Hepatic stellate cells (HSCs) form a functional unit with endothelia and hepatocytes in the liver to play a pivotal role in heterotypic cellular communication. To investigate this role of HSCs, it is of great benefit to establish a triculture model that forms the functional unit from proximal layers of hepatocytes, HSCs, and endothelial cells (ECs). Here, we established a three-dimensional triculture model, using a microporous membrane to create the functional unit. HSC behavior was controlled by the membrane pore size, which was critical for achieving proximal cell layers. With a specific pore size, the HSCs intercalated between layers of hepatocytes and ECs, due to the limitation on HSC behavior. When only cytoplasmic processes of quiescent HSCs were adjacent to ECs, while the HSC bodies remained on the side of the hepatocytes, the ECs changed morphologically and were capable of long-term survival. We confirmed that HSCs mediated the communication between hepatocytes and ECs in terms of EC morphogenesis. This triculture model allows us to investigate the roles of HSCs as both facilitators and integrators of cell-cell communication between hepatocytes and ECs, and is useful for investigating heterotypic cellular communication in vitro.
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