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

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周时细胞长成单层。②兔骨髓基质干细胞传代培养情况:刚传代的骨髓基质干细胞呈圆形,数小时后迅速贴壁,重新成为梭形。之后细胞呈长梭形均匀分布生长,形态比原代培养更均一,细胞生长旺盛、增殖迅速。连续传代22代无明显变化。但随传代次数增多,细胞形态出现多样性,逐渐呈老化现象。③不同温度不同冻存时间下复苏后的兔骨髓基质干细胞存活率的比较:兔骨髓基质干细胞液氮保存1年,存活率为79%;-60℃保存1,3,8个月的存活率分别为67%,38%和0%;不宜于4℃及-20℃保存。④复苏后的骨髓基质干细胞再培养情况:复苏后的骨髓基质干细胞细胞形态及生长状况与冻存前无明显差异,呈长梭形生长,增殖旺盛。结论:分离纯化的兔骨髓基质干细胞生长增殖能力强,短期保存可用-60℃冻存,长期需液氮保存,为择期进行骨髓基质干细胞实验和应用提供实验依据。

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 37 ℃ and shaken lightly, then thawed quickly about one minute later. After diluted by the complete culture medium of five times larger, the cryopreservation liquid containing BMSCs was suspended and centrifugated, and the revival rate of BMSCs was detected. The thawed BMSCs were subcultured by the complete culture medium of 1×10^4 cells/cm^2. The form change and the proliferation of BMSCs were observed. RESULTS： Totally 5 New Zealand rabbits of three months were involved in the result analysis. ①The primary culture status of BMSCs in rabbits： When cultured for three days, BMSCs decreased in number and showed the scattered distribution in short fusiform shape. At one week after culture, the cells formed mass microcolonies with the increasing length. Then the colony cells increased remarkably and gradually converged into pieces, with the uniform long fusiform shape and whirlpool arrangement.②The subcultured status of BMSCs in rabbits： The passaged BMSCs were round and pasted to the wall several hours later, and shaped the fusiform again. Then with the long fusiform shape that was more uniform than that by primary culture, the cells growed productively and showed active proliferative capacity. However, the shape of cells turned to diversity and gradually aged phenomena, with the passage time increased. ③Comparison of survival rates of revivified BMSCs at different cryopreserved temperatures and times： When BMSCs were cryopreserved in liquid nitrogen for one year, the viability was 79%; when stored at -60 ℃ for 1, 3, and 8 months, the viabilities were 67%, 38%, and 0% respectively; It was not suited to store BMSCs at 4 ℃ or -20 ℃.④The subculture status of thawed BMSCs： There was no significant difference in the shape and growth of thawed BMSCs compared with before cryopreservation, the BMSCs with the high prolifera7 tion were in the fusiform shape. CONCLUSION： The isolated and purified BMSCs have active proliferative capacity, and can be cryopreserved in liquid nitrogen for long term and at -60 ℃ for short term, which provides experimental data for the timechoice of the BMSCs experiment and BMSCs application.