Ficoll密度梯度离心法分离培养纯化猪的骨髓间充质干细胞

Isolation, cultivation and identification of porcine bone marrow mesenchymal stem cells using Ficoll density gradient centrifugation method

背景:研究报道显示猪骨髓间充质干细胞体外分离方法、效率、培养条件各不相同,并且尚无统一的鉴定标准。目的:建立一种高效、经济的猪骨髓间充质干细胞分离培养及鉴定方法。方法:采集健康猪股骨骨髓,Ficoll密度梯度离心法分离单个核细胞,进行原代和传代培养,流式细胞仪检测细胞膜表面抗原表达情况,并观察细胞成骨、成脂及成软骨分化的能力。结果与结论:分离培养的单个核细胞活力旺盛,体外生长良好,细胞呈梭形、纺锤形和小多角形,具有稳定的增殖分裂能力;细胞膜表面抗原CD29、CD44、CD90呈阳性表达,而CD14、CD34、CD45、CD166、HLA-DR呈阴性表达;在特定诱导条件下,该细胞可分化为成骨细胞、脂肪细胞和软骨细胞。说明采用Ficoll密度梯度离心法成功分离培养出纯化的猪骨髓间充质干细胞,其生长状态良好,具有多项分化潜能,可作为组织工程的种子细胞。

BACKGROUND： Culture condition, isolation method and efficiency of porcine bone marrow mesenchymal stem cells are different, and there is a lack of unified identification standards. OBJECTIVE： To establish a high-efficiency and economical method of isolating, culturing and identifying porcine bone marrow mesenchyrnal stem ceils. METHODS： The bone marrow was collected from healthy porcine femur to isolate mononuclear cells by Ficoll density gradient centrifugation method. After primary and subculture, the cell surface antigens were detected by flow cytometery, and the osteogenic, adipogenic and chondrogenic differentiation abilities of the cells were observed. RESULTS AND CONCLUSION： The isolated mononuclear cells showed exuberant viability, and grew well in vitro. They were spindle- or small polygonal-shaped and had a stable doubling time on culture. Flow cytometric analysis revealed that they were positive for CD29, CD44, CD90, and negative for CD14, CD34, CD45, CD166, HI_A-DR. Furthermore, they were able to differentiate into osteogenic, adipogenic and chondrogenic lineages under specific conditions in vitro. Porcine bone marrow mesenchymal stem ceils isolated by Ficoll density gradient centrifugation method can be purified, grow well in vitro, and have multi-lineage differentiation potentials, which can act as seed cells for tissue engineering research.