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Layered patterning of hepatocytes in co-culture systems using microfabricated stencils
Cheul H. Cho*, Jaesung Park**, Arno W. Tilles, François Berthiaume, Mehmet Toner, and Martin L. Yarmush
Center for Engineering in Medicine and Department of Surgery, Massachusetts General Hospital, Harvard Medical School, and the Shriners Hospitals for Children, Boston, MA, USA
BioTechniques, Vol. 48, No. 1, January 2010, pp. 47–52
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Supplementary Material

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 ┬Ám. 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.


Hepatic tissue engineering has emerged as a novel therapeutic approach to treat damaged or diseased liver tissue (1-3). The reconstruction of functional hepatic tissue is dependent on the ability to control factors that influence the cell environment, including cell-matrix interactions, soluble stimuli, and cell-cell interactions (4-8). Cell-cell interactions play a critical role in tissue morphogenesis, embryogenesis, and organ development. In vivo, the liver is a structurally and functionally heterocellular construct, composed of primary hepatocytes, endothelial cells, Kupffer cells, stellate cells, and fibroblasts (4). In an effort to reconstruct liver tissue in vitro for therapeutic applications, several studies have demonstrated that co-culturing primary hepatocytes with non-parenchymal cells such as fibroblasts or endothelial cells maintains hepatocyte viability and function, whereas hepatocytes cultured alone rapidly lose their function (9-12). However, these conventional co-culture methods (i.e., mixing the two cells types at random) are not able to control the cellular interactions and spatial signaling that occur in the in vivo hepatic microenvironment.

Microfabrication and micropatterning technologies have been used to control cell-cell and cell-surface interactions for applications in tissue engineering (13-18). Various techniques have been used to create micropatterned cells including photolithography, elastomeric stencils, and cell printing. Two-dimensional spheroid microarrays using microfabrication techniques have been utilized to mimic in vivo-like tissue structure (19-21). These cell micropatterning techniques allow co-cultures to be created in which the cell density and the total length of contact between the two cell populations (“heterotypic interface”) can be controlled independently of the cell-cell ratio. Using photolithography to create micropatterned islands of hepatocytes with surrounding 3T3-J2 fibroblasts, we and others demonstrated that albumin and urea levels increased as the length of the heterotypic interface increased (13,22). In addition, intracellular albumin staining of the hepatocytes by day 6 in the micropatterned co-cultures revealed that albumin induction was strongest in hepatocytes that were in close proximity to fibroblasts (i.e., at the heterotypic interface) compared with that in hepatocytes that were far (i.e., >5 cell diameters) from the heterotypic interface. These studies indicate that heterotypic cell-cell interactions play an important role in the maintenance of hepatocellular function.

In the present study, we present a novel culture method to generate layered, patterned hepatocytes on micropatterned fibroblast feeder layers that significantly increases the heterotypic interface using microfabricated polydimethylsiloxane (PDMS) stencils. Based on the previous studies, we hypothesize that hepatocellular structure and function can be improved with increasing heterotypic interface between hepatocytes and fibroblasts by the layered cell patterning method. Micropatterned PDMS stencils were fabricated using a soft lithography approach. The morphologic, phenotypic, and functional characteristics of hepatocytes in the micropatterned co-cultures were evaluated. Results of this study indicate that increasing the heterotypic interface using the layered cell patterning technique in micropatterned hepatic co-cultures significantly enhances the liver-specific functions of hepatocytes, including intracellular albumin staining, urea synthesis, albumin secretion, E-cadherin expression, and glycogen storage. This cell culture technique can be useful for generating a stable in vitro hepatic co-culture model with enhanced function, enabling better understanding of cellular interactions.

Materials and methods

Cell isolation and culture

Hepatocytes were isolated from adult female Lewis rats (Charles River Laboratories, Wilmington, MA, USA) using a two-step collagenase perfusion procedure as described previously (23). Hepatocyte viability was greater than 90% as determined by trypan blue exclusion. Hepatocyte culture medium consisted of DMEM supplemented with 10% fetal bovine serum (Gibco, Gaithersburgh, MD, USA), 7 ng/mL glucagon (Bedford Laboratories, Bedford, OH, USA), 7.5 µg/mL hydrocortisone (Pharmacia Corporation, Kalamazoo, MI, USA), 0.5 U/mL insulin (Eli Lilly, Indianapolis, IN, USA), 20 ng/mL epidermal growth factor (Sigma-Aldrich, St. Louis, MO, USA), 200 U/mL penicillin, and 200 µg/mL streptomycin (Gibco). Culture medium was changed daily and medium samples were collected for functional analysis.

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