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Microfluidic confinement enhances phenotype and function of hepatocyte spheroids.

A number of cell culture approaches have been described for maintenance of primary hepatocytes. Forming hepatocytes into 3D spheroids is one well accepted method for extending epithelial phenotype of these cells. Our lab has previously observed enhanced function of 2D (monolayer) hepatocyte cultures in microfluidic devices due to increased production of several hepato-inductive growth factors, including hepatocyte growth factor (HGF). In the present study we wanted to test a hypothesis that placing hepatocytes spheroids (3D) into microfluidic devices will also result in enhanced phenotype and function. To test this hypothesis, we fabricated devices with small and large volumes. Both types of devices included a microstructured floor containing arrays of pyramidal wells to promote assembly of single hepatocytes into spheroids with individual diameter of ~100 µm. The hepatocyte spheroids were found to be more functional in low volume compared to large volume devices. Importantly, high functionality of spheroid cultures correlated with elevated levels of HGF secretion. While decay of hepatic function (albumin secretion) was observed over the course three weeks, this behavior could be abrogated by inhibiting TGF-β1 signaling. With TGF-β1 inhibitor, microfluidic hepatocyte spheroid cultures maintained high and stable levels of albumin synthesis over the course of four weeks. To further highlight utility of this culture platform for liver disease modeling, we carried out alcohol injury experiments in microfluidic devices and tested protective effects of interleukin (IL)-22 - a potential therapy for alcoholic hepatitis.

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