Our laboratory has investigated heterotopic hepatocyte transplantation on biodegradable polymer matrices as an experimental treatment for end-stage liver disease. One of the limitations has been survival of sufficient cell mass after transplantation. We hypothesize that in vitro conditioning of cells within polymer matrices prior to implantation may increase hepatocyte survival and function. In this preliminary study we investigated the effect of continuous flow on hepatocytes and sinusoidal 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 were isolated from adult, male Lewis rats (200-300 g) using a two-step collagenase digestion. Sinusoidal endothelial cells were isolated using a two-step collagenase digestion, differential sedimentation, Percoll gradient centrifugation, and selective adherence. PLLA discs were seeded with hepatocytes alone or with co-cultures of hepatocytes and sinusoidal 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 static culture conditions served as controls. Specimens seeded with only hepatocytes were harvested at 24 hours, 48 hours, and 168 hours after seeding. Co-culture specimens were harvested after 168 hours. Specimens were viewed under phase-contrast microscopy and then formalin-fixed and prepared for histologic sectioning. Sections were stained with Hematoxylin and Eosin and then analyzed with light microscopy. Hepatocytes under flow conditions formed spheroidal aggregates of cells of 50 to 200 microns in diameter by 24 hours in culture. Hepatocytes in static conditions showed decreased aggregation of cells and spheroid formation was absent. Co-cultured specimens under flow also showed spheroid formation with endothelial cells lining the outside of hepatocyte spheroids. Co-cultured specimens in static culture showed no spheroid formation and no organization between sinusoidal endothelial cells and hepatocytes. These results suggest that continuous flow increases organization of hepatocytes cultured within biodegradable polymer matrices.

Background: An optimal method for hepatocyte transplantation is not yet determined. With the principles of tissue engineering in vitro conditioning of hepatocytes on biodegradable polymer in a flow bioreactor before implantation forming spheroids may achieve increased cell mass and function to replace lost organ function in vivo. Methods: Biodegradable poly-L-lactic (PLLA) polymer discs were seeded with rat hepatocytes in a concentration of 10 × 10⁶ cells per ml and exposed to a medium flow of 24 ml/min for 1, 2, 4 and 6 days. The number and diameter of spheroidal aggregates was measured by phase-contrast microscopy. H&E histology was performed. Albumin production as hepatocyte specific function was determined by ELISA. Results: Spheroids of viable hepatocytes of 50–200 µm in diameter were formed. Both the number and diameter of the spheroids increased during the first 2 days and then remained constant until day 6. Albumin production was maintained throughout the culture period. Conclusion: Short (2– 3 days) pre-transplant conditioning of hepatocytes in a flow bioreactor on biodegradable PLLA resulted in formation of spheroids with a liver-like morphology and preserved specific metabolic function. Tissue engineered hepatocyte spheroids on polymer may represent a functionally active and easy transplantable neotissue and may serve as an in vivo substitute for lost liver function.