HETEROTOPICALLY TRANSPLANTED HEPATOCYTES PRODUCE LIVER NEO-TISSUE WITH PROLIFERATIVE ACTIVITY AND EXPRESSION OF A MATURE BILE CANALICULI NETWORK

Abstract

P1218 Aims: As previously reported, we performed flow culture of hepatocytes on a biodegradable polymer matrix and observed formation of spheroids, which in vitro created a bile canaliculi network. We were now interested in their in vivo behavior. Therefore we designed a long term implantation study and compared hepatocyte transplantation without pre-culture with pre-cultured hepatocyte spheroid transplantation in a heterotopic matrix based transplantation model. Methods: Primary hepatocytes were isolated from male Lewis rats using a two step collagenase digestion method. Highly porous poly(L-lactic acid) (PLLA) sponges were seeded with 4 x 106 hepatocytes. They were transplanted onto the mesentery of Lewis rats either immediately after cell seeding, or after 3 days of culture. For hepatotrophic stimulation a porto-caval shunt operation was performed 2 weeks before hepatocyte transplantation. After 1 week, 1, 3, or 6 months the specimens were harvested and underwent histological, immunohistochemical, immunofluorescent and morphometric analysis. For statistical analysis the nonparametric Kruskal-Wallis test was used, assuming p< 0,1 as statistically significant. Results: Newly formed liver tissue could be found in all implants and was positive for PAS, Hepar 1, and Cytokeratin 18. Phaloidin immunofluorescent studies showed expression of a liver like bile canaliculi network in the spheroid group after 1 week in vivo. The non pre-cultured group expressed only some bile canaliculi at that time point. After 1 month no difference in expression between the two groups could be observed. The HE staining showed no sign of intracellular cholestasis throughout the observation period of 6 months after transplantation. Proliferation activity of the hepatocytes was demonstrated in both groups by PCNA stain and morphometric analysis, which showed an increase of the total heterotropic hepatocyte area. With p=0,023 (pre-cultured spheroids) and p=0,051 (non pre-cultured) for the monte-carlo-significance a statistically significant difference was shown with increasing rank order over the incubation time. After 6 months, the transplantation efficiency was twice as high in the spheroid group (90 ± 39%) compared to the non pre-cultured group (40 ± 25%). Conclusions: Matrix based heterotopic hepatocyte and hepatocyte spheroid transplantation led to liver neo-tissue with in vivo formation of a bile canaliculi network and long term proliferation activity. As there are no signs of cholestasis within the heterotopically formed liver neo-tissue even after 6 months in vivo, a removal of bile must be assumed. Transplantation of pre-cultured hepatocyte spheroids led to a higher efficiency and proliferation, compared to non pre-cultured hepatocyte transplantation. Pre-implantation tissue assembly, including the formation of a bile canaliculi network could possibly explain this advantage.