梯度交联水凝胶用于骨髓间充质干细胞的三维培养与分化

Three-dimensional cell culture and differentiation of bone marrow-derived mesenchymal stem cells based on gradient hydrogel

目的:研究骨髓间充质干细胞(MSCs)的三维培养体系,探索梯度交联水凝胶对MSCs生长及分化的影响。方法:采用贴壁分离培养的方法分离纯化大鼠MSCs,传至第3代后接种在水凝胶的表面,对细胞骨架进行免疫荧光染色,以此观察细胞在梯度交联水凝胶和普通水凝胶里的生长迁移情况;后基于同样的观察方法研究不同接种浓度的细胞在梯度交联水凝胶内的三维(3D)生长;最后对MSCs进行碱性磷酸酶染色和油红染色,观察干细胞在3D空间里的成骨和成脂分化。结果:梯度交联水凝胶更适合MSCs的3D生长,3 d后细胞可以贯穿长入水凝胶内部约200μm处;随着细胞接种浓度的增大,贯穿长入水凝胶内部的细胞数量在增多;基于梯度交联水凝胶建立的3D培养微环境中,MSCs向成骨和成脂分化的潜能没有受到影响。结论:应用梯度交联水凝胶可进行MSCs的3D培养,并且依此培养的干细胞的分化潜能得到很好的维持。

Objective： To investigate three-dimensional（ 3D） cell culture system of bone marrow-derived mesenchymal stem cells（ MSCs）,and to explore the differentiation of MSCs based on gradient hydrogel. Methods： MSCs derived from rat bone marrow were seeded on the gradient hydrogel. The 3D growth of MSCs migrate into the gel was examined by immunofluorescent staining of actinin. And the osteogenic and adipogenic differentiation of MSCs were tested by ALP and Oil Red staining. Results： MSCs seeded on the gradient gels immediately adopted a typical3D phenotype characterized by a spindlelike shape. MSCs invaded the gradient gels over time,reaching 200 μm within 3 days after seeding. MSCs keep their pluripotency during the invasion of the gradient gels,as MSCs were differentiated toward adipogenic and osteogenic phenotypes. Conclusion： The presented gradient hydrogel provides a simple and new method for 3D culture of MSCs,and MSCs keep their pluripotency during the invasion.