The Use of Microcarrier-Roller Bottle Culture for Large-Scale Production of Porcine Hepatocytes

Microcarrier-attached hepatocytes have applications in both artificial liver support and cell transplantation therapies. In either scenario, maintenance of the cells' in vitro metabolic functions is of primary importance. We are reporting the potential to scale up production and enhance detoxification function of porcine hepatocytes for use in cell-based liver support systems, such as those currently in clinical trials. Previous studies of anchorage-dependent cells in vitro have suggested a correlation between tridimensional morphology and the maintenance of function. In this study, four different microcarrier-roller bottle cultures of porcine hepatocytes were compared for their influence on aggregate formation, benzodiazepine metabolism, and cell proliferation. Porcine hepatocytes were seeded on microcarriers at cell density normalized to surface area by adjustment of bead number (6.25 cm²/1 × 10⁶ cells) and maintained in roller bottle culture for 6 days. Microcarrier sizes ranged from 90 to 1000 µm. Mean diazepam metabolic production ranged from 18 to 31 µg/1 × 10⁶ cells. The highest diazepam metabolic activity and DNA content were measured on day 4 cultures. Ratios based on aggregate size (number of microcarriers per aggregate) were similar for most microcarriers, but the aggregation characteristics of surface-treated polystyrene (Biosilon) beads were higher. The increased cell contact and tissuelike structure observed in those cultures were reflected in improved cell proliferation, as measured by DNA content, but not in increased detoxification function, as evaluated by diazepam metabolite production. In conclusion, microcarrier-roller bottle culture of porcine hepatocytes allows large numbers of cells to be maintained with greater ease than in monolayers and enhances cell proliferation but, under these study conditions, did not improve individual cell function.