TABLE 3.

Physical treatments used for decellularization.

Category	Definition	Advantages	Shortcomings	References
Freeze-thaw	An ice crystal structure is formed to increase cytoplasm concentration and cause cell dissolution	-Less effective on the structural and mechanical properties	-Still contains cell components	Teo et al. (2011); Lu et al. (2012); Burk et al. (2014); Cheng et al. (2019); Fernandez-Perez and Ahearne (2019)
		-Significant effect of chemical methods after freeze-thaw	-Requires further decellularization by chemical methods or enzymes
High hydrostatic pressure	The cell membranes are damaged by deformation under pressure higher than 600 MPa	-No chemical reagent added	-Damages the collagen and elastin to affect mechanical properties	Diehl et al. (2005); Morimoto et al. (2015); Morimoto et al. (2017); Kurokawa et al. (2021); Matsuura et al. (2021)
		-Avoids damage from the toxic effects of solvents
Supercritical fluids	Cell residues can be removed when supercritical carbon dioxide passes through tissues at a controlled rate similar to critical point drying	-Less impact on the mechanical properties	-Poor solubility for macromolecules and polar substances	Kim et al. (2013); Huang et al. (2021); Kim et al. (2021); Reis et al. (2022)
		-Much simpler procedure	-Easily introduces new impurities after adding entrainment agents to improve solubility
Mechanical Agitation	Uses a magnetic stirring tray or shaker throughout the decellularization process	-Facilitates the full contact and penetration of chemicals into the tissue	-Poor effect when used singly	Yang et al. (2010); Syedain et al. (2011); Sarig et al. (2012); Boriani et al. (2017); Duisit et al. (2018)
		-Significantly improves the efficiency