Cryopreservation (−20°C) of equine corneoscleral tissue: Microbiological, histological, and ultrastructural study

Objective: To evaluate microbiological, histological, and ultrastructural characteristics of short-term cryopreserved (STC) equine corneoscleral tissue (7 years). Animals studied: Thirty-four healthy equine globes. Procedure: After a decontamination protocol, globes were enucleated and stored at −20°C in broad-spectrum antibiotics. Corneoscleral tissue was evaluated at different storage periods: 1 month-1 year (20 eyes) and 7-9 years (12 eyes). Two eyes were used as controls. Microbiologic study included direct (blood, McConkey, and Sabouraud agars) and enrichment (brain-heart infusion broth) cultures. Cryopreservation artifacts were evaluated by hematoxylin-eosin. Corneoscleral collagen organization and number of normal and dead keratocytes were established by transmission electron microscopy. Results: All microbiologic direct cultures were negative. Enrichment cultures were positive in 12.5% of corneal and 59.4% of scleral tissues (pcornea = 0.136; psclera = 1.000). Cryopreservation artifacts were most commonly observed in LTC tissues (P = 0.002). Normal keratocytes were predominant in STC corneas (STC 60% and LTC 0%) and apoptotic ones in LTC (STC 40% and LTC 90%), whereas necrotic keratocytes were only seen in LTC (LTC 10%) (P = 0.001). No structural differences were detected in collagen organization between STC and LTC (pcornea = 1.000; psclera = 0.703). Conclusions: Cryopreservation of equine corneoscleral tissue did not yield direct bacterial contamination. Apoptosis is the main cause of death of cryopreserved equine keratocytes. Based on the lack of significant structural differences between STC and LTC samples, these cryopreserved tissues could potentially be used for tectonic support for at least 9 years without structural or microbiological impediment.