【摘要】 目的 :优化原代大鼠肝细胞 (PRH)分离方法与冻存条件。方法 :建立低浓度胶原酶原位循环灌流法分离肝细胞 ;分别采用含 2 %与l0 %DMSO的冻存液于 - 70℃或液氮中冻存PRH ,并比较各组冻存后PRH活力。结果 :1.该分离方法肝细胞产量为 1.5 84± 0 .5 2 5× 10 8/10 0g大鼠体重 ,存活率达 95 .2± 2 .9% ,肝细胞纯度 >95 % ,胶原酶用量减少为 4 .5mg/10 0 g大鼠体重。 2 .含 2 %DMSO冻存液于 - 70℃组与液氮组PRH存活率均可达 90 %～ 95 % ;ALB冻存 14d - 70℃组PRH明显高于液氮组与新鲜分离组 ,LDH值于冻存 7d的 - 70℃组与液氮组明显低于新鲜分离组 ,其它各组间均无显著差异。结论 :低浓度胶原酶原位循环灌流法为高效的PRH分离技术 ;含 2 %DMSO冻存液于 - 70℃可作为原代肝细胞冻存的理想条件。更多还原

【Abstract】 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.更多还原