Purpose Cardiac tissue engineering is suggested as a promising approach to overcome problems associated with impaired myocardium. This is the first study to investigate the use of BC and gelatin for cardiomyocyte adhesion and growth. Methods Bacterial cellulose (BC) membranes were produced by Komagataeibacter xylinus and coated or mixed with gelatin to make gelatin-coated BC (BCG) or gelatin-mixed BC (mBCG) scaffolds, respectively. BC based-scaffolds were characterized via SEM, FTIR, XRD, and AFM. Neonatal rat-ventricular cardiomyocytes (nr-vCMCs) were cultured on the scaffolds to check the capability of the composites for cardiomyocyte attachment, growth and expansion. Results The average nanofibrils diameter in all scaffolds was suitable (~ 30–65 nm) for nr-vCMCs culture. Pore diameter (≥ 10 μm), surface roughness (~ 182 nm), elastic modulus (0.075 ± 0.015 MPa) in mBCG were in accordance with cardiomyocyte requirements, so that mBCG could better support attachment of nr-vCMCs with high concentration of gelatin, and appropriate surface roughness. Also, it could better support growth and expansion of nrvCMCs due to submicron scale of nanofibrils and proper elasticity (~ 0.075 MPa). The viability of nrvCMCs on BC and BCG scaffolds was very low even at day 2 of culture (~ ≤ 40%), but, mBCG could promote a metabolic active state of nr-vCMCs until day 7 (~ ≥ 50%). Conclusion According to our results, mBCG scaffold was the most suitable composite for cardiomyocyte culture, regarding its physicochemical and cell characteristics. It is suggested that improvement in mBCG stability and cell attachment features may provide a convenient scaffold for cardiac tissue engineering.

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