兔外周血间充质干细胞的分离培养及冻存

目的建立兔外周血间充质干细胞（MSCs）体外分离培养及冻存的方法。方法用密度梯度离心法及贴壁培养法分离纯化兔外周血MSCs，MSCs经液氮冻存半年复苏，观察细胞冻存前后的生长状况，并用台盼蓝法测定其存活率。结果MSCs于体外培养2h开始贴壁生长，细胞形态为梭形、多角形及纺锤状等。传代MSCs变为形态均一、排列有序的长梭形细胞，增殖活跃；MSCs经6mo冻存后复苏培养，台盼蓝法计数存活率达95％以上，细胞形态及生长状况与冻存前无明显差别，均呈长梭形，细胞生长增殖旺盛。结论采用密度梯度离心法和贴壁法可以有效获得应用G-CSF动员后的外周血MSCs，且细胞纯度高，生长增殖活性好，采用液氮冻存MSCs的方法十分有效可行。

Isolated culture and cryopreservation of rabbit mesenchymal stem cells (MSCs) from peripheral blood

Objective To explore the optimal methods of isolating, puring, and cryopreservating of rabbit mesonchymal stem cells from peripheral blood. Methods Isolate and culture the MSCs from the mobilized peripheral blood of rabbit by density gradient centrifugation and adhesive culture methods;Store MSCs in liquid nitrogen for six months then thaw it. Oserve the growth condition of MSCs before and after cryopreserved,then detect their survival rate by trypan blue staining test.Results MSCs not only had the characteristics of adherence growing after cultured for 2h but also morphologically became spindle, polygon and fusiform. Subeultured MSCs grew uniform spindle and showed actively proliferative capacity. After MSCs were cryopreserved in liquid nitrogen for six months, their viability was 95% by trypan blue staining test. The cell morphous and growth condition had no obvious difference after thawing. Conclusions High pured MSCs can be obtained by gradient centrifuging and adherent method from peripheral blood that are mobilized by G-CSF. Futhermore,the MSCs show active proliferative capacity,and it can be stored by cryopreserved.