新生大鼠许旺细胞体外培养纯化的最佳培养条件

Optimized conditions for growth and in vitro purification of neonatal rat Schwann cells

背景：许旺细胞对促进外周神经的生长、发育和修复有重要作用，目前对于许旺细胞体外培养方法的优劣情况报道不一。目的：筛选许旺细胞最佳体外培养条件。设计、时间及地点：细胞学体外观察，于2008-06／10在武汉大学人民医院中心实验室完成。材料：三四天龄SD大鼠24只，由武汉大学医学部实验动物中心提供。方法：无菌剥离大鼠双侧坐骨神经，应用低浓度酶快速消化法、差速贴壁法分离培养纯化许旺细胞。取处于指数生长期的细胞，调整密度为1×10^7L^-4接种到6孔培养板，然后分组实验。设定4个条件：培养基pH（6．8-8．8）、胎牛血清体积分数（2％-20％）、培养箱温度（34-39℃）、培养箱CO2浓度（3％-8％）。首先固定胎牛血清体积分数、培养箱温度和培养箱CO2浓度，改变培养基pH对细胞进行培养，从而确定最适培养基pH。然后依次改变胎牛血清体积分数、培养箱温度以及培养箱CO2浓度，进而得到其他3个指标的最佳值。镜下观察见明显克隆形成时终止培养，以含5个以上细胞的细胞团作为1个克隆，在倒置显微镜下进行克隆计数。主要观察指标：通过许旺细胞长势及克隆形成情况，筛选许旺细胞体外培养扩增的最适pH、胎牛血清体积分数、培养温度和培养箱C02浓度。结果：在以DMEM／F12（1：1）为基础培养基的情况下，培养基pH为7．0-7．2时克隆形成最多，〈7．0或〉7．2时克隆形成明显下降：胎牛血清体积分数为10％许旺细胞长势较好且克隆数明显增加，当血清浓度过高或过低时克隆形成相对较差：34-36℃细胞生长受到明显抑制，克隆形成较少，37℃时克隆形成最多，〉37℃细胞长势开始变差，克隆形成下降，至39℃时细胞已无法贴壁；培养箱CO2浓度为3％～8％时许旺细胞克隆形成无明显变化。结论：许旺细胞体外培养的最佳条件为pH7．2的DMEM／F12（1：1）细胞培养基、胎牛血清体积分数10％、培养箱温度37℃、培养箱CO2浓度5％。

BACKGROUND： Schwann cells can promote the growth, development and repair of peripheral nerve. The reports on the method of Schwann cells cultured in vitro are various presently. OBJECTIVE： To study the optimized conditions of Sohwann cells cultured in vitro. DESIGN, TIME AND SETTING： The cytology in vitro study was performed at the Central Laboratory of Renmin Hospital of Wuhan University from June to October in 2008. MATERIALS： Twenty-four 3-4-day-old Sprague-Dawley rats were provided by Experimental Animal Center, Medical College, Wuhan University. METHODS： Bilateral sciatic nerve was sterilety dissociated from rats. Schwann cells were prepared and purified by low-concentration enzyme fast digestion and differential adherence. At exponential growth phase, cells at 1 ×10^7/L were incubated in 6-well plate. There were 4 conditions： medium pH （6.8-8.8）, fetal bovine serum （2% 20%）, incubator temperature （34-39℃）, and incubator CO2 concentration （3%-8%）. Firstly, volume fraction of fetal bovine serum, incubator temperature and incubator CO2 concentration were fixed. Medium pH value was changed for cell culture to determine an optimal pH value. Subsequently, volume fraction of fetal bovine serum, incubator temperature and incubator CO2 concentration were changed in order to determine optimal values. Clone formation was observed under a microscope. One clone contained at least five cells. Cell clone was counted under an inverted microscope. MAIN OUTCOME MEASURES： The following parameters were measured： Schwann cell growth and clone formation, optimal pH value, volume fraction of fetal bovine serum, incubator temperature and incubator CO2 concentration. RESULTS： The cell-state of purified Schwann cells cultured in DMEM/F12 medium （1：1） was good, at pH value of 7.0 7.2. At ph value of 〈7.0 or 〉7.2, clone formation significantly decreased. Schwann cells with 10% fetal bovine serum had good growth, and clone number obviously increased. When serum concentration was higher or lower, clone formation would be poor. Cell growth was markedly inhibited between 34-36℃, with a few clones. At 37℃, clones were the most. At 〉 37℃, cell growth became poor, and clone formation decreased. At 39℃, cells could not adhere. Under incubator CO2 concentration between 3% and 8%, there was no significant difference in cell clone formation. CONCLUSION： The optimized culture condition of Schwann cells were 7.2 pH, 10% fetal bovine serum, 37℃ of temperature and 5% of C02（v/v） of incubator.