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The objective of this study was to fabricate 3-dimensional (3D) chitosan nanofiber scaffolds using an electrospinning technique and explore its potential for cardiac tissue engineering. Three culture conditions were tested: cardiomyocytes only, cardiomyocytes-fibroblasts cocultures, and cardiomyocyte-endothelial cells cocultures. The cells were seeded on 2-dimensional (2D) chitosan films and 3D chitosan nanofibers. Cellular morphology and functionality were assessed using immunofluorescent staining for alpha-sarcomeric actin (SA) and gap junction protein, connexin-43 (Cx43). In both the 2D and 3D scaffolds, only the cardiomyocyte-fibroblasts cocultures resulted in polarized cardiomyocyte morphology, fibril SA expression and Cx43 expression was higher compared to the other two conditions. In addition, the fibroblasts cocultures demonstrated synchronized contractions involving large tissue-like cellular networks. To our knowledge, this is the first attempt to utilize 3D chitosan nanofibers as cardiomyocyte scaffolds. Our results demonstrate that chitosan nanofibers can serve as a potential scaffold that can retain cardiomyocyte morphology and function.