also transfected with pmaxGFP plasmid vector to show that the cells on collagen micropatterns could also used in transfection studies. Taking these together, this novel method is promising for efficient cell culture studies on micropatterened surfaces in the future.
    Full-text · Article · Feb 2013
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    • "Our laboratory has recently reported that HGF molecules co-printed with ECM proteins (e.g. collagen (I), (IV), laminin) are retained on printed spots for several days under culture conditions [34]. Importantly, primary hepatocytes cultivated on top of the printed HGF/ECM arrays remained functional after 10 days in culture. "
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    ABSTRACT: The success in driving embryonic stem cells towards hepatic lineage has been confounded by the complexity and cost of differentiation protocols that employ large quantities of expensive growth factors (GFs). Instead of supplementing culture media with soluble GFs, we investigated cultivation and differentiation of mouse embryonic stem cells (mESCs) on printed arrays of GFs. Hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF) and bone morphogenetic protein (BMP4) were mixed in solution with fibronectin and collagen (I) and then printed onto silane-modified glass slides to form 500 μm diameter protein spots. mESCs were cultured on top of GF spots for up to 12 days and analyzed by RT-PCR and immunostaining at different time points. The stem cells residing on HGF-containing combinations of GFs exhibited requisite features of hepatic differentiation including pronounced loss in pluripotency (Oct4), transient (up and down) expression of endoderm (Sox17) and upregulation of early hepatic markers--albumin and alpha-fetoprotein. The hepatic differentiation was enhanced further by adding hepatic stellate cells to surfaces that already contained mESCs on GF spots. A combination of co-culture with non-parenchymal liver cells and the optimal GF stimulation was found to induce endoderm and hepatic phenotype earlier and to a much greater extent than the GF arrays or micropatterned co-cultures used individually. While this paper investigated hepatic differentiation of mouse ESCs, our findings and stem cell culture approaches are likely to be relevant for human ESC cultivation. Overall, the platform combining printed GF arrays and heterotypic co-cultures will be broadly applicable for identifying the composition of the microenvironment niche for ESC differentiation into various tissue types.
    Full-text · Article · Dec 2010 · Biomaterials
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    • "Furthermore, heparin-based hydrogel may be fabricated into miniature constructs suitable for cell delivery/transplantation [30] and was found to cause little or no inflammation and hemolysis in vivo [28]. As a step towards development of heparin hydrogel scaffolds for hepatocyte or stem cell transplantation, the present study sought to characterize cultivation of primary rat hepatocytes in this gel and to test the effects of incorporating hepatocyte growth factor (HGF) [31] [32] [33]-a potent liver morphogen – into the hydrogel. Future applications of heparin hydrogel as a microenvironment niche for hepatocyte or stem cell differentiation and as a vehicle for cell transplantation are envisioned. "
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    ABSTRACT: Primary hepatocytes are commonly used as liver surrogates in toxicology and tissue engineering fields, therefore, maintenance of functional hepatocytes in vitro is an important topic of investigation. This paper sought to characterize heparin-based hydrogel as a three-dimensional scaffold for hepatocyte culture. The primary rat hepatocytes were mixed with a prepolymer solution comprised of thiolated heparin and acrylated poly(ethylene glycol) (PEG). Raising the temperature from 25 degrees to 37 degrees C initiated Michael addition reaction between the thiol and acrylated moieties and resulted in formation of hydrogel with entrapped cells. Analysis of liver-specific products, albumin and urea, revealed that the heparin hydrogel was non-cytotoxic to cells and, in fact, promoted hepatic function. Hepatocytes entrapped in the heparin-based hydrogel maintained high levels of albumin and urea synthesis after three weeks in culture. Because heparin is known to bind growth factors, we incorporated hepatocyte growth factor (HGF)-an important liver signaling molecule - into the hydrogel. HGF release from heparin hydrogel matrix was analyzed using enzyme linked immunoassay (ELISA) and was shown to occur in a controlled manner with only 40% of GF molecules released after 30 days in culture. Importantly, hepatocytes cultured within HGF-containing hydrogels exhibited significantly higher levels of albumin and urea synthesis compared to cells cultured in the hydrogel alone. Overall, heparin-based hydrogel showed to be a promising matrix for encapsulation and maintenance of difficult-to-culture primary hepatocytes. In the future, we envision employing heparin-based hyrogels as matrices for in vitro differentiation of hepatocytes or stem cells and as vehicles for transplantation of these cells.
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