利用细胞外基质大规模扩增临床级人脂肪间充质干细胞

背景：如何运用无血清培养体系大规模扩增处于未分化状态的脂肪间充质干细胞，并使其保持“干性”是脂肪间充质干细胞临床应用转化中急待解决的难题。目的：建立含细胞外基质的脂肪间充质干细胞体外培养系统，验证其扩增细胞的高效性、有效性及安全性。方法：在无血清培养条件下，将体外分离得到的脂肪间充质干细胞分别接种在包被细胞外基质的培养板和传统二维塑料培养板上。经过体外扩增后，比较2种条件下细胞数量、细胞表面标记物表达、细胞衰老状况以及体外多向分化能力（诱导成脂、成骨和成软骨）的差异，考察脂肪间充质干细胞在含细胞外基质的培养条件下扩增后的临床安全性。结果与结论：脂肪间充质干细胞扩增到第5代时，包被细胞外基质培养板的细胞产量已经是传统二维塑料培养板的10倍以上，流式细胞检测表明，在含细胞外基质条件下扩增的脂肪间充质干细胞仍保持了干细胞表面特定标记物的表达。细胞衰老检测结果显示，使用包被细胞外基质的培养板扩增脂肪间充质干细胞至第15代时，细胞仍几乎无老化现象，而使用二维塑料培养板扩增的细胞在第5代时已出现明显老化，传代后细胞增殖能力明显下降。体外多向诱导分化实验显示，脂肪间充质干细胞在含细胞外基质条件下扩增至第15代时仍具有成脂、成骨和成软骨的分化功能，并且与第5代时没有明显差异，同时显著优于传统培养条件下的第5代脂肪间充质干细胞。染色体核型分析及小鼠成瘤性试验结果表明，脂肪间充质干细胞在含细胞外基质条件下扩增后仍具备临床应用的安全性。以上结果证实，含细胞外基质的无血清培养系统可以更高效且安全地扩增出具有临床应用潜能的脂肪间充质干细胞。

Large-scale expansion of clinical-grade human adipose-derived stem cells using the extracellular matrix

BACKGROUND：Large-scale expansion of undifferentiated and multipotential adipose-derived stem cells using serum-free culture system is a difficult issue to be resolved. OBJECTIVE：To establish an in vitro culture system combined with the extracellular matrix in order to investigate the efficiency, effectiveness and security of extracellular matrix on expanding adipose-derived stem cells. METHODS：In vitro isolated adipose-derived stem cells were seeded in traditional two-dimensional plastic plates and extracellular matrix-coated plates supplemented with serum-free medium respectively. After in vitro＆amp;nbsp;expansion, total cellnumber, expression of cellsurface markers, cellsenescence degree and multipotent differentiation ability （adipogenic, osteoblastic and chondrogenic differentiation） of adipose-derived stem cells cultured under both conditions were detected and compared. Moreover, the clinical safety of adipose-derived stem cells expanded in extracellular matrix-coated plates was investigated. RESULTS AND CONCLUSION：Total cellnumber of passage 5 adipose-derived stem cells cultured in extracellular matrix-coated plates was 10 times more than that in traditional two-dimensional plastic plates. Flow-cytometric analysis showed that adipose-derived stem cells cultured with extracellular matrix expressed stem cellsurface markers. cellular senescence examination showed that almost al of passage 15 adipose-derived stem cells cultured with extracellular matrix showed no aging, while most passage 5 adipose-derived stem cells cultured by the two-dimensional system aged and lost their proliferation ability. Multidirectional induction of adipose-derived stem cells showed that passage 15 adipose-derived stem cells cultured with extracellular matrix could stil differentiate into adipocytes, osteoblasts and chondrocytes as passage 5 adipose-derived stem cells did, which performed much better than the induced differentiations of passage 5 adipose-derived stem cells cultured by the two-dimensional system. Karyotype analysis and in vivo invasion experiment insured the clinical safety of adipose-derived stem cells expanded with extracellular matrix. Al above results suggest a safe and more efficient expansion system of extracellular matrix for clinical application using the serum-free culture system combined with extracellular matrix.