传代种植密度对人间充质干细胞增殖及成骨分化的影响

Effects of cell density on proliferation and osteogenic differentiation of mesenchymal stem cells in vitro

目的 :研究人间充质干细胞 (mesenchymalstemcells ,MSCs)传代培养时 ,细胞种植密度对MSCs增殖及成骨诱导分化影响。方法 :将第 2代MSCs以 8× 10 3 /cm2 、 3× 10 3/cm2 、 8× 10 2 /cm2 密度接种 ,分析其生长曲线 ,并扩增培养 18d ,记录细胞扩增数量 ,再分析扩增后细胞的生长曲线和成骨诱导能力。结果 :人骨髓MSCs阴性表达ALP、CD3 4;在 8× 10 3/cm2 种植密度下 ,细胞倍增时间 40h ,18d后细胞数量扩增 (5 1± 13 )倍 ;在 3× 10 3 /cm2 种植密度下 ,细胞扩增 (2 8± 6)倍 ;在 8× 10 2 /cm2 种植密度下 ,细胞总数仅增加 (5± 3 )倍。在低密度下获得的细胞增殖能力强于高密度组 ,两组细胞均具有成骨诱导能力。结论 :种植密度对MSCs增殖有明显影响 ,快速扩增MSCs作为骨组织工程种子细胞 ,宜选择 8× 10 3 /cm2 种植密度。

Objective: To evaluate the proliferation and osteogenic differentiation of human mesenchymal stem cells (MSCs) plated at different cell density and provide seeding cells for regeneration tissue-engineering bone by culture-expanded MSCs.Method:Cells were cultured from bone marrow after density fractionation and identified the expression of alkaline phosphatase and CD34.Then MSCs were plated at 8×10 3cm -2 、 3×10 3cm -2 or 8×10 2cm -2 for expansion.After 18 days,proliferation and differentiation of three groups were evaluated by analysis of number of MSCs,growth curves and phenotypes of osteoblasts.Result:Human MSCs from bone marrow were CD44 negative and expressed low levels of alkaline phosphatase.Population doubling time was 40 hour as MSCs were plated at 8×10 3cm -2 and cells were expanded(51±13)time after 18 days.MSCs were expanded(28±6)time after 18 days while cells were plated at 3×10 3cm -2 .While cells were plated at 8×10 2cm -2 ,many cells couldn't attach to culture flask,but could form colony.At last proliferation of cells from 8×10 2cm -2 group were superior to cells from 8×10 3cm -2 group.MSCs could be induced to differentiate into the osteocytic lineages after plated at not only 8×10 3cm -2 but also 8×10 2cm -2 .Conclusion:In the expanded cultures,MSCs should be plated at 8×10 3cm -2 for regeneration tissue-engineered bone and MSCs should be plated at 8×10 2cm -2 for acquired cells capable to form colony.