Establishing a reproducible method for the culture of primary equine corneal cells.

This study aimed to devise a reliable method for isolating and culturing various types of cells from the corneas of horses. The conditions for preserving these cells were also established. The researchers were able to successfully culture these cells and state their characteristics for differentiation purposes. This represents the first time such a process has been reported in veterinary corneal cell culture.

Methods

• The researchers collected corneas from eight horses that had been euthanized for reasons unrelated to the study.
• The corneas were aseptically and enzymatically separated into three individual layers to isolate the cells: these were the corneal epithelial cells, keratocytes, and endothelial cells.
• The respective cells were cultivated and allowed to grow over several passages.
• Eventually, the characteristics of each cell type were assessed using morphology and immunocytochemical staining.

Results

• All three types of equine corneal cells were successfully cultured.
• The corneal endothelial cells were identified as large, hexagonal cells demonstrating moderate growth rates.
• Keratocytes were highlighted as small, spindloid cells with rapid growth.
• Epithelial cells presented a diverse morphology and grew at a slower pace.
• The endothelial cells and keratocytes stained positively for vimentin, a protein involved in maintaining cellular integrity, while the epithelial cells stained positively for cytokeratin, a protein found in the intracytoplasmic cytoskeleton of epithelial tissue.
• The cryopreservation and subsequent recovery of keratocytes and endothelial cells were successful, with the cells maintaining their morphological and immunocytochemical features post-recovery.

Conclusions

• The study successfully established reproducible methods for the isolation and culture of equine corneal keratocytes and endothelial cells.
• The morphology and cytoskeletal element expression for equine corneal epithelial cells, keratocytes, and endothelial cells were also described, adding to the existing body of knowledge regarding these cell types.
• The research additionally demonstrated the ability to preserve keratocytes and endothelial cells for extended periods, rendering them useful long after the primary-cell collection. This feature has not been previously reported in veterinary corneal cell culture.