不同冻存时间与温度对兔骨髓基质干细胞存活率的影响  

Effects of different cryopreserved time and temperatures on survival rate of bone mesenchymal stem cells in rabbits

作　　者：张文元[1] 杨亚冬[1] 房国坚[1] 陈勇[1] 

机构地区：[1]浙江省医学科学院生物工程研究所,浙江省杭州市310013

出　　处：《中国临床康复》2006年第13期44-46,i0003,共4页Chinese Journal of Clinical Rehabilitation

基　　金：浙江省科技计划重大项目(2004F11018)~~

摘　　要：目的:以二甲基亚砜作为保护剂,采用慢冻快融法观察不同温度及不同时间冻存的兔骨髓基质干细胞复苏后存活率的变化。方法:实验于2003-09/2005-06在浙江省医学科学院生物工程所完成。①选取清洁级3月龄新西兰大白兔5只,抽取兔髂骨及胫骨的骨髓液,密度梯度离心法结合贴壁培养法分离纯化出兔骨髓基质干细胞,并进行增殖。②取第3代骨髓基质干细胞,经消化后离心重悬,用完全培养液调整细胞浓度为6×109L-1。将质量浓度为250g/L的二甲基亚砜缓慢滴入同体积含骨髓基质干细胞的上述培养液中,混匀,二甲基亚砜的终浓度为125g/L,骨髓基质干细胞的终密度为3×109L-1,1mL/安瓿冻存。③4℃冻存直接将安瓿置4℃冰箱中;-20℃及-60℃冻存,则将安瓿置于有脱脂棉的聚丙烯泡沫盒中,壁厚1.5cm,扎紧密封,各置-20℃及-60℃冰箱过夜,取出后分别直接放于-20℃及-60℃冰箱保存;-196℃液氮冻存,则将置有安瓿的泡沫盒于-60℃冰箱过夜后,取出冻存安瓿直接放于液氮中保存。骨髓基质干细胞分别于4℃、-20℃、-60℃、-196℃条件下进行3d,10d,1,3,8个月以及1年的冻存。④经不同温度及不同时间冻存后,给予复苏。复苏时将冻存安瓿从液氮或冰箱中取出,立即置37℃水浴并轻轻摇动,约1min迅速解冻。含骨髓基质干细胞的冻存液经5倍于冻存液量的完全培养液稀释后,离心重悬,进行骨髓基质干细胞存活率测定。复苏后的骨髓基质干细胞以1×104/cm2用完全培养液再培养,观察骨髓基质干细胞的形态变化及生长活性情况。结果:实验选取3月龄新西兰大白兔5只,全部进入结果分析。①兔骨髓基质干细胞原代培养情况:培养3d时,骨髓基质干细胞数量较少,散在分布,呈短梭形形态。培养1周时细胞形成大量的小集落,细胞变长。此后集落细胞迅速增多,逐渐汇合成片,形态为均一的长梭形,呈旋涡状排列,2周时�AIM： To observe the change in the survival rate of rabbits＇ bone mesenchymal stem cells （BMSCs） cryopreserved at different temperatures and times by freeze-thaw method and dimethyl sulfoxide （DMSO） as protective agent. METHODS： The expe.riment was conducted in the Institute of Biological Engineering, Zhejiang Academy of Medical Sciences between September 2003 and June 2005. ①Five New Zealand rabbits aged three months, of cleaning grade were selected and the bone marrow fluid was extracted from ilium and tibia. The BMSCs were isolated and purified by a combination of density gradient centrifugation and adhesive culture, and the proliferation was performed.②The BMSCs of third passages were obtained and harvested by centrifugation and suspended after digestion, with the concentration adjusted to 6×10^9 L^-1 by complete culture medium. The DMSO with the concentration of 250 g/L was slowly dribbled into the culture medium containing BMSCs of same volume. After the mixture, the final concentration of DMSO was 125 g/L and BMSCs density was 3×10^9 L^-1,cryopreservated in 1 mL/ampule.③ The 4℃ cryopreservation： The ampule was kept for cryopreservation in the refrigerator at 4 ℃;The cryopreservation at -20 ℃ and -60 ℃： The ampule was put in the polypropylene bubble box containing absorbent cotton, with the width of 1.5 cm and enclosed tightly. The ampule stayed overnight and then preserved in refrigerators at -20 ℃ and -60 ℃ respectively; The -196 ℃ liquid nitrogen cryopreservation： The bubble box containing ampule stayed overnight in refrigerator at -60 ℃, and then the freezed ampule was taken out and preserved in the liquid nitrogen. The BMSCs were cryopreserved at 4 ℃,-20 ℃,-6 ℃,-196 ℃ and for different times respeetively （3 days, 10 days, l month,3 months,Smonths and 1 year）. ④ The ampule was thawed after cryopreservations of different temperatures and times. Taken out of liquid nitrogen or refrigerator, the freezed ampule was immediately put in water bath at 3

关 键 词：干细胞 骨髓细胞/细胞学 低温保藏 二甲亚砜 复苏术 存活率 兔 

分 类 号：R394.2[医药卫生—医学遗传学]