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设计了一种细胞间距可调的transwell共培养方法,以研究脐带血来源的造血干/祖细胞(HS/PCs)和人脂肪干细胞(human-adipose derived stem cells,h-ADSCs)体外共培养时细胞间作用距离对造血干细胞扩增能力和脂肪干细胞在共培养后干细胞特性的影响。采用不同规格的砂纸打磨孔板的上壁面,经高精度游标卡尺测量,使两种细胞之间的有效接触间距为10~450μm不等。然后以h-ADSCs为基质细胞,分别考察HS/PCs和h-ADSCs在不同的作用距离下的共培养情况。其间每24小时对细胞进行计数,观察细胞形态。培养7天后对MNCs表面抗原CD34+、CFU-GM集落扩增倍数进行了检测;同时也对h-ADSCs表面抗原(CD13、CD29、CD34、CD44、CD45、CD73、CD105、CD166、HLA-DR)及其多向分化潜能(成骨、成软骨、成脂肪)进行分析以鉴定其干细胞性能。结果表明,通过transwell共培养,细胞之间在作用距离为350μm时HS/PCs的扩增效果最好,其中造血MNCs扩增了15.1±0.2倍,CD34+扩增了5.0±0.1倍;扩增的h-ADSCs表达CD29、CD44、CD166,而不表达CD34、CD45,且在适当诱导条件下可以向成骨细胞、软骨细胞、脂肪细胞分化。
A co-culture method of transwell with adjustable intercellular distance was designed to study the co-culture of HS / PCs and h-ADSCs in vitro Effect of Intercellular Distance on Amplification Ability of Hematopoietic Stem Cells and Stem Cell Characteristics of Adipose Stem Cells after Co - culturing. Using different specifications of sandpaper polished plate on the wall, measured by high-precision vernier caliper, the effective contact between the two cells spacing of 10 ~ 450μm range. Then h-ADSCs were used as stromal cells to investigate the co-culture of HS / PCs and h-ADSCs under different distance of action. During this period, the cells were counted every 24 hours to observe the cell morphology. After cultured for 7 days, the number of MNCs surface antigen CD34 + and CFU-GM colonies were detected, and the expression of hs-CRSCs (CD13, CD29, CD34, CD44, CD45, CD73, CD105, CD166, HLA-DR) Its multidrug differentiation potential (osteogenic, chondrogenic, adipogenic) was analyzed to identify its stem cell properties. The results showed that HS / PCs had the best amplification effect by transwell co-culture at a distance of 350μm. The hematopoietic MNCs amplified 15.1 ± 0.2 times and CD34 + amplified 5.0 ± 0.1 times. The amplified h-ADSCs express CD29, CD44, CD166, but do not express CD34, CD45, and can differentiate into osteoblasts, chondrocytes and adipocytes under appropriate inducing conditions.