兔骨髓基质细胞的体外培养及鉴定

背景：骨髓基质细胞的分离纯化、细胞培养、细胞标记、诱导因子、基因转染对细胞生物学影响、细胞载体构建及细胞复合物回植时间的选择等尚处于实验研究阶段。 目的：摸索一种较为简便且可有效获得骨髓基质细胞的体外培养方法。 设计、时间及地点：细胞学体外实验，于2006-06/2007-01在中国科学院北京基因组研究所完成。 材料：SPF级6周龄新西兰大白兔8只，由中国科学院遗传发育研究所实验动物中心提供。 方法：无菌条件下抽取兔骨髓1 mL，D-Hanks液稀释后，离心弃上清，DMEM培养基重悬，制备单细胞悬液，叠加在密度为1.077的等体积淋巴细胞分离液的液面上，离心后取界面云雾状的单个核细胞层，用含有20%胎牛血清的DMEM培养基重悬。以1×10/cm2接种后贴壁法纯化细胞，待70%~80%长满单层后消化传代。 主要观察指标：倒置显微镜下观察细胞生长情况，锥虫蓝染色计数活细胞，苏木精-伊红染色对细胞进行鉴定，MTT法检测细胞活性，观察传代培养的细胞冻存后复苏情况。 结果：接种24 h后细胞开始贴壁，呈短梭形或三角形，且有长短不等的细胞突起；3 d后贴壁细胞开始分裂增殖，部分区域形成细胞团簇；1周后大部分细胞呈成纤维状散落在培养瓶底生长；传代后细胞均匀分布，形态呈细长梭形，并沿一定方向排列，1 mL骨髓基质细胞悬液传3代后的贴壁细胞数可达5×106~1×107数量级。骨髓基质细胞成活率约为90%，经鉴定为单核细胞，接种后1~6 d细胞持续旺盛生长，至第8天细胞活性最高，随后生长活性下降。复苏冻存56 d的细胞，其生长良好，增长迅速。 结论：以淋巴细胞分离液梯度离心后可获得纯度较高的骨髓基质单核细胞，经体外扩增培养能够得到足够数量的种子细胞，冻存复苏后细胞活性无变化。

Culture and identification of rabbit marrow stromal cells in vitro

BACKGROUND: The study of isolation, purification, culture, cell labeling, inducing factors, effects of gene transfection on cytobiology, cell carrier construction, and time window for back transplantation of cell compound pertaining to marrow stromal cells (MSCs) is still in its infancy. OBJECTIVE: To search for an in vitro culture method that can be simply and effectively obtained . DESIGN, TIME AND SETTING: The present cytological in vitro experiment was performed at the Beijing Institute of Genome, Chinese Academy of Sciences between June 2006 and July 2007. MATERIALS: Eight specific pathogen-free New Zealand rabbits, aged 6 weeks, were provided by the Laboratory Animal Center, Institute of Genetics and Development, Chinese Academy of Sciences. METHODS: Under sterile condition, 1 mL rabbit bone marrow was taken and diluted with D-Hanks solution. Following centrifugation and subsequent supernatant removal, bone marrow was re-suspended using dulbecco's modified eagle's medium (DMEM) for single cell suspension. Next, single cell suspension was dropped onto the liquid surface of equal-volume lymphocyte separation medium (density: 1.077). Subsequent to centrifugation, cloudlike mononuclear cell layer was collected and re-suspended with DMEM containing 20% fetal bovine serum. The cells were inoculated at 1×10/cm2 and purified by adherent method. When 70%-80% of flask bottom was covered, cell digestion and passage was performed. MAIN OUTCOME MEASURES: Cell growth was observed with an operating microscope. Surviving cells were counted by Trypan blue viability test. Cell identification was performed by hematoxylin-eosin staining. Through the use of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay, cell viability was detected to observe the cellular resuscitation of the cultured cells following cryopreservation. RESULTS: Twenty-four hours after inoculation, cells began to adhere to the wall, exhibiting short shuttle- or triangle- shaped appearance with different sizes of cellular processes. Three days later, adherent cells began to divide, and cell clusters could be found in some areas; One week later, most of cells exhibited scattered fibroblast-like growth; After passage, cells were evenly distributed with long shuttle-shaped appearance and arranged orderly. Following 3 passages, there wound be 5×106-1×107 adherent cells in 1 mL MSC suspension. Approximately 90% of MSCs survived and identified as mononuclear cells. Cells vigorously grew at days 1-6 after inoculation and reached a peak level at day 8, followed by a viability decline. After 56 days of resuscitation, frozen cells displayed a good and rapid growth. CONCLUSION: Highly purified MSCs can be acquired by gradient centrifugation of lymphocyte separation medium. Enough seeded cells can be obtained by in vitro culture and the cellular viability does not change after frozen preservation and resuscitation.