骨髓间充质干细胞无血清培养

In vitro culture of bone marrow-derived mesenchymal stem cells in a chemically-defined serum-free medium

为建立一种化学成分明确的、能用于体外扩增骨髓间充质干细胞的无血清培养基,且骨髓间充质干细胞经无血清培养扩增后仍能保持其多向分化的潜能。采用密度梯度离心结合贴壁法从1月龄新西兰大白兔股骨中分离骨髓间充质干细胞,比较在含10%胎牛血清的培养基(SCM)和自制的化学成分明确的无血清培养基(CDSFM)中骨髓间充质干细胞的形态、增殖能力,以及扩增后的骨髓间充质干细胞的细胞周期、集落形成能力和成骨、成脂肪分化能力。经过10d的培养,骨髓间充质干细胞在自制的无血清培养基中扩增了50倍,在含10%胎牛血清的培养基中扩增了40倍。在无血清和有血清培养基中扩增后的细胞中G0/G1期比例分别为(80.31%±0.58%)和(75.24%±4.05%),两者无显著差异(P>0.05)。无血清培养扩增后的骨髓间充质干细胞集落形成率(12.7%±4.0%)低于有血清培养组(28.7%±4.2%),两者比较差异显著(P<0.01)。经过无血清培养扩增的骨髓间充质干细胞在成骨、成脂肪诱导分化培养基中能够分化成成骨和脂肪细胞。自制的化学成分明确的无血清培养基能够在体外培养扩增骨髓间充质干细胞,并且维持其干细胞特性,可以用于细胞治疗以及生物医学研究。

This study is aimed to design a chemically-defmed serum free medium （CDSFM） to support in vitro culture of bone marrow-derived mesenchymal stem cells （BM-MSCs）. BM-MSCs were isolated from the femoral bones of one month old New Zealand Rabbits with density gradient centrifugation. We compared the proliferation capability, cell cycle, colony-forming efficiency, osteogenic and adipogenic differentiation capabilities of BM-MSCs cultured in CDSFM with those cultured in serum-containing medium （SCM）. After 10 days culture, BM-MSCs were expanded by 50 folds in CDSFM, while only 40 folds in SCM. EGF, bFGF and hy-drocortisone were the most important additives and significantly stimulated BM-MSCs proliferation. The percentage of cells at G0-G1 cell cycle was 80.31% ±0.58% after CDSFM culture, with no significant difference （P〉0.05） compared to 75.24% ± 4.05% for SCM culture. However, the cloning efficiency of BM-MSCs cultured in CDSFM was significantly lower than that in SCM （P〈0.01）. The expanded BM-MSCs in CDSFM preserved differentiation potentials into mesenchymal lineages in vitro, including adipocytes and osteoblasts. We have designed a chemically-defined serum free medium that could support in vitro proliferation and maintain the properties of BM-MSCs as stem cells, which could be applied to cell-based therapy and biomedical research.