| Objective To investigate the influence of HSC on the growth, morphology and metabolism of hepatocytes by microcarrier-attached-coculture hepatocytes with HSC, accordingly harvesting the nutrition support data for primary cultured hepatocyte in vitro. To evaluate the therapeutic effects of intraperitoneal transplantation of microcarrier-attached hepatocytes with HSC to rats with acute liver failure (ALF) induced by D-galactosamine (D-GalN), supporting clinic application of hepatocyte transplantation and bioartificial liver theoretically.Methods We cultivated hepatocytes isolated from SD rats'livers by means of semi-in-situ enzyme separation, and observed their growth, morphology and metabolism so as to ascertain the optimal time for HCT. And then we intraperitoneally transplantated HC and HSC to rats with ALF which were induced by D-GalN. The effects of intraperitoneal transplantation were evaluated by comparing their liver function and the pathologic changes of hepatic and peritoneum tissue. We also compared the survival rate of the three groups during two weeks after transplantation.Results We could collected (13.4±4.3)×107 hepatocytes per rats with a high viability(89.2%±9.6%)owing to semi-in-situ enzyme separation method. The morphologial characterstics and synthesis abilities of blbumin and urea can maintain for above one week. There is the least LDH leakage level and highest albumin and urea level on the third day. So we chose the hepatocytes with HSC which were cultured in vitro for three days for HCT. The survival rate of group HCT was much higher than those of the group ST during two weeks after transplantation, and group HCT-HCT's was the highest, the discrepancy between three groups was significant (P<0.05). During 7 days after transplantation, the hepatic function of group HCT and group HSC-HCT was significant better than group ST(P<0.05). Three days after intraperitoneally transplantation , the livers of rats in group ST show unconspicuous pathological recovery. Pathological recovery occurs to some extent in the livers of model rats in groupHCT and group HSC-HCT. Peritoneum tissues with HE staining prove that hepatocytes and HSC can survive about a week in peritoneal cavity.Conclusion Isolating hepatocytes from SD rats by semi-in situ enzyme separation method is reliable. In the microcarrier-attached culture system, there is a least LDH leakage and highest albumin and urea level on the third day, so the third day may be the optimal time of applying microcarrier culture hepatocytes for HCT. Co-cultured with HSC, the function of the liver can maintain on a higher level for a longer time. No matter intraperitoneally transplanted microcarrier attached cultured HC or HC/HSC, it could provide sufficient metabolic support to the damaged recipient liver, so it relieved the damage to the liver and obviously improved the hepatic function and increased the survival rates of rats with ALF. Comparing between two groups, the effect of the one intraperitoneally transplanted microcarrier attached co-cultured HC with HSCs was better.
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