Comparing and combining bovine cornea decellularization methods for ocular surface biomaterials and tissue engineering applications

Abstract

Aims/Purpose: The purpose of this research was to evaluate the decellularization efficiency of various individual protocols and their combinations for bovine cornea intended for ocular surface biomaterial applications.Methods: Two fundamental decellularization protocols, namely sodium chloride (NaCl) and Ammonium & Triton X‐100 were taken as benchmark and their combinations with freeze–thaw and lyophilization, were assessed whether, the decellularization efficiency improved (n = 3). Overall, protocols that were investigated were as follows: (1) Lyophilization followed by NaCl, (2) Freeze–Thaw followed by NaCl, (3) Lyophilization, Ammonium and Triton X‐100 (1%), (4) Ammonium and Triton X‐100 (5%), (5) Freeze–thaw, Ammonium and Triton X‐100 (1%), (6) Lyophilization, Ammonium and Triton X‐100 (5%), (7) Freeze–thaw and (8) NaCl. These methods were assessed using gel electrophoresis for qualitative DNA analysis and NanoDrop for quantitative DNA content. Efficient decellularization was achieved in the presence of DNA content below 50 ng/mg ECM dry weight and <200 bp DNA fragment length.Results: Gel electrophoresis revealed DNA content over 200 bp in groups 1, 2,7 and 8 while other groups fulfilled the criterion for efficient decellularization. DNA content as measured by NanoDrop indicated efficient decellularization in all groups, except group 7.Conclusions: Our study provides valuable insights into the effectiveness of combined and individual decellularization protocols for bovine cornea. Our findings suggest, that NaCl and its combinations with lyophilization and freeze–thaw yield in ineffective decellularization. On the other hand, Ammonium and Triton X‐100, individually or in combinations, result in favourable decellularization.