Comparison of porcine corneal decellularization methods and importance of preserving corneal limbus through decellularization

Abstract

Background The aim of this study is to compare the three previously applied, conventional porcine corneal decellularization methods and to demonstrate the importance of preserving the corneal limbus through decellularization. Methods Fresh, wild-type (with or without) limbus porcine corneas were decellularized using three different methods, including (i) sodium dodecyl sulfate (SDS), (ii) hypertonic saline (HS), and (iii) N2 gas (NG). Post-treatment evaluation was carried out using histological, residual nuclear material, and ultrastructural analyses. Glycerol was used to help reduce the adverse effects of decellularization. The corneas were preserved for two weeks in cornea storage medium. Results All three decellularization methods reduced the number of keratocytes at different rates in the stromal tissue. However, all methods, except SDS, resulted in the retention of large numbers of cells and cell fragments. The SDS method (0.1% SDS, 48h) resulted in almost 100% decellularization in corneas without limbus. Low decellularization capacity of the NG method (<50%) could make it unfavorable. Although HS method had a more balanced damage-decellularization ratio, its decellularization capacity was lower than SDS method. Preservation of the corneoscleral limbus could partially prevent structural damage and edema, but it would reduce the decellularization capacity. Conclusion Our results suggest that SDS is a very powerful decellularization method, but it damages the cornea irreversibly. Preserving the corneoscleral limbus reduces the efficiency of decellularization, but also reduces the damage.

A Decellularized Human Limbal Scaffold for Limbal Stem Cell Niche Reconstruction

“…Similarly, the DHL preserved radially oriented vascular channels, which may support revascularization and nutrition after transplantation. It has been reported that sodium dodecyl sulfate (SDS) disrupted the ultrastructure of the cornea most severely, resulting in the disorganization of collagen fibrils in the stroma of the cornea and limbus [ 37 ]. Similar results have been reported for SDS-treated corneas, which had the greatest loss of transparency with most ECM disruption [ 23 ].…”

“…For decellularization, we used whole corneoscleral tissue to simplify handling and avoid structural damage [ 37 ]. Decellularization was carried out as published previously [ 24 ].…”

Generation of a Biomimetic Substitute of the Corneal Limbus Using Decellularized Scaffolds

“…Pharmaceutics 2021, 13, x FOR PEER REVIEW 3 of 19 obtaining adequate limbal scaffolds for use in tissue engineering. In fact, some preliminary reports using SDS and NaCl decellularization protocols have described the efficient generation of limbal substitutes for use in regenerative medicine [11,28,30].…”

“…Physical methods are mostly based on freezethawing, osmotic pressure, and lyophilization, whereas biological protocols make use of enzymes such as trypsin, DNAse, and RNAse [24,27]. Although little clinical experience is available for the use of decellularized corneal xenografts, some preliminary clinical trials have pointed out the biosafety and functionality of decellularized porcine corneas in patients subjected to lamellar keratoplasty [26][27][28][29].…”

“…However, the use of decellularization methods applied to the native limbi provides the specific morphology, structure, and protein composition of the corneal limbus [30], and offers the opportunity of obtaining adequate limbal scaffolds for use in tissue engineering. In fact, some preliminary reports using SDS and NaCl decellularization protocols have described the efficient generation of limbal substitutes for use in regenerative medicine [11,28,30].…”