LONG-TERM PRIMARY CULTURES OF ADULT HUMAN HEPATOCYTES

In this work we have investigated a system of long-term primary cultur es of adult human hepatocytes which, in contrast to those previously d escribed, has the advantage of requiring neither the use of additive c ells as in co-cultures, nor of matrix component preparations like Matr igel or collagen sandwich. This system has been used previously for lo ng-term cultures of hepatocytes from young baboon, and some modificati ons have been introduced here to take into account the specificity of adult human hepatocytes. In this system, hepatocytes are plated at con fluence 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, alph a-1 antitrypsin, plasminogen, fibrinogen, lipoproteins ApoA1 and ApoB1 00, have been quantified in the extracellular medium as a function of time, either by immunoblot or ELISA. In addition, the expression and i nducibility of CYP proteins of the CYP1, CYP2 and CYP3 families in res ponse to their prototypical inducers including 2,3,7,8-tetrachlorodibe nzo(p)dioxin and rifampicin, have been evaluated by immunoblot analysi s 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 extracell ular medium. Our results show that, under these culture conditions, ad ult 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. (C) 1997 Elsevier Science Ireland Ltd.