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One of the leading trends in the modern tissue engineering is the development of new effective methods of decellularization aimed at the removal of cellular components from donor tissue reducing its immunogenicity and the risk of rejection. Supercritical CO2 (scCO2) extraction can significantly improve the outcome of decellularization, reduce contamination and time costs. The resulting products can serve as personalized tools for tissue-engineering therapy of various somatic pathologies. However, decellularization of complex 3D structures, such as the aortic root, requires optimization of the parameters, including preconditioning medium composition, type of co-solvent, values of pressure and temperature insight the scCO2 reactor, etc. In our work, using an ovine aortic root model, we performed a comparative analysis of the efficiency of decellularization using approaches based on various combinations of these parameters. The protocols were based on combinations of treatment in alkaline, ethanol or detergent solutions with scCO2 extraction at different modes. Based on a histological analysis, we have selected an optimal protocol for the decellularization of ovine aortic root employing preconditioning in a detergent solution. The positive effects of scCO2 on the decellularization extent, cytotoxicity and histoarchitecture of the tissue were demonstrated.