||Decellularization by various detergents such as sodium dodecyl sulfate (SDS) and triton X-100 can remove the cell nuclei in tissue organs. However, this leads to ECM structure denaturation, less presence of various ECM proteins and cytokines and loss of mechanical properties. To overcome these limitations, we developed a supercritical carbon dioxide and ethanol co-solvent(scCO2-EtOH) decellularization method with rat heart tissues, which is a detergent-free system that prevents ECM structure disruption and retains various angiogenic proteins in the heart dECM. scCO2-EtOH contained more ECM components such as collagen, GAGs, collagen I, laminin and fibronectin as well as angiogenic factors in comparison to the Detergent. In addition, to estimate angiogenesis of the dECM hydrogels, the gels were injected in a rat subcutaneous layer. Consequently, blood vessel formation and density of vWF and α-SMA in scCO2-EtOH were significantly greater than the Collagen.