TABLE 2.
Summary of several available methods for tissue decellularization.
| Method | Main characteristics | Disadvantages | Decellularized tissues | Ref |
|---|---|---|---|---|
| Chemical Methods | ||||
| (i) Collagen and glycosaminoglycans (GAGs) damage | (Ott et al., 2008) | |||
| Acid—base | Cell membrane solubilization | (ii) Insufficient cell removal | Rat heart | (Syed et al., 2014) |
| Peracetic acid | Disruption of cytoplasmic components and nucleic acids by utilizing charges | (iii) Increased ECM stiffness | Small intestine submucosa | Gilpin and Yang, (2017) |
| Ethylenediaminetetra-acetic acid (EDTA) | (iv) Decreases salt- and acid-soluble ECM proteins | Urinary bladder | ||
| Reversible alkaline swelling | (v) Alters mechanical properties | |||
| Triton X (100 or 200) | Disruption of lipid–lipid and lipid–protein unions, while leaving protein interactions | (i) Not recommended for ECM when lipids and GAGs are important components | (Wagner et al., 2014); Rieder et al. (2004) | |
| Very effective in some tissues | (ii) Limited potential by immunogenicity in vivo | Normal and emphysematous human lungs | ||
| Less damaging to tissue structures than ionic surfactants | (iii) Triton X-200 needs to be combined with a zwitterionic detergent to be effective | Porcine heart valves | ||
| (iv) Triton X-200 damages the matrix similar to SDS | ||||
| Sodium dodecyl sulfate (SDS) | Liquefaction of internal and external cell membranes | Tends to denaturalize proteins and induce nuclear and cytoplasmic waste in the remaining matrix (i) Cytotoxic: requires extensive washing steps | Rat forearm | (Yang et al., 2015); (Gilpin and Yang, 2017); Wang et al. (2010) |
| (ii) Alters microstructure (i.e., collagen fibers) | Porcine tissues (cornea, myocardium, heart valve, small intestine, kidney) | |||
| Human vein, lungs and heart | ||||
| Witterionic, nondenaturing detergent, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) | Properties of ionic and nonionic detergents | (i) Similar damage as Triton X-100 | Human and porcine-derived lung tissues | (Gilpin and Yang, 2017); O'Neill et al. (2013) |
| Maintenance of structural ECM proteins and ultrastructure | (ii) Remanent cytoplasmic proteins | Rat lungs | ||
| Tributyl phosphate (TBP) | Destructor of protein structures | Variable results, leads to collagen degradation but keeping the mechanical properties | Equine flexor tendons, ligaments and articular cartilage | (Deeken et al., 2011); Elder et al. (2009) |
| Disruption of protein–protein interactions | ||||
| Hypertonic and hypotonic solutions | Solutions with a higher/lower solute concentration than that in cells | High amount of cell waste in the remaining matrix | Bovine vessel nerve, small intestinal and submucosa | (Zhang et al., 2022); Kim et al. (2016) |
| Cell lysis, cell dehydration and cell death because of their osmotic pressure | ||||
| Enzymatic Methods | ||||
| Trypsin | Digestion of membrane proteins leading to cell dead | (i) Can damage the proteins in the ECM, in particular laminin and GAGs | Porcine pulmonary valves and trachea | Giraldo-Gomez et al. (2016) |
| Commonly used with EDTA | (ii) Breaks cell-matrix adhesions | |||
| Pepsin | It targets peptide bonds | Causes high damage in the ECM proteins if left for long periods of time | Porcine lung and liver | (Pouliot et al., 2020); Coronado et al. (2017) |
| nuclease | Hydrolysis of DNA and RNA | Further cleaning and enzyme removal is required, as they may promote immune response | Bovine osteochondral plugs, human corneal limbus and porcine dermis | (Greco et al., 2015); Fermor et al. (2015) |
| Physical Methods | ||||
| Thermal shock (freeze-thaw cycle) | Disruption of tissue and organ cells | (i) The freeze-thaw cycle causes a small degradation in the structure of the ECM, due to the crystal shape that may damage the scaffold, with little effect on the mechanical properties of ECM | Tendon fragments (large), fibroblast sheets, lumbar vertebrae cells, kidneys, lungs and adipose tissues | (Rabbani et al., 2021); Zhao et al. (2019) |
| Frozen water crystals occupy the volume inside the cell and cause the membrane to burst | (ii) The heat shock cycle alone is not capable of removing sensitive cellular components | |||
| Force | Mechanical pressure can be enough to induce cell lysis | (i) Limited to tissues with hard structures, as it can damage the ECM structure | Liver, lung | Gilpin and Yang, (2017) |
| (ii) The amount of required force must be precise since both the underlying structure and membrane attachment are vulnerable to any kind of direct mechanical stress | ||||
| Immersion and agitation | It is commonly used to facilitate chemical agent infiltration to induce cell lysis | Aggressive processes, such as sonication, can damage the ECM. | Submucosal substrate, laryngeal and intestine tissues | Keane et al. (2015) |
| The immersion time and intensity of agitation depend on the thickness and density of the tissue | ||||
| Vacuum-assisted decellularization (VAD) | VAD would accelerate and improve the delivery and efficiency of detergents into the deepest parts of the tissue | It is not a decellularization method but a facilitator | Porcine tracheal specimen and fresh porcine costal cartilage | Alizadeh et al. (2019) |
| Removal of detergents from a decellularized tissue is the other application of the VAD methodology | ||||
| Hydrostatic pressure (water is sprayed with pressure on the target tissue) | Application of high pressure (>600 MPa) to the tissue and induction of cell lysis | (i) Excessive pressure can damage the structure | Porcine retinal specimen, porcine artery, porcine meniscus and rat uterine | Rabbani et al. (2021) |
| (ii) The formation of ice crystals caused by the presence of water may damage the ECM structure | ||||
| (iii) Increasing the temperature during the process may suppress the creation of the ice crystals, but may increase the entropy and lead to the ECM vulnerability | ||||
| (iv) Residue of DNA fragments | ||||
| (v) Denatures ECM proteins | ||||
| Nonthermal irreversible electroporation | Microsecond electrical pulses are applied throughout a tissue, causing micropores in the cell membrane | The relatively small electrodes that limit the size of the tissue for decellularization | Carotid arteries of rat, liver of porcine and myocardial muscle tissue | Rabbani et al. (2021) |
| Ultrasonic waves (sonication) | High-power waves are capable of disrupting intermolecular bonds, disrupting the cell membrane, and removing its internal components | The physical phenomenon of cavitation during the process is unavoidable, but uncontrolled cavitation can severely damage the structure and mechanical properties of the tissue | Aortic tissues, small intestine, cartilage tissue and meniscus | Forouzesh et al. (2019) |
| Pressure gradient | Induction of a pressure gradient can help the enzyme-mediated decellularization method | To be determined | Embryonic veins, tendon, and aortic tissue | Sierad et al. (2015) |
| Supercritical fluid | Removal of cell debris. It is used in combination with detergents | To be determined | Porcine pericardium, aorta and retinal tissues | Guler et al. (2017) |
| Reduction of the detrimental effect on the ECM mechanical properties | ||||
| Perfusion | The organ is completely separated from its main blood vessel and the chemical agents are injected into its vascular system after being washed with detergents | (i) The required pressure to drive the agent along the vascular system can cause the capillaries and small vessels to tea | Heart muscle, lung, liver, kidney, pancreas, small intestine, skeletal muscle, coronary artery | Tajima et al. (2020) |
| (ii) The flow rate control is crucial | ||||