Hepatocytes isolated from adult rats were cultured for 2 to 3 weeks on collagen in a modified, serum-free Waymouth medium containing fatty acids and varying concentrations of glucocorticoid, insulin and glucagon. (ii) In the presence of all three hormones, it was possible to maintain the content of DNA, the activity of glucokinase, pyruvate kinase, hexokinase and lactate dehydrogenase at initial levels for 2 to 3 weeks. The activity of glucokinase and pyruvate kinase was affected by the concentration of insulin. (iii) The activity of alcohol dehydrogenase was stable for 3 days and declined to about 25% of the initial level after 2 weeks of culture, irrespective of the presence of hormones. (iv) Maintenance of albumin secretion was dependent on the presence of glucocorticoid, and glucocorticoid and insulin showed an additive or, at some time points, a synergistic effect on its secretion. (v) The content of cytochrome P-450 could be kept at 65% of the initial level, provided that a relatively high concentration of dexamethasone was present (lo-' M). (vi) In the absence of hormones, urea synthesis was 70% of initial levels throughout the experimental period. With insulin and glucocorticoid present, a high concentration of glucagon M) was required to maintain the synthesis of urea at this level. (vii) It is concluded that hepatocyte cultures as described in the present study may be a useful, well-defined system for long-term metabolic, pharmacologic and toxicologic studies.
Primary cultures of hepatocytes can easily be established from suspensions of hepatocytes as demonstrated by Bissell and coworkers (1) in 1973. This model has since been used in several metabolic, pharmacologic and toxicologic studies. However, the model has limited application, as the survival of hepatocytes in primary cultures nearly invariably have been reported to be short. Hepatocytes loose their differentiated functions after a few days of culture (2), and the cells tend to detach from the culture dishes (3). The reason for this degenerative course is uncertain, but three factors must principally be