The Preliminary Study Of Establishing A Hepatocyte Bank

Objective:To explore a hepatocyte isolation technique of benign lesions of the adult liver with surgical excision,in order to improve the output of adult primary hepatocytes,purity,and viability.To detect the biochemical changes of adult primary hepatocytes in the process of cultivation and hepatocytes transdifferentiation to bililary epithelial cell line(BEC s).To explore the appropriate cryopreservation protocol.Methods:Isolate hepatocytes from the livers of patients with intra-and extrahepatic bile duct stones with surgical excision,and fresh adult primary hepatocytes are counted by blood count plate,trypan blue staining technology and flow cytometry instrument living cell fluorescence staining technology detect the viability of fresh adult primary hepatocytes.We use an improved two-step extracorporeal collagenase perfusion technique for isolatation.Fully automatic biochemical analyzer test biochemical changes of adult primary hepatocytes in the process of cultivation.Cell immunofluorescence technique and Bile acid analogues of fluorescent tags CDFDA detect hepatocytes transdifferentiation to bililary epithelial cell line(BECs).Freeze-stored liquid With different concentrations of DMSO and serum cryopreserve adult primary hepatocytes,trypan blue staining technology and flow cytometry instrument living cell fluorescence staining technology detect cryopreserved adult primary hepatocytes activity after recovery.Results:The quantity of fresh adult primary hepatocytes count up to 109,viability detection of fresh adult primary hepatocytes with trypan blue staining was 85%,and with the flow cytometry instrument living cell fluorescence staining was 84%.in the process of cultivation,ALT,AST and LDH leakage rate rise after a downward trend,in 3 days,to reach the lowest,albumin secretion reached the highest in 3 days.fluorescent substances completely Can't see in 4 days with immunofluorescence technique,fluorescent clues appear in uniform distribution in 5 days,fluorescent clues became wider in 6 days.fluorescent scattered point appear in 4 days with CDFDA,fluorescent clues appear in uniform distribution in 5 days,fluorescent clues became wider in 6 days.The activity after recovery of the level of serum 20%+DMSO10%+culture medium group,is much higher than the other conditions.Conclusion:1.Improved two-step extracorporeal collagenase perfusion technique for adult primary hepatocytes isolatation is simple,short,efficient,inexpensive,2.Biochemical properties of adult primary hepatocytes in the process of cultivation is best on the third day and The fourth day,suitable for bioartificial liver and hepatocyte transplantation.3.Adult primary hepatocytes in the process of cultivation made transdifferentiation to bililary epithelial cell line(BEC s),preliminary formation in the fifth day,the sixth day further.4.The viability of adult primary hepatocytes significantly decreased after recovery,The viability of hepatocytes in the level of serum 20%+DMSO10%+culture medium group is highest.