Biologic scaffold materials composed of the extracellular matrix (ECM) are typically derived by processes that involve decellularization of tissues or organs. All decellularization methods result in the ECM architecture disruption and a potential loss of surface structure and composition. Freeze-thaw processing effectively lyses cells and permits to diminish amounts of chemical lysing agents henceforth. However, it also causes certain disruptions of the ECM ultrastructure. In order to diminish these adverse effects we suggested using extracellular cryoprotectants (namely 5 % trehalose) to preserve the ECM molecular network without impeding the cell lysis. The original optimization of the perfusion-mediated decellularization method to comprise the single freeze-thaw processing cycle and subsequent perfusion with chemical agents' solution is presented here.