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The liver carries out a variety of essential functions regulated in part by autocrine signaling, including hepatocyte-produced growth factors and extracellular matrix (ECM). The local concentrations of autocrine factors are governed by a balance between receptor-mediated binding at the cell surface and diffusion into the local matrix and are thus expected to be influenced by the dimensionality of the cell culture environment. To investigate the role of growth factor and ECM-modulated autocrine signaling in maintaining appropriate primary hepatocyte survival, metabolic functions, and polarity, we created three-dimensional cultures of defined geometry using micropatterned semisynthetic polyethylene glycol-fibrinogen hydrogels to provide a mechanically compliant, nonadhesive material platform that could be modified by cell-secreted factors. We found that in the absence of exogenous peptide growth factors or ECM, hepatocytes retain the epidermal growth factor (EGF) receptor ligands (EGF and transforming growth factor-α) and the proto-oncogenic mesenchymal epithelial transition factor (c-MET) ligand hepatocyte growth factor (HGF), along with fibronectin. Further, hepatocytes cultured in this three-dimensional microenvironment maintained high levels of liver-specific functions over the 10-day culture period. Function-blocking inhibitors of α5β1 or EGF receptor dramatically reduced cell viability and function, suggesting that signaling by both these receptors is needed for in vitro survival and function of hepatocytes in the absence of other exogenous signals.

Autocrine-Controlled Formation and Function of Tissue-Like Aggregates by Primary Hepatocytes in Micropatterned Hydrogel Arrays