title: Establishing a reproducible method for the culture of primary equine corneal cells.
abstract: OBJECTIVE
  To establish a reproducible method for the culture of primary equine corneal epithelial cells, keratocytes, and endothelial cells and to describe each cell's morphologic characteristics, immunocytochemical staining properties and conditions required for cryopreservation.
  
  
  METHODS
  Corneas from eight horses recently euthanized for reasons unrelated to this study were collected aseptically and enzymatically separated into three individual layers for cell isolation. The cells were plated, grown in culture, and continued for several passages. Each cell type was characterized by morphology and immunocytochemical staining.
  
  
  RESULTS
  All three equine corneal cell types were successfully grown in culture. Cultured corneal endothelial cells were large, hexagonal cells with a moderate growth rate. Keratocytes were small, spindloid cells that grew rapidly. Epithelial cells had heterogeneous morphology and grew slowly. The endothelial cells and keratocytes stained positive for vimentin and were morphologically distinguishable from one another. The epithelial cells stained positive for cytokeratin. Keratocytes and endothelial cells were able to be cryopreserved and recovered. The cryopreserved cells maintained their morphological and immunocytochemical features after cryopreservation and recovery.
  
  
  CONCLUSIONS
  This work establishes reproducible methods for isolation and culture of equine corneal keratocytes and endothelial cells. Cell morphology and cytoskeletal element expression for equine corneal epithelial cells, keratocytes, and endothelial cells are also described. This has not previously been reported for equine corneal cells. This report also demonstrates the ability to preserve equine keratocytes and endothelial cells for extended periods of time and utilize them long after the primary-cell collection, a feature that has not been reported for veterinary corneal cell culture.
authors.affiliation: Department of Small Animal Medicine and Surgery, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA. rmathes@uga.edu
authors.forename: Rachel L
authors.initials: RL
authors.lastname: Mathes
authors.forename: Ursula M
authors.initials: UM
authors.lastname: Dietrich
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Krunkosky
authors.forename: David J
authors.initials: DJ
authors.lastname: Hurley
authors.forename: Adrian J
authors.initials: AJ
authors.lastname: Reber
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000662:veterinary*; D003315:Cornea / Q000166:cytology* / Q000502:physiology; D015925:Cryopreservation / Q000662:veterinary; D042783:Endothelial Cells / Q000502:physiology*; D004847:Epithelial Cells / Q000502:physiology*; D005434:Flow Cytometry; D006736:Horses; D013997:Time Factors