FORMATION OF SPHEROIDAL AGGREGATES OF HEPATOCYTES ON BIODEGRADABLE POLYMERS UNDER CONTINUOUS-FLOW BIOREACTOR CONDITIONS

Our laboratory has investigated heterotopic hepatocyte transplantation on biodegradable polymer matrices as an experimental treatment for en d-stage river disease. One of the limitations has been survival of suf ficient cell mass after transplantation. We hypothesize that in vitro conditioning of cells within polymer matrices prior to implantation ma y increase hepatocyte survival and function. In this preliminary study we investigated the effect of continuous flow on hepatocytes and sinu soidal endothelial cells on poly-L-lactic acid (PLLA) discs in vitro. Highly porous PLLA discs were manufactured measuring 18 mm diameter by 1 mm thickness using previously described techniques. Hepatocytes wer e isolated from adult, male Lewis rats (200-300 g) using a two-step co llagenase digestion. Sinusoidal endothelial cells were isolated using a two-step collagenase digestion, differential sedimentation, Percoll gradient centrifugation, and selective adherence. PLLA discs were seed ed with hepatocytes alone or with co-cultures of hepatocytes and sinus oidal endothelial cells. Seeded discs were then secured within a flow bioreactor chamber and exposed to continuous flow of culture media at a rate of 20 ml/minute through the chamber. Seeded discs placed in sta tic culture conditions served as controls. Specimens seeded with only hepatocytes were harvested at 24 hours, 48 hours, and 168 hours after seeding. Go-culture specimens were harvested after 168 hours. Specimen s were viewed under phase-contrast microscopy and then formalin-fixed and prepared for histologic sectioning. Sections were stained with Hem atoxylin and Eosin and then analyzed with light microscopy. Hepatocyte s under flow conditions formed spheroidal aggregates of cells of 50 to 200 mu m in diameter by 24 hours in culture. Hepatocytes in static co nditions showed decreased aggregation of cells and spheroid formation was absent. Co-cultured specimens under flow also showed spheroid form ation with endothelial cells lining the outside of hepatocyte spheroid s. Go-cultured specimens in static culture showed no spheroid formatio n and no organization between sinusoidal endothelial cells and hepatoc ytes. These results suggest that continuous flow increases organizatio n of hepatocytes cultured within biodegradable polymer matrices.