Influence of Different Substrates in Detoxification Activity of Adult Rat Hepatocytes in Long-Term Culture: Implications for Transplantation

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Abstract
Substrates used to immobilize hepatocytes for transplantation govern attachment and long-term metabolic activity of these cells. The choice of these substrates is based on the familiarity with proteinaceous materials that are constituents of the extracellular matrix. The use of substrates that recognize carbohydrates on the cell surface may provide an alternative method to attach adult mammalian hepatocytes. In this study, immobilized lectins on tissue culture plasticware were used to support hepatocyte attachment. Long-term cultures with these substrates were compared with control cultures seeded on a mixture of collagen types I and III (Vitrogen). To evaluate the attachment efficiency and long-term maintenance of diazepam metabolic activity of hepatocytes seeded on different commercially available plasticware, four different types of polymers (supplied as 60-mm dishes) were tested. Diazepam, a benzodiazepine metabolized by the P450 intracytoplasmic pathway, is associated with a synaptic receptor (GABA-benzodiazepine receptor) which plays an important role in hepatic coma. Polymethylpentene, a derivative of polypropylene treated by plasma discharge, was the best polymer to maintain P450 phenotypic expression, although other polymers provided similar cell attachment efficiencies. The amounts of adsorbed concanavalin A, Arachis hypogaea (peanut), Lens culinaris, and Pisum sativum agglutinin correlate with the percentage values of hepatocyte attachment. Cell attachment to wheat germ agglutinin increased with increased lectin concentrations in spite of constant amounts of adsorbed lectin, whereas hepatocyte attachment to Bandieraea simplicifolia agglutinin was lower and did not change at different lectin concentrations. Long-term cultures of hepatocytes seeded on Vitrogen, concanavalin A, or wheat germ agglutinin showed similar diazepam metabolic activities up to the 10th day, but by day 25, cells seeded on Vitrogen metabolized diazepam at higher values. This study showed that a polymer used for the manufacture of tissue culture plasticware, which permits a better exchange of gases, contributes to the long-term expression of P450 activity. Lectins proved to be nontoxic for hepatocyte survival, maintained hepatocyte viability, and can be used as an alternative substrate to immobilize hepatocytes to be transplanted in animal models of acute or chronic liver failure.