Furthermore, biological properties of cardiogel such as the cytocompatibility, potential for cardiomyogenic differentiation and angiogenesis were evaluated to validate cardiogel as a potential scaffold for cardiac regeneration. This scaffold has been shown to promote proliferation, adhesion, cardiomyogenic differentiation of BMSCs and provide protection against oxidative stress, but the composition of this complex matrix has not yet been completely characterized. In this study, comparative proteomic analysis of cardiogel was performed to obtain a detailed characterization of this nanomatrix. However, literature describing the extraction of an ECM from in vitro cultured cells and its proteomic analysis is limited. An accurate and reliable proteomic analysis of any ECM requires a substantial amount of protein, absence of any interfering substances such as detergents and intracellular contaminations, and complete solubilization of the fibrous proteins in the matrix. These exacting requirements necessitate an optimized protocol for the efficient decellularization, extraction and solubilization of an ECM. Decellularization protocols using several non-enzymatic agents have been widely used to generate ECM scaffolds; but, protocols using cation chelating agents such as EDTA do not remove the cells completely while ECM extracted with methods using NH4OH and TX100 have been reported to contain intracellular impurities. Capsaicin Therefore, in the present study, four different decellularization protocols with various combinations and concentrations of the above reagents were used for generation of cardiogel. Protocol IV resulted in complete decellularization with maximum yield of ECM and was chosen for further studies. Previous studies have used gelatin as a substrate for cardiogel isolation. To understand the effect of gelatin during decellularization of cardiac fibroblasts, four different protocols were carried out on cells cultured on gelatin coated and non-coated plates. It was observed that gelatin coating not only Vernakalant preserved the structure of the matrix but also yielded higher amount of protein and collagen compared to gelatin non-coated plates.