Control of hepatocyte function on collagen foams: sizing matrix pores toward selective induction of 2-D and 3-D cellular morphogenesis

While microporous biopolymer matrices are being widely tested as cell culture substrates in hepatic tissue engineering, the microstructural basis for their control of cell differentiation is not well understood. In this paper, we studied the role of void size of collagen foams in directing the induction of liver-specific differentiated morphology and secretory activities of cultured rat hepatocytes. Hepatocytes cultured on collagen foams with subcellular sized pore diameters of 10 μm assumed a compact, cuboidal cell morphology, rapidly achieving monolayer coverage, and secreted albumin at the rate of 40±8 pg/cell/d. Increasing the pore size to 18 μm elicited a distinctly spread cellular phenotype, with discontinuous surface coverage, and albumin secretion rates declined precipitiously to 3.6±0.8 pg/cell/d. However, when collagen foams with an even higher average void size of 82 μm were used, hepatocytes exhibited high degree of spreading within an extensive three-dimensional cellular network, and exhibited high albumin secretory activity (26±0.6 pg/cell/d). The effect of void geometry on cellular ultrastructral polarity was further analyzed for the three void size configurations employed. The distribution of the cell–cell adhesion protein, E-cadherin, was primarily restricted to cell–cell contacts on the 10 μm foams, but was found to be depolarized to all membrane regions in cells cultured on the 18 and 82 μm foams. Vinculin-enriched focal adhesions were found to be peripherally clustered on cells cultured on 10 μm foams, but were found to redistribute to the entire ventral surface of cells cultured on the 18 and 82 μm foams. Overall, we demonstrate the significance of designing pore sizes of highly adhesive substrates like collagen toward selective cell morphogenesis in 2-D or 3-D. Subcellular and supercellular ranges of pore size promote hepatocellular differentiation by limiting 2-D cell spreading or effecting 3-D intercellular contacts, while intermediate range of pore sizes repress differentiation by promoting 2-D cell spreading.