生物反应器内再造组织工程化心肌的实验研究

Experimental Study of Cardiac Muscle Tissue Engineering in Bioreactor

目的在体外模拟微重力条件下构建心肌细胞-胶原复合体,探索组织工程化心肌组织体外再造的可行性。方法采用顺序消化及差速贴壁法从1～2d龄新生大鼠的心肌组织中分离心肌细胞,并将其与多孔胶原复合形成复合体。复合体在旋转生物反应器(rotarycellculturesystem,RCCS)中进行培养,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常规培养的复合体进行比较。结果在RCCS中培养的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,与静止培养的复合体细胞相比,细胞代谢更加旺盛。结论采用组织工程技术可以在RCCS中培育出组织工程化心肌组织。

Objective This study investigates construction of cardiac muscle cell-porous collagen scaffold complex in a bioreactor so as to unveil the possibility of generating3-dimensional cardiac muscle tissue under the environment that mimics microgravity in vitro.Methods 1～2-day old neonatal rat cardiac muscle cells were isolated by sequential digestion and pre-plating methods,then seeded onto porous collagen scaffold.The cell-collagen complex was transferred into rotary cell culture system(RCCS)and incubated for7days.Cells cultured in75ml flasks and constructs cultures on plates served as control.Morphological changes of the cells were observed by light microscope and metabolic rate was recorded.Ultrastructure of the cells growing in porous collagen was observed by transmission electron microscopy.Content of total DNA and protein in the newly-formed tissue were analyzed.H-E and anti-sarcomericα-actin stains were performed in comparison with native cardiac muscle.Results The isolated cardiac muscle cells adhered to the bottom of the flasks24hours after plating and began to beat spontaneously.When incubated for7days in RCCS,cell-collagen constructs of form a continuous outer tissue layer containing cells aligned with each other.The cell population in the interior of the construct was less in density than the outer part.Transmission electron microscopy demonstrated that subcellular elements characteristic of cardiac myocytes were in the outermost layer of constructs.A strongly positive stains of anti-sarcomericα-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 and approximately40% of that in adult rat ventricles.Construsts in plates contained a few of cells which were less than those in RCCS.Metabolic activity of cells cultured in RCCS was higher than that in flasks and plates.Conclusions Dissociated cardiac muscle cells cultured on3-dimensional scaffolds in RCCS under favorable conditions can form engineered constructs with structural and functional features resembling those of native cardiac tissue.