We have fabricated a hepatic tissue construct using a multilayer photopatterning platform for embedding cells in hydrogels of complex architecture. We first explored the potential of established hepatocyte culture models to stabilize isolated hepatocytes for photoencapsulation (e.g., double gel, Matrigel, cocultivation with nonparenchymal cells). Using photopolymerizable PEG hydrogels, we then tailored both the chemistry and architecture of the hydrogels to further support hepatocyte survival and liver-specific function. Specifically, we incorporated adhesive peptides to ligate key integrins on these adhesion-dependent cells. To identify the appropriate peptides for incorporation, the integrin expression of cultured hepatocytes was monitored by flow cytometry and their functional role in cell adhesion was assessed on full-length extracellular matrix (ECM) molecules and their adhesive peptide domains. In addition, we modified the hydrogel architecture to minimize barriers to nutrient transport for these highly metabolic cells. Viability of encapsulated cells was improved in photopatterned hydrogels with structural features of 500 µm in width over unpatterned, bulk hydrogels. Based on these findings, we fabricated a multilayer photopatterned PEG hydrogel structure containing the adhesive RGD peptide sequence to ligate the ₅₁ integrin of cocultured hepatocytes. Three-dimensional photopatterned constructs were visualized by digital volumetric imaging and cultured in a continuous flow bioreactor for 12 d where they performed favorably in comparison to unpatterned, unperfused constructs. These studies will have impact in the field of liver biology as well as provide enabling tools for tissue engineering of other organs.--Liu Tsang, V., Chen, A. A., Cho, L. M., Jadin, K. D., Sah, R. L., DeLong, S., West, J. L., Bhatia, S. N. Fabrication of 3D hepatic tissues by additive photopatterning of cellular hydrogels.