分离培养方法及冻存技术对犬骨髓间充质干细胞生长和增殖的影响

Effects of isolation and culture method and frozen technique on the growth and proliferation of dog bone marrow mesenchymal stem cells

背景:骨髓间充质干细胞在体外长期培养易发生自发分化,而失去多分化潜能,因此必须对培养的细胞及时冻存,需要时再进行复苏。目的:建立犬骨髓间充质干细胞体外分离、培养、增殖、冻存的方法。设计、时间及地点:对照观察细胞学实验,于2005-06/2008-06在浙江中医药大学附属第一医院骨伤研究所完成。材料:健康成年雄性Beagle犬用于骨髓间充质干细胞的提取。方法:采用密度梯度离心法结合贴壁培养法分离纯化骨髓间充质干细胞,并进行传代扩增培养。将第2代骨髓间充质干细胞与12.5%DMSO混匀置于冻存管中,采用慢冻快融法进行冻存和复苏:将冻存管置于-4℃冰箱2h,然后移入-30℃冰箱2h,再置于厚壁塑料泡沫盒中,扎紧密封,置-80℃冰箱过夜后,取出冻存管直接放入液氮中保存,或放入-80℃冰箱保存。复苏时将冻存管从液氮或-80℃冰箱中取出,立即置37℃水浴并轻轻摇动,1～2min内迅速解冻。主要观察指标:复苏前后细胞成生长活性及形态变化。结果:分离的骨髓间充质干细胞为以均一的梭形的成纤维细胞样贴壁生长,贴壁及增殖能力强,传代细胞贴壁较快。经过液氮长期冻存后,将复苏后的骨髓间充质干细胞再培养,细胞形态及生长状态与冻存前无差异,均呈长梭形均匀分布生长,细胞生长增殖旺盛。结论:采用密度梯度离心法结合贴壁培养法可获得犬骨髓间充质干细胞,应用慢冻快融技术进行冻存和复苏,较好的保持了冻存细胞的活力和功能。

BACKGROUND： Bone marrow mesenchymal stem cells （BMSCs） cultured in vitro are easy to spontaneously differentiate, and lose the potential of multi-directional differentiation. Thus, cultured BMSCs should be frozen in time, and were resuscitated when needed. OBJECTIVE： To explore the optimal methods of isolating, culturing, proliferating and freezing of dog BMSCs in vitro. DESIGN, TIME AND SETTING： The control cytology experiment was performed at the Institute of Orthopadedics, First Hospital Affiliated to Zhejiang University of Traditional Chinese Medicine from June 2005 to June 2008. MATERIALS： Healthy adult masculinity Beagle dogs was used for extracting BMSCs. METHODS： BMSCs were purified by density gradient centrifugation and adhesive culture methods, which were cultured and expanded in flasks. At the second passage, BMSCs were incubated in 12.5% dimethyl sulphoxide （DMSO） in a freezing tube. Freezing and resuscitation were performed by slow freezing and rapid thawing method. Freezing tube was placed at -4 ℃ for 2 hours, and at -30 ℃ for 2 hours, and then sealed up in a thick plastic foam box at -80 ℃, overnight. Freezing tube was kept in liquid nitrogen or at -80 ℃. During resuscitation, freezing tube was immediately stored at 37 ℃ in a water bath, slightly shake, rapidly thawed within 1-2 minutes. MAIN OUTCOME MEASURES： Growth activity and morphological changes before and after resuscitation. RESULTS： BMSCs in culture were uniformly spindle in appearance and showed active proliferative capacity. Passage cell adhered fast. After long-term freezing in liquid nitrogen, BMSCs were incubated after resuscitation. No significant difference in cell morphology and growth state was detected compared with that before freezing, showing spindle shape and active proliferation. CONCLUSION： Dog BMSCs can be collected using density gradient centrifugation and adhesive culture methods. Freezing and resuscitation are conducted by slow freezing and rapid thawing method, which greatly keeps the activity and function of frozen cells.