Cardiac tissue engineering has major applications in regenerative medicine, disease modeling and fundamental biological studies. Despite the significance, numerous questions still need to be explored to enhance the functionalities of the engineered tissue substitutes. In this study, three dimensional (3D) cardiac micro-tissues were developed through encapsulating co-culture of cardiomyocytes and cardiac fibroblasts, as the main cellular components of native myocardium, within photocrosslinkable gelatin-based hydrogels. Different co-culture ratios were assessed to optimize the functional properties of constructs. The geometry of the micro-tissues was precisely controlled using micro-patterning techniques in order to evaluate their role on synchronous contraction of the cells. Cardiomyocytes exhibited a native-like phenotype when co-cultured with cardiac fibroblasts as compared to the mono-culture condition. Particularly, elongated F-actin fibers with abundance of sarcomeric α-actinin and troponin-I were observed within all layers of the hydrogel constructs. Higher expressions of connexin-43 and integrin β1 indicated improved cell-cell and cell-matrix interactions. Amongst co-culture conditions, 2:1 (cardiomyocytes: cardiac fibroblasts) ratio exhibited enhanced functionalities, whereas decreasing the construct size adversely affected the synchronous contraction of the cells. Therefore, this study indicated that cell-cell ratio as well as the geometrical features of the micropatterned constructs are among crucial parameters, which need to be optimized in order to enhance the functionalities of engineered tissue substitutes and cardiac patches.
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