犬骨髓基质干细胞片层制备及向成骨细胞的分化(英文)

Preparation of canine bone marrow stromal stem cell sheets and investigation on their osteoblastic differentiation

背景:目前传统的胰酶消化法和单细胞悬液转移细胞的方法存在诸多缺点,限制了骨组织工程学的发展。目的:培养骨髓基质干细胞并向成骨细胞诱导分化,制备细胞片层。方法:密度梯度离心法分离犬骨髓基质干细胞,接种于DMEM成骨诱导培养基,向成骨细胞诱导分化,采用细胞片层技术,利用温度反应性培养基的温度变化,收获完整的细胞片层。结果与结论:刚接种至培养皿的原代细胞在培养瓶底散在分布,呈圆形,胞体透亮,折光性好,12d时细胞呈长梭形,完全融合,呈旋涡状生长;成骨诱导后的骨髓基质干细胞大部分变为方形、多角形和鳞片形,21～28d形成明显的圆形或卵圆形钙化结节;温度反应性培养皿中的间充质干细胞降温至临界温度32℃以下时,细胞便逐步从培养皿底分离,形成完整的骨髓基质干细胞片层。实验结果证实密度梯度离心法可成功分离培养犬骨髓基质干细胞并向成骨细胞诱导分化,应用细胞片层技术可获取完整的骨髓基质干细胞片层。

BACKGROUND：Conventional methods,including trypsin digestion and cells transfer using single cell suspension,have many drawbacks,which limit the development of bone tissue engineering.OBJECTIVE：To culture bone marrow stromal stem cells,induce osteoblastic differentiation,and prepare cell sheets.METHODS：Canine bone marrow stromal cells were isolated by density gradient centrifugation technique,inoculated into DMEM medium,and induced to differentiate into osteoblasts.Complete cell sheets were harvested by cell sheet engineering based on the temperature change of temperature-responsive medium.RESULTS AND CONCLUSION：Immediately after inoculation,primary cells were scattered on the bottom of culture flask,presenting a transparent spherical body with a good refractive capacity.At 12 hours,cells exhibited a long shuttle shape,reached complete confluency,and grew in a whirlpool-like fashion.After osteoblastic induction,the majority of bone marrow stromal stem cells appeared tetragonal,polygonal,and squamose.At 21-28 days,round or oval-shaped calcified nodules formed.When the bone marrow stromal stem cells in the temperature-responsive culture dishes were cooled below the critical temperature 32 ℃,cells were gradually detached from the bottom of culture flask and formed complete bone marrow stromal stem cell sheets.These findings indicate that density gradient centrifugation technique can be used to successfully isolate and culture canine bone marrow stromal stem cells to differentiate into osteoblasts and cell sheet engineering enables to harvest complete bone marrow stromal stem cell sheets.