Three dimensional culture of neonatal rat cardiac cells in vitro | 乳鼠心肌细胞的体外三维培养

Chinese. 探讨应用PLGA泡沫支架材料，在体外建造组织工程化心肌组织的可行性。采用顺序消化及差速贴壁法分离纯化乳鼠心肌细胞，将分离所得的心肌细胞接种于PLGA多孔支架材料上，观测复合体内心肌细胞的生长状况、超微结构、细胞代谢率及细胞组分的变化，并将其与正常心肌和二维培养的心肌细胞进行比较。复合体内的细胞具有心肌细胞的特殊超微结构，免疫组化显示其中的α-横纹肌肌动蛋白染色呈强阳性，与二维培养的细胞相比，细胞代谢更加旺盛。采用组织工程技术有可能在体外培育出组织工程化心肌组织。

English. This study investigates construction of cardiac muscle cell-porous PLGA scaffold complex so as to unveil the possibility of generating 3-dimensional cardiac muscle tissue in vitro. 1 day old neonatal rat cardiac muscle cells were isolated by sequential digestion and pre-plating methods, then seeded onto porous PLGA scaffold, and incubated for 7 days. Cells cultured in plates and native cardiac muscle served as control. Morphological changes of the cells in constructs were detected by light microscope and metabolic rate was recorded. Ultrastructure of the cells growing in porous PLGA was observed by transmission electron microscopy. Content of total DNA and protein in the newly-formed tissue were analyzed. H-E and anti-sarcomeric α-actin stains were performed in comparison with native cardiac muscle. When incubated for 7 days, cell-PLGA constructs formed a continuous outer tissue layer containing cells aligned with each other. The cell population in the interior part of the construct was less in density than the outer part. Transmission electron microscopy demonstrated subcellular elements characteristic of cardiac myocytes in the outermost layer of the constructs. Strongly positive stains of anti-sarcomeric α-actin suggested presence of cell population of differentiated cardiac myocytes in these constructs. Construct biomass was not significantly different from that in neonatal rat ventricle. Metabolic activity of cells in constructs was higher than that in plates. It was possible to form cardiac muscle cells-porous PLGA scaffold constructs with structural and functional features resembling those of native cardiac tissue in vitro.