Table 4.

Different physical methods used for cardiac tissue decellularization.

Physical Decellularization Techniques	Mechanism	General Disadvantages	Study Findings
Temperature	crystals created in the freezing process disrupt cellular membranes causing cell lysis	may cause certain disruptions of the ECM ultrastructure ineffective at removing cells and genetic material, therefore used in combination with enzymes and detergents	pre-treatment of adult porcine hearts with low temperature (−80 °C for at least 16 h) assisted in cell lysis [15] a single freeze–thaw cycle could reduce adverse immune responses such as leukocyte infiltration of decellularized vascular allografts [103] extracellular cryoprotectants (5% trehalose) prevented freeze–thaw cycles to cause certain disruptions of the ECM ultrastructure [104]
Pressure	applying pressure destroys the cellular membrane	can damage the ECM components	HHP treatment showed excellent decellularization efficiency of porcine aortic blood vessels; an allogenic transplantation study showed that the acellular scaffolds reduced the host immune response, endured the arterial blood pressure, with no clot formation on the luminal surface [105] creating pressure gradients across the aortic valve to keep it closed improved coronary perfusion efficiency of SDS and provided a whole decellularized human heart [56]
Non-thermal irreversible electroporation (NTIRE)	formation of micropores in the cell membrane leads to cell death	electrical field oscillation can disrupt ECM	NTIRE proved to be safe and efficient for in vivo myocardial decellularization (it might become a viable means for scaffold creation via organ decellularization) [110]
Perfusion	it establishes a channel for circulating detergents through the intrinsic vascular system of the organs	tissues without innate vasculature cannot be decellularized	decellularization by coronary perfusion with 1% SDS of cadaveric rat hearts preserved the underlying ECM and maintained intact the vascular architecture and chambers geometry [11,127] decellularized cardiac ECM decellularized using a combination of enzymatic and chemical treatments via pulsatile retrograde aortic perfusion supported the formation of organized chicken cardiomyocyte sarcomere structure in vitro [15] efficient decellularization of heart-lung blocs perfused via the ascending aorta as well as via the trachea with 1% SDS could be the first step on the pathway to creating bioengineered transplantable heart-lung scaffolds [128] pressure-controlled perfusion decellularization enables whole-organ tissue engineering at a clinically relevant scale [111,129]
Immersion and agitation	causes cell lysis, facilitates chemical exposure and cellular components removal	severe stirring can damage the ECM needs complementary treatments to assess effective decellularization	immersion of porcine hearts in a decellularization chamber using a modified Langendorff Radnoti system produced acellular whole heart scaffolds [18,115] detergent-based decellularization of porcine pulmonary valves under continuous shaking conditions for 24 h delivered proper dECM for efficient recellularization with human endothelial cells [113]
Sonication	ultrasonic waves can disrupt the cellular membrane and release intracellular components	uncontrolled lower frequencies can damage tissue’s structure and mechanical properties	sonication treatment significantly influenced the detergent-based decellularization efficiency of thick tissues (porcine aortic wall) compared to conventional ways of shaking [116,117] decellularized porcine aortic scaffolds using a closed sonication system (170 kHz) in 0.1% and 2% SDS showed a minimal inflammatory response after subcutaneous implantation in a rat model [118] sonication-assisted decellularization provided an acellular vascular scaffold with in vitro cytocompatibility and in vivo biocompatibility in a rat abdominal aorta implantation model [119]
Supercritical fluid technology	facilitates chemical exposure, leading to cell removal	initial pretreatment with chemical agents is required	hybrid decellularization with chemical agents and scCO2 offered significantly reduced treatment times [121,122,123,124,125] scCO2 was found efficient in providing 100% sterility of the porcine decellularized aortic valves [120]