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

This research evaluates and compares the microbiological, histological, and ultrastructural characteristics of short-term and long-term cryopreserved equine corneoscleral tissue. Findings indicate no significant structural differences or bacterial contamination between either type of sample, suggesting the potential for cryopreserved tissues to be used for up to 9 years without any impediments.

Study Methods

• Thirty-four healthy equine globes were used in the study. These globes were enucleated and kept at a temperature of -20°C in broad-spectrum antibiotics.
• The corneoscleral tissue harvested from these globes was evaluated at different storage periods: short-term (1 month to 1 year – 20 eyes) and long-term (7 – 9 years – 12 eyes).
• A pair of eyes were reserved for control purposes. The tissues were examined microbiologically using direct cultures (blood, McConkey, Sabouraud agars) and enrichment cultures (brain-heart infusion broth).
• A histological evaluation was performed to identify any cryopreservation artifacts, using hematoxylin-eosin staining.
• The tissues were also assessed ultrastructurally via transmission electron microscopy. The organization of corneoscleral collagen, as well as the count of normal and dead keratocytes, were noted.

Key Findings

• All direct microbiologic cultures were devoid of bacterial contamination – an important assurance for any form of transplant or use in procedures.
• 12.5% of corneal tissues and 59.4% of scleral tissues tested positive in enrichment cultures, although this was not statistically significant.
• Long-term cryopreserved tissues exhibited a higher incidence of cryopreservation artifacts compared to short-term cryopreserved ones – a finding that was statistically significant.
• Keratocytes (cells in the cornea) majored in normal form in short-term cryopreserved tissues, whereas long-term cryopreserved tissues mainly comprised apoptotic (programmed cell death) keratocytes. Necrotic (injured leading to cell death) keratocytes were seen only in long-term samples – with a statistically significant difference seen in this distribution.
• There were no detectable structural differences in collagen organization between the short- and long-term cryopreserved samples.

Conclusions

• The research concludes that cryopreservation of equine corneoscleral tissue does not result in direct bacterial contamination.
• Apoptotic processes were identified as the main cause of keratocyte death in the cryopreserved samples. In other words, cell death is not due to injury but is a programmed natural event.
• Considering negligible structural differences between short-term and long-term cryopreserved samples, the study posits that these cryopreserved tissues could potentially be deployed for tectonic support (used in corrective surgical procedures) for up to 9 years, without any structural or microbiological hindrance.