梯度血清递减体外培养人脐带间充质干细胞

Human umbilical cord mesenchymal stem cells cultured in serum-decremental medium in vitro

背景：人脐带间充质干细胞的扩增与培养条件密切相关,而含体积分数10%-20%胎牛血清的培养基可促进细胞生长。目的：建立梯度血清递减扩增培养脐带间充质干细胞的培养技术。方法：采用胶原酶Ⅱ消化分离获得脐带间充质干细胞悬液,并通过贴壁培养进行纯化,细胞贴壁后采用梯度血清递减方法,即第1代80%含血清培养基,20%无血清培养基;第2代50%含血清培养基,50%无血清培养基;第3代20%含血清培养基,80%无血清培养基;第4代100%无血清培养基。另一种传代中始终采用含体积分数10%胎牛血清的α-MEM完全培养液。用流式细胞仪检测细胞的表面标记,进行成骨诱导试验,同时与经典的含10%胎牛血清的α-MEM培养体系进行比较。结果与结论：梯度血清递减培养法与经典α-MEM培养体系所获得的细胞在扩增能力、细胞形态、免疫表型等方面相似。细胞在两种培养体系中均能保持良好的分化潜能。但梯度血清浓度法培养间充质干细胞可使用更少量胎牛血清,提高临床应用安全性。

BACKGROUND： The proliferation of human umbilical cord mesenchymal stem cells （hUCMSCs） strongly depends on the culture conditions. Medium containing 10-20% fetal bovine serum （FBS） can promote cell growth. OBJECTIVE： To build the serum-decremental technology for UCMSCs culture. METHODS： hUCMSCs were separated using collagenase Ⅱ digestion method and purified by adherent culture. The cells were cultured and expanded by the serum-decremental method： media 20% serum-free medium for the primary culture, 50% serum-free medium for the second passage, 80% serum-free medium for the third passage, and 100% serum-free medium for the fourth passage. Cells were cultured in α-MEM medium containing 10% FBS as control. Cell surface markers were tested by flow cytometry. Osteogenic induction test was performed. RESULTS AND CONCLUSION： Cells cultured in the both media were similar in proliferative ability, morphology, and immunophenotype. Their differentiative potential kept well in both media. However, less FBS was used in serum-decremental medium. Serum-decremental medium improves the safety of hUCMSCs culture in clinical practice.