A Study On The Large-scale Cryopreservation Of Microencapsulated Hepatocytes For Application In Bioartificial Livers

Acute liver failure, mainly caused by viral or toxin-induced hepatitis in China, remains associated with an unacceptably high mortality rate. Bioartificial liver (BAL), known as a "bridging" device, has been under intense investigation over the years as a way to bridge patients to liver transplantation or spontaneous recovery. A liver cell bank is urgently needed, to provide a sufficient, readily available hepatocyte supply for the application of BAL. It is believed that cryopreservation remains the only viable option for long-term storage of hepatocytes.'Microencapsulated hepatocytes have not only been proposed as promising bioactive agents, for packed-bed or fluidized-bed bioartificial liver assist devices (BLADs), but have also been considered as one of the best ways for hepatocyte cryopreservation currently.The purpose of this thesis is to explore an optimal method for cryopreseravtion of alginate-chitosan (AC) microcapsulated primary porcine hepatocytes and reversibly immortalized human hepatocytes (HepLi4). Additionally, to develop a protocol for large-scale cryopreservation of microencapulated primary porcine hepatocytes and HepLi4 cells.Primary porcine hepatocytes were isolated using a modified four-step collagenase perfusion method, whilst the reversibly immortalized human hepatocytes (HepLi4) were thawed and cultured. These two types of hepatocytes were then microencapsulated. Microencapulated primary porcine hepatocytes were cryopreservated in liquid nitrogen for 40 days with University of Wisconsin (UW) solution, fetal calf serum and 5 different concentrations of DMSO (5%,10%,15%,20%,25%) respectively. After thawing, microencapsulated primary porcine hepatocytes were evaluated on their integrity, mechanical stability, viability and functionality. Microencapulated HepLi4 cells were cryopreservated in liquid nitrogen for 40 days with University of Wisconsin (UW) solution, fetal calf serum and 3 different concentrations of DMSO (10%,15%,20%) respectively. After thawing, the microencapsulated HepLi4 cells were evaluated on their integrity, mechanical stability and viability. Results showed that 15% DMSO with 20% fetal calf serum and 65% UW solutions could be an optimal freezing solution for cryopreseravtion of AC microcapsulated primary porcine hepatocytes and HepLi4 cells.We further studied the large-scale cryopreservation of microencapulated primary porcine hepatocytes and HepLi4 cells using a freezing solution of 15%DMSO with 20% fetal calf serum and 65% UW solutions. Microencapsulated primary porcine hepatocytes and HepLi4 cells were cryopreserved in liquid nitrogen for 40 days in 1.8 ml and 50 ml vials, respectively. After thawing, we compared the microencapsulated primary porcine hepatocytes in two vials on their integrity, mechanical stability, viability and functionality. In order to test functionality, microencapsulated primary porcine hepatocytes were assessed on their capacity for albumin secretion, ammonia removal and diazepam clearance. Similarly, microencapsulated Hepli4 cells were tested for mRNA expression of GS,GST,ALB and HBCF-X. Resluts showed that microencapsulated hepatocytes cryopreserved in the 50 ml vial may be a better option for large-scale cryopreservation.In this study, we have elucidated an optimal freezing solution for cryopreseravtion of AC microcapsulated hepatocytes and presented a protocol for the large-scale cryopreservation of microencapsulated hepatocytes. This work contributes to the development of a BAL system which may be readily applied to a variety of situations and locations.