TABLE 1.
Methods for cornea decellularization and associated mechanisms, advantages, and disadvantages.
| Methods/Techniques | Mechanism of action | Advantages | Disadvantages |
|---|---|---|---|
| Biological | |||
| Enzymatic Agents | |||
| Trypsin Zhang et al., 2007; Gilpin and Yang (2017); Isidan et al. (2019) | Hydrolyzes protein and disrupts protein-protein interactions | Breaks cell-matrix interactions | An extended exposure can disrupt the collagen structure |
| Dispase Gonzalez-Andrades et al. (2011) | Cleaves peptides associated with basement membrane proteins | Can aid the decellularization process by initially removing epithelium and endothelium | May cause damage to the basement membrane |
| Phospholipases A2 (PLA2) Wu et al. (2009) | Hydrolyzes phospholipid components of cells | Effective at the removal of DNA and residual cellular components that tend to adhere to ECM proteins | — |
| Helps maintain collagen and proteoglycans in the corneal tissue | |||
| Nucleases (RNase and DNase) Cebotari et al. (2010) | Cleaves nucleic acids and aid in their removal | Effective at the removal of DNA and residual cellular components that tend to adhere to the stroma’s ECM proteins | Incomplete removal of the enzymes may impede recellularization and successful transplantation |
| Sera Wu et al. (2009) | Serum nucleases degrade DNA and RNA. | Effectively removes cells while maintaining tissue transparency | The use of non-human sera carries a risk of cross-species transmission of pathogens |
| Non-enzymatic Agents | |||
| EDTA Alhamdani et al. (2010) | Dissociates cells by separating metal ions | Can be used for effective when combined with other agents | Ineffective at cell removal when used unaccompanied |
| Chemical | |||
| Alcohols | |||
| Ethanol Ponce Márquez et al. (2009), Wilson et al. (2013) | Dehydrates and lyses cells | More effective in removing lipids from tissues than lipase | Can cause damage to the ultrastructure of tissue |
| Removes lipids from tissues | Antimicrobial, antifungal, and antiviral properties | ||
| Glycerol Lynch and Ahearne (2013), Wang et al. (2022b) | Dehydrates and lyses cells | Can maintain or restore corneal transparency | Can cause damage to the ultrastructure of tissue |
| Removes lipids from tissues | Cryoprotectant for long-term cornea storage | ||
| Acids and Alkalis | |||
| Peracetic acid Ponce Márquez et al. (2009), Gilpin and Yang (2017) | Solubilizes cytoplasmic components of cells | Acts to simultaneously sterilize tissue | Ineffective decellularization that can also disrupt the ECM |
| Removes nucleic acids via hydrolytic degradation | |||
| Ammonium hydroxide Choi et al. (2010), Dai et al. (2012) | Hydrolytic degradation of biomolecules | Results in complete DC with little effect on collagen architecture | Can eliminate GFs and reduce mechanical properties |
| Ionic Detergents | |||
| Sodium dodecyl sulfate(SDS) Ponce Márquez et al. (2009), Du and Wu (2011) | Solubilizes cell membranes and dissociates DNA from protein | Complete removal of cells can be achieved | Can be highly detrimental to ECM structure including disorganization of collagen fibrils and loss of GAGs |
| Disrupts protein-protein interactions | Loss of tissue transparency | ||
| Sodium deoxycholate Blum et al. (2022) | Solubilizes cell membranes and dissociates DNA from protein | Complete removal of cells can be achieved when used with other agents | Less effective at removal of cells |
| Cebotari et al. (2010), Wang et al. (2022b) | Disrupts protein-protein interactions | ||
| Non-ionic Detergents | |||
| Triton X-100 Cebotari et al. (2010) | Breaks up lipid-lipid and lipid-protein interactions | Mild and non-denaturing | Less effective than ionic detergent treatments |
| Can cause damage to the ECM | |||
| Zwitterionic Detergents | |||
| CHAPS Alhamdani et al. (2010), Keane et al. (2015) | Has properties of non-ionic and ionic detergents | Better cell removal than non-ionic detergents | Poor cellular removal |
| Improved preservation of the ECM ultrastructure than ionic detergents | Very disruptive to stromal architecture | ||
| Hypo- and Hypertonic Solutions | |||
| Sodium Chloride (NaCl) Alhamdani et al. (2010), Ekser et al. (2012), Wilson et al. (2013) | Detaches DNA from proteins | Can maintain optically clarity | Does not remove cellular residues |
| Ability to maintain the stromal architecture and retain GAG content | Mixed reports on the success of cell removal efficiency | ||
| Tris-HCl Alhamdani et al. (2010), Wilson et al. (2013) | Lyses cells by osmotic shock | Reduces decellularization time | Mixed reports on cell removal |
| Physical | |||
| Freeze-thawing Crapo et al. (2011), Wang et al. (2022b) | Ice crystal formation causes cell lysis | Effectively destroys tissue and organ cells | Expensive |
| Needs subsequent treatment to remove cells | |||
| Enhanced pore formation and disruptions to ECM | |||
| Hydrostatic Pressure Crapo et al. (2011), Wilson et al. (2013), Gilpin and Yang (2017), Wang et al. (2022b) | Increase in pressure results in cell lysis | Effectively decellularizes while maintaining collagen fibril structure | Expensive |
| Kills bacteria and viruses | |||
| Sonication and Mechanical Agitation Xu et al. (2008) | Cell lysis and removal | Does not remove DNA remnants from the corneal tissue | Only effective with enzymatic treatments |