Objective:To optimize the techniques for isolation and cryopreservation of primary rat hepatocytes.Methods:By improving traditional isolation methods and establishing an in situ recirculating collagenase perfusion through the portal vein

we want to improve the yield

viability and purity of primary rat hepatocytes and to reduce the cost of collagenase.Primary rat hepatocytes were cryopreserved at 1×10 7/ml in a suspension buffer containing 2% DMSO or 10% DMSO and were stored for 7 days

14 days or 2 months at -70℃ or in liquid nitrogen.After rapidly thawed hepatocytes were cultured for 7 days

their viabilities were measured by trypan blue exclusion (TBE) and MTT method

and the albumin (ALB) and lactodehydrogenase (LDH) in the supernatant of hepatocytes were analyzed.Results:By using the new technique

the hepatocyte yields have been improved to 1.584±0.525×10 8/100g rat body weight (BW)

the viability of the hepatocytes is 95.2±2.9%

and purities better than 95%.At the same time

the cost of the collagenase has been reduced to 4.5mg/100g rat BW.The efficiency of cryopreservation is:(1)The viabilities of hepatocytes preserved in buffer containing 2% DMSO or 10% DMSO were ≥90% and ≥85% respectively.(2)The viabilities of hepatocytes cryopreserved in buffer containing 2% DMSO at -70℃ or in liquid nitrogen were all 90%-95%.(3)ALB synthesis was unaffected by different cryopreservation methods.(4)LDH in the supernatants of freshly cultured hepatocytes was equivalent to that of cryopreserved hepatocytes.Conclusion:Isolation technique of primary rat hepatocytes has been successfully improved.Freshly isolated hepatocytes suspended in buffer containing 2% MDSO can be cryopreserved for 2 moths at -70℃.These results have showed so far that the methods of isolation and cryopreservation of primary rat hepatocytes are optimized.