Long-term primary cultures of adult human hepatocytes

In this work we have investigated a system of long-term primary cultures of adult human hepatocytes which, in contrast to those previously described, has the advantage of requiring neither the use of additive cells as in co-cultures, nor of matrix component preparations like Matrigel or collagen sandwich. This system has been used previously for long-term cultures of hepatocytes from young baboon, and some modifications have been introduced here to take into account the specificity of adult human hepatocytes. In this system, hepatocytes are plated at confluence on collagen-coated dishes and cultured in a serum-free medium consisting of Williams'E supplemented with hormones and growth factors. Proteins secreted specifically by the liver, including albumin, alpha-1 antitrypsin, plasminogen, fibrinogen, lipoproteins ApoA1 and ApoB100, have been quantified in the extracellular medium as a function of time, either by immunoblot or ELISA. In addition, the expression and inducibility of CYP proteins of the CYP1, CYP2 and CYP3 families in response to their prototypical inducers including 2,3,7,8-tetrachlorodibenzo(p)dioxin and rifampicin, have been evaluated by immunoblot analysis of microsomes or cell lysates. Moreover, the oxidative metabolism of cyclosporin A, a monoxygenase activity depending on CYP3A4, has been monitored directly on the cultured cells by HPLC analysis of extracellular medium. Our results show that, under these culture conditions, adult human hepatocytes retain these phenotypical characteristics for at least 35 days. This system meets the requirements for use as a model for screening CYP protein inducers.