Desenvolvimento de método de criopreservação de córneas para potencial aplicação em teste de permeabilidade e opacidade da córnea bovina (BCOP)

Abstract

Currently, biological assays must be used to assess the health safety of products before they go to market. The Bovine Corneal Opacity and Permeability Test (BCOP) is an alternative method to the use of animals that uses fresh bovine corneas to assess the irritant and corrosive potential of any substance at the ocular level. The need to always have to use viable corneas makes scaling this test more difficult. To overcome this challenge, corneal cryopreservation can be a possibility to have corneal banks available at any time. Despite the success obtained in the freezing of human corneas, the freezing process can damage the endothelium and efficient protocols for this purpose are not known in cattle. This work aims to develop a method of cryopreservation of bovine corneas for application in the BCOP test. Initially, the corneas were cryopreserved in DMEM with cryoprotectants (CPA) dimethylsulfoxide (DMSO) and ethylene glycol (EG), in combinations with two adjuvants, bovine serum albumin (BSA) and bovine serum (SB). The control was made from fresh corneas and corneas cryopreserved with DMEM and adjuvant. The tumescence test was performed to verify the resistance of the endothelium to CPAs. Afterwards, they were frozen at two temperatures: -20°C and -80°C. After freezing, macroscopic analysis of corneal opacity and cell viability was performed using the trypan blue (AT) exclusion method. Endothelial cell area and cell morphology were assessed by staining with alizarin red (VA). Furthermore, histological sections stained with hematoxylin and eosin were performed to assess the integrity of the different layers of the cornea after the application of the studied cryopreservation methods. The tumescence and cell viability data were evaluated using linear models following the Akaike criterion (AICc) and R² values (R² = 1 – relative error). The results revealed that the corneas only increased in weight, meaning damage to the endothelial layer, after 6 hours in contact with the CPAs (p < 0.05). Treatments with CPAs were different from the control (p < 0.05) and the different adjuvants kept the weight stabilized, differing from each other (p < 0.05). SB, which has BSA in its composition in addition to other proteins beneficial to cryopreservation, was chosen as an adjuvant to continue freezing. Macroscopic analysis revealed that DMSO had better corneal transparency. Endothelial cell viability was higher in the control treatment, followed by DMSO and EG, at both freezing temperatures. Freezing at -20°C had a better response when compared to -80°C for viability after cryopreservation. The damages of the EG treatment are evidenced in the morphological analysis and in the histological analyses. Thus, the data suggest that freezing DMSO has potential for the cryopreservation of bovine corneas, as it showed the best results in the parameters evaluated in the present work. Additionally, freezing at -20°C showed potential use for the maintenance of bovine corneas, with the advantage of facilitating the storage of these materials in common refrigerators.