用MDCK细胞为种子细胞体外构建组织工程化肾小管片层的实验研究

Reconstruction of tissue-engineered renal tubules using MDCK cells

目的以MDCK细胞为种子细胞、胶原凝胶复合Matrigel为支架材料,采用组织工程技术体外构建组织工程化肾小管片层,观察不同浓度Matrigel以及静态拉伸作用对MDCK细胞在三维支架中极性重建并形成小管样结构的影响。方法将培养的MDCK细胞与添加不同浓度Matrigel的液态Ⅰ型胶原混合,在12孔板内铸模形成MDCK细胞-胶原片层。通过相差显微镜、HE染色、免疫荧光染色等方法对细胞的生长情况及形成的三维肾小管结构进行比较和鉴定。在此实验的基础上,对片层施加静态拉伸力,观察静态拉伸作用对细胞生长、小管样结构形成的影响。结果随培养时间的延长,各实验组片层内的MDCK细胞逐渐增殖、黏附、聚集。单纯以胶原为支架材料的片层内MDCK形成囊腔状结构;添加Matrigel的胶原片层内MDCK细胞可形成管腔样结构,且低浓度Matrigel的胶原片层内细胞存活率高。静态拉伸作用能够刺激细胞的生长、管腔样结构的形成。结论本研究应用组织工程技术,以MDCK细胞为种子细胞、添加Matrigel的液态Ⅰ型胶原为支架材料,培养过程中施加静态拉伸力,体外成功构建出组织工程化肾小管胶原组织片层,肾脏小管上皮细胞可在该支架材料中完成极性重建的自组装过程。

Objective To reconstruct tissue-engineered renal tubules in vitro by tissue engineering technique using MDCK cells as seeding cells and liquid collagen combined Matrigel as scaffold, and observe the effects of Matrigel in several concentrations and static stretch on the polarity remodeling of MDCK cells in three-dimensional （3-D） scaffold and on the formation of structures. Methods The cultured MI）CK cells were mixed with liquid collagen supplementing with Matrigel of different concentrations and loaded in 12-well plates to reconstruct MDCK cell-collagen sheet. Observation with phase-contrast microscope was done, routine H＆E staining and imrnunofluorescent staining were used to analyze and identify the growth of cells and the formation of 3-D renal tubule structures. On the basis of this study, the static stretch was exerted onto the constructs during culture time to observe the effects of static stretch on the growth of cells and the formation of tubules. Results With the culture time going on, MDCK cells of every experimental group proliferated, adhered and aggregated gradually. MDCK cells formed cysts in collagen scaffold, while the ones in collagen supplementing with Matrigel formed tubule like structures, and there were more living cells in low concentration Matrigel group. The static stretch could promote the growth of cells and the formation of tubular structures. Conclusion The engineered renal tubule tissue has been successfully reconstructed in vitro in present study by tissue engineering technique using MDCK cells as seeding cells and liquid collagen supplementing with Matrigel as scaffold. During the culture time, the static stretch is exerted onto the collagen sheet. Renal tubular epithelial cells could restore their polarity and self-assemble into tubular structures in 3-D collagen/Matrigel scaffold.