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In vitro culture of bone marrow-derived mesenchymal stem cells under serum-free condition

机译:在无血清条件下的骨髓衍生间充质干细胞的体外培养

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The aim of this work is to establish a chemically-defined serum free medium (CDSFM) supports in vitro proliferation and maintains the multilineage differentiation potential of bone marrow-derived mesenchymal stem cells (BM-MSCs). BM-MSCs were isolated from the femoral bone of one-month-old New Zealand rabbits with combined density gradient centrifugation (Ficoll, γ 1.073) and attachment culture method. And compared the proliferation capability, cell cycle, colony-forming efficiency and osteogenic, adipogenic differentiation capability of BM-MSCs cultured in the CDSFM and serum-containing medium (SCM). Based on α -MEM, the SCM was supplied with 10% fetal bovine serum, the CDSFM was supplied with bFGF (10 mg/L), EGF (10 mg/L), bovine serum albumin ( BSA, 5 g/L ) , reduced glutathione ( 1.5 mg/L ) , β -mercaptoethanol ( 0.1 mM ) , SITE ( 1%, v/v ),dexamethasone ( 10nM ) and hydrocortisone (10 mg/L). The culture dishes were coating with rat tail collagen type I (0.1 mg/cm2). Over 10 days' culture, resulting in 50-fold increases in cell number in the CDSFM compared to 40-fold in the SCM. bFGF, EGF and hydrocortisone were the most important additives that significantly stimulated BM-MSCs proliferation. The percentage of cells at G0-G1 cell cycle was (80.31±0.58)% in the CDSFM compare to (75.24±4.05)% in the SCM without significant difference (P>0.05). However, the cloning efficiency of BM-MSCs in the CDSFM was lower than that in the SCM with significant difference (p<0.01). The expanded MSCs in the CDSFM preserved differentiation potentials into mesenchymal lineages in vitro, including adipocytes and osteoblasts. A chemically-defined serum free medium was designed to support in vitro proliferation and maintains the stem cells' characters of BM-MSCs, which could be applied to cell-based therapy and biomedical research.
机译:这项工作的目的是建立化学定义的血清培养基(CDSFM)支持体外增殖,并保持骨髓衍生的间充质干细胞(BM-MSC)的多线分化潜力。 BM-MSCs与单个新西兰兔子的股骨骨分离,具有组合密度梯度离心(Ficoll,γ1.073)和附着培养方法。并比较了CDSFM和含血清培养基(SCM)培养的BM-MSCs的增殖能力,细胞周期,菌落形成效率和成骨,促进分化能力。基于α-MEM,SCM配备10%胎牛血清,CDSFM用BFGF(10mg / L),EGF(10mg / L),牛血清白蛋白(BSA,5g / L)提供,降低谷胱甘肽(1.5mg / L),β-巯基乙醇(0.1mm),位点(1%,v / v),地塞米松(10nm)和氢化环(10mg / L)。培养皿用大鼠型胶原I型(0.1mg / cm 2)涂布。超过10天的培养物,导致CDSFM中的细胞数增加50倍,而SCM中的40倍。 BFGF,EGF和氢化酶是最重要的添加剂,可显着刺激BM-MSCs增殖。在CDSFM中,G0-G1细胞周期的细胞的百分比(80.31±0.58)%,在SCM中的(75.24±4.05)%,而无明显差异(p> 0.05)。然而,CDSFM中BM-MSCs的克隆效率低于SCM中具有显着差异的(P <0.01)。 CDSFM中的扩展MSC将分化电位保存在体外的间充质谱系中,包括脂肪细胞和成骨细胞。设计了一种化学定义的血清游离培养基,以支持体外增殖并保持BM-MSCs的干细胞的特性,其可以应用于基于细胞的治疗和生物医学研究。

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