乳猪肝细胞-196℃保存的初步研究  

A preliminary study of cryopreservation of suckling porcine hepatocytes at -196℃

作　　者：刘鸿凌[1] 王英杰[1] 郭海涛[1] 王宇明[1] 于乐成[1] 刘俊[1] 谭朝霞[1] 

机构地区：[1]第三军医大学附属西南医院全军感染病研究所,重庆400038

出　　处：《第三军医大学学报》2003年第6期472-474,共3页Journal of Third Military Medical University

基　　金：国家自然科学基金资助项目 ( 30 0 2 70 0 1) ;全国优秀博士基金资助项目 ( 19994 7)

摘　　要：目的　探索适合于乳猪肝细胞 -196℃冷冻保存的条件和方法。方法　体外分离新生乳猪肝细胞 ,以含不同浓度二甲亚砜 (DMSO)的冻存液及不同的细胞浓度在液氮中保存。 2个月后复苏接种培养 ,对其存活率、贴壁率、尿素合成能力、猪白蛋白mRNA的表达等进行检测 ,并观察其形态结构变化。结果　不同DMSO浓度保存猪肝细胞复苏后的活力及形态均有所不同 ,其中含 5 %DMSO冻存液组保存效果最差 ;10 %组次之 ;15 %组最佳。 15 %DMSO冻存液组复苏后肝细胞的存活率为 ( 83± 4) % ,贴壁率是 ( 81± 5 ) % ,细胞形态与未冻存组相似 ,均保持较强的尿素合成能力及猪白蛋白mRNA的表达 ,较 5 %与 10 %DMSO冻存液组有显著性差别 (P <0 .0 5 )。冻存时肝细胞浓度为 ( 5～ 10 )× 10 6个 /ml组复苏后细胞存活率明显优于 ( 1～ 2 .5 )× 10 6个 /ml组。结论　 15 %DMSO浓度适合于乳猪肝细胞的长期保存。Objective To explore the conditions and methods for cryopreservation of suckling porcine hepatocytes at -196 ℃. Methods Porcine hepatocytes were harvested by modified two step perfusion collagenase method, and then stored in 5%, 10%, 15% or 20% DMSO cryopreservation medium at different concentrations of hepatocytes. After 2 months of cryopreservation, the hepatocytes were thawed and inoculated for culture and then the viability, attachment rate, drug metabolic function and expression of porcine albumin mRNA were detected and the intracellular structure changes were observed with light microscope. Results There were differences in cell viability and forms between different concentrations of DMSO for the cryopreservation of porcine hepatocytes. After storage in 5% DMSO cryopreservation medium, cell viability and attachment rate were significantly reduced, but the hepatocytes stored in 15% DMSO cryopreservation solution kept their function well. Porcine albumin mRNA, extracellular LDH and drug metabolic reaction were close to those found in fresh hepatocytes. Significant difference was found between 15% DMSO group and 5% and 10% DMSO groups( P <0.05). Conclusion The ideal concentration of DMSO for -196 ℃ cryopreservation of porcine hepatocytes is 15%. The cryoprotective effect of hepatocytes in high concentration is better than that in low concentration.

关 键 词：猪肝细胞 培养 冻存保护剂 二甲亚砜 

分 类 号：R318.14[医药卫生—生物医学工程] R318.52[医药卫生—基础医学]