Improved maintenance of adult rat hepatocytes in a new serum-free medium in the presence or absence of barbiturates

Summary For serum-free primary culture of adult rat hepatocytes, a synthetic medium DM-160 and rat-tail collagen were selected for the basal medium and for the culture substratum, respectively. Barbiturates, such as phenobarbital and 1-ethyl-5-isobutylbarbiturate, efficiently supported survival of hepatocytes and maintained their morphologic features at lower concentrations under the serum-free conditions than under the serum-supplemented conditions. However, the hepatocyte survival rates under the serum-free conditions were lower than those under the serum-supplemented conditions in the presence or absence of barbiturates. Supplementation of the basal medium with a combination of five groups of factors (5Fs), such as eight amino acids (Ala, Arg, Gly, Ile, Met, Phe, Pro, and Trp), two unsaturated fatty acids (linoleate and oleate), a protease inhibitor (aprotinin), three vitamins (A, C, and E), and five trace elements (Mn, Fe, Cu, Zn, and Se), improved the hepatocyte survival under the serum-free conditions in the presence or absence of barbiturates. In other words, the serum could be completely substituted by the 5Fs. Hepatocyte cultures maintained in the 5Fs-suppelemented basal medium showed excellent induction of tyrosine aminotransferase activity in response to dexamethasone in the presence or absence of barbiturates. The efficiency of the 5Fs-supplemented basal medium for maintaining hepatocytes was not inferior to those of other media in common use with hepatocytes, such as Williams' medium E and Waymouth's medium MB-752/1. In conclusion, maintenance of functional hepatocytes in serum-free primary culture could be improved by use of the new medium preparation (the 5Fs-supplemented DM-160) in the presence of barbiturates. This work was supported by a grant no. 61771923 from the Ministry of Education, Science and Culture of Japan.