Isolation and Long-Term Maintenance of Adult Rat Hepatocytes in Culture

Abstract

Long-term and stable hepatocyte culture systems have a wide variety of uses, both in basic science and in the development of hepatocyte-based applications. In most cases, long-term cultures of hepatocytes are superior to traditional cultures in collagen-coated dishes, which only transiently express a low level of liver-specific function during the first wk in culture (1,2). The collagen sandwich provides a system capable of maintaining long term and stable function of hepatocytes with which to study liver physiology (3,4). This system is now used along with several other long-term hepatocyte culture techniques that have been developed since the mid 1970s. These other methods include the use of special extracellular matrix (ECM) materials, such as an extract from the Engelbreth-Holm-Swarm sarcoma grown in mice [5] (under the commercial appellations of Matrigel and Biomatrix, Biomedical Technologies, Stoughton, MA), co-culture with mesenchymal, endothelial, or epithelial cells (6–8), special culture media (e.g., dimethyl sulfoxide supplementation or arginine-free formulas), and culture at high seeding densities. In the context of studying liver physiology and morphogenesis of the liver plate, the sandwich culture system appears to be particularly well-suited, since it exhibits in vivo-like ECM geometry, has relatively flexible medium requirements, and individual cell morphology and structure can be easily visualized. One disadvantage, however, is that, in current practice, the ECM layer on top of the cells may present a transport barrier that can slow down the exchange of nutrients, products, and chemical signals with the bulk of the medium.