A Culture Method of Rat Hepatocytes Using the Three-dimensional Cell Culture Scaffold "Cellbed" and Its Application for Liver Toxicity Testing

Abstract

There are some cases that the results of in vitro liver toxicity testing can't correlate with the results of clinical studies or animal experiments because of difficulty in maintaining viability and activity of normal hepatocytes under in vitro culture conditions. In addition, based on the three Rs of animal experiments, the development of a culture system that can replace animal experiments is desired. In this study, we aimed to optimize a culture method using the three-dimensional cell culture scaffold "Cellbed^®" that maintains the liver functions of rat hepatocytes longer than traditional monolayer culture on type I collagen-coated dishes, by coating Cellbed with various types of extracellular matrix (type I collagen, fibronectin, and laminin). As a result, culture conditions using laminin-coated Cellbed presented the most favorable effect in terms of maintenance of liver functions (based on albumin and CYP3A expression), and this was superior to monolayer culture using any other coating. As such, it was indicated that a culture method using laminin-coated Cellbed contributes to the long-term maintenance and improvement of rat hepatocyte functions. This is expected for the application of Cellbed to hepatic metabolism studies and long-term in vitro liver toxicity testing.