Journal of Bioprocessing & Biotechniques

Effects of Culture Conditions on a Micropatterned Co-culture of Rat Hepatocytes with 3T3 cells

Abstract

Kohji Nakazawa, Yukako Shinmura, Ami Higuchi and Yusuke Sakai

We investigated the effect of culture conditions on the micropatterned co-culture of rat hepatocytes with 3T3 cells. A micropatterned chip was prepared using polydimethylsiloxane (PDMS) microstencil such that the chip contained 724 hepatocyte islands, each 500 μm in diameter, in a triangular arrangement with 800-μm pitch, in which hepatocytes were co-cultured with 3T3 cells. The hepatocytes in micropatterned co-culture exhibited hepatocellular morphology, and the micropatterned configuration of hepatocyte islands was maintained for several weeks of culture by supporting the heterotypic interface between the hepatocytes and 3T3 cells. The albumin secretion activity of hepatocytes was highest in the micropatterned co-culture but decreased in the random co-culture, micropatterned mono-culture (hepatocytes only), and random mono-culture (hepatocytes only) in that order. Furthermore, earlier formation of co-culture promoted higher functional activity of hepatocytes as compared to later formation, and hepatocyte functions were induced with an increasing the density of inoculated 3T3 cells. These results suggest that the formation of a heterotypic interaction at an early stage is important for maintaining high levels of hepatocyte functions. The findings of this study will provide information useful for designing co-culture conditions for liver tissue engineering and pharmacological and toxicological studies.