Neonatal Rat Hepatocytes Transplantation The Joint Ralr Treatment Of Severe Hepatitis

Objective: Lots of literatures had reported the methods that digested the liver from mammals through non-perfusion for hepatocyte preparation, but hepatocyte viability and yield obtained by above methods were not satisfactory,moreover, contaminated with lots of other cells so that further research was hampered. So, we tried to improve method of isolation and purification in order to establish the foundation for hepatocyte transplantation and quested the best way of hepatocyte cryopreservation and revivfication and assessed the viability of hepatocyte cryopreserved in order to resolve the shortage of donor cell in future clinic. Methods: Performing enzymatic digestion of newborn rat liver tissues by three sequential steps in vitro and gaining supernatant, we applied discontinous gradient centrifugation method to purify hepatocytes, and the living times of hepatocytes were compared when cultured with rALR or not.We analyzed the histogram of DNA of newborn rat hepatocyte and primary adult rat hepatocyte for cell cycle study by flow cytometer. We used different concentrations DMSO as cryopreservation solutions for hepatocyte, revived hepatocytes at various intervals in our experiment, and assessed the viability of hepatocyte cryopreserved by trypan blue exclusion. Results: The hepatocyte viability using our non-perfusion methodwas 85% approximately by trypan blue exclusion,and the hepatocyte purification reached above 98% to avoid the contamination with mesothelial cell. Through the comparison of adding rALR to culture or not,we found out the living time of hepatocyte cultured with rALR was 5 days longer than without rALR. The histogram of DNA showed that the percent for G2-M phase of newborn rat hepatocyte cultured with rALR was higher (13.49% VS 11.56%) compared to that without rALR.At the same time, the results for primary adult rat hepatocyte was 4.57%vs4.12% . The function of albumin synthesis could have kept at the high levels for 9 days. The best way for cryopreservation was adding 10% DMSO in the medium for 3 weeks. Conclusions: The newborn rat's hepatocyte isolated and purified by our method had a good function, which could be used as donors for hepatocyte transplantation. It may be useful for the near future clinic applications that the investigation of hepatocyte's cryopreservation and revivfication. KEY WORDS: newborn rat;hepatocytes;isolation and purificatrion;cryopreservation...