悬浮集簇培养与悬浮分散培养对肌源性干细胞增殖的影响

Effects of suspension cluster culture and suspension dispersion culture on multiplication of muscle-derived stem cells

目的在悬浮培养肌源性干细胞(MDSC)的基础上初步探讨集簇与分散培养对MDSC增殖活性的影响。方法采取2周龄C57BL/6小鼠四肢骨骼肌,用机械法与多步酶消化法制备细胞悬液进行悬浮培养,在培养过程中分别吹打(分散培养组)与静置(集簇培养组)。光镜观察与细胞计数统计增殖数量。流式细胞检测与分化实验验证细胞干性。结果集簇培养组MDSC呈团簇状,细胞表面光滑,呈小球形,折光率高;分散培养组细胞呈单个或小团簇状,细胞表面光滑,小球形,高折光,偶见细胞皱缩。细胞计数结果显示悬浮集簇培养pp2细胞数量(4.0±0.5)×10~5个,悬浮分散培养pp2细胞数量(4.2±0.7)×10~5个,悬浮集簇培养pp6细胞数量(14.9±0.7)×10~5个,悬浮分散培养pp6细胞数量(10.5±1.3)×10~5个,分别增加至约3.7倍和2.5倍,悬浮集簇培养增殖量约是分散培养组的1.7倍,差异有统计学意义(P<0.05)。流式细胞检测发现集簇培养表面干性标记物表达量更高。结论悬浮集簇培养较分散培养获得细胞量更多,细胞干性更好。

ObjectiveTo investigate the effects of suspension cluster culture and suspension dispersion culture on multiplication of muscle-derived stem cells（MDSCs）. MethodsMDSCs were separated from 2 weeks old C57BL/6 mice; cell suspension was prepared through mechanical process and multistep enzyme digestion; cells in dispersion culture group were agitated during culture and cells in cluster culture group were cultured in static state. Multiplication number of MDSCs was observed and counted under microscope. Cell viability was tested by flow cytometry and differentiation assay. ResultsMDSCs in cluster culture group were cluster-like, with smooth cell surface, small sphere and high refraction. MDSCs in dispersion culture group showed single or small clusters, smooth surface, small sphere, high refraction and occasional cell wrinkling. The number of PP2 cells was（4.0±0.5）×105 in cluster culture group and （4.2±0.7）×105 in dispersion culture group; the number of pp6 cells was （14.9±0.7）×105 in cluster culture group and （10.5±1.3）×105 in dispersion culture group; the cell number in cluster culture group was 1.7 multiples more than that in dispersion culture group（P〈0.05）. Conclusionsuspension cluster culture of MDSCs can obtain more multiplications and better viability than dispersion culture.