兔骨髓间充质干细胞的体外分离培养、低温冻存及复苏研究

Isolation,biocytoculture and cryopreservation of rabbit bone marrow-derived mesenchymal stem cells in vitro

目的寻求体外培养、冻存、复苏兔骨髓间充质干细胞(bone marrow mesenchymal stem cell,BMMSC)的方法,观察体外培养BMMSC的形态及生长特性,研究低温冻存对兔BMMSC生长的影响,为兔BMMSC进一步的实验研究打下基础。方法取新西兰白兔胫骨穿刺获取骨髓液,密度梯度离心法联合贴壁培养法体外纯化扩增,并行液氮冻存3个月。通过倒置显微镜观察其生物学表现,并进行细胞增殖活性分析,绘制生长曲线。结果兔BMMSC为贴壁生长,形态为均匀成纤维细胞样,增殖能力强,传代及冻存后细胞仍然保持其生物学特性。结论兔BMMSC可采取密度梯度离心法联合贴壁培养法进行较好地纯化和扩增,并可长期保存于液氮中,在一定的条件下可复苏存活,冻存不影响细胞的增殖能力。

Objective To explore an optimal isolation, purification and cytopreservation method for rabbit bone mar- row mesenchymal stem ceils. Methods Bone marrow mesenchymal stem cells were obtained from bone marrow cavity and purified via density gradient centrifugation and cultured. The culture-expanded bone marrow mesenchymal stem cells were copreserved in - 196 - liquid nitrogen for 3 months. Following recovery from cryopreservation, cell growth ca- pacity was determined. Light microscopy was used to study the morphologic features of bone man＇ow mesenchymal stem cells. The owth curve of cell proliferation was obtained based on the observation of the proliferation status of l st to 3th generation and recovered bone marrow mesenchymal stem cells . Results The isolated bone man＇ow mesenchymal stemcells were well grown mid adhered to wall 48h after culture in a fibroblastic morphology.. After cry. opreservation and tha- wing, the bone marrow mesenchymal stem cells grew in a fibroblastic morphology with a similar growth curve to that be- fore cryopreservation. Conclusion Bone marrow mesenchyma stem cells of rabbit were isolated successfully using den- sity gradient centrifugation combined with adherence method. Bone marrow mesenchymal stem cells of rabbit can maintain their biological features after cryopreservation and thawing.