The influence of medium composition and matrix on long-term cultivation of primary porcine and human hepatocytes

Abstract

The differentiated hepatocyte phenotype remains difficult to maintain in culture. The duration over which phenotypically stable hepatocytes can be cultured ranges from a couple of days to a few weeks. Shortcomings in medium formulation may be a factor in this lack of success. We have investigated effects of medium formulation on primary porcine and human hepatocyte cultures. We tested seven culture medium compositions (DMEM, ExCell 400, HepatoZYME-SFM, L-15 Leibovitz, SF-3, Waymouth, and Williams’ E) and the effects of serum, fibronectin and biomatrix in a sandwich culture configuration. Albumin, urea, cholesterol, GOT, GPT, LDH and triglyceride concentrations were measured over 14 days. For both human and porcine cultures, the best results were obtained with SF-3 medium. Cells cultivated with Williams’ E medium and FCS had good morphology and synthetic function during the first days of culture. However, continued addition of serum, was associated with a subsequent loss of differentiated phenotype. Addition of fibronectin was associated with improved function in cultures maintained in SF-3 medium whilst biomatrix had no effect. In contrast, addition of fibronectin did not influence cultures maintained in Williams’ E medium, but cultures with biomatrix were associated with improved function at longer time points.