Isolation and cultivation of equine corneal keratocytes, fibroblasts and myofibroblasts.
OBJECTIVE
To establish an in vitro model for the investigation of equine corneal wound healing. To accomplish this goal, a protocol to isolate and culture equine corneal keratocytes, fibroblasts and myofibroblasts was developed. ANIMAL MATERIAL: Equine corneal buttons were aseptically harvested from healthy research horses undergoing humane euthanasia for reasons unrelated to this study. Slit-lamp biomicroscopy was performed prior to euthanasia by a board-certified veterinary ophthalmologist to ensure that all samples were harvested from horses free of anterior segment disease.


METHODS
Equine corneal stroma was isolated using mechanical techniques and stromal sub-sections were then cultured. Customized media at different culture conditions was used to promote growth and differentiation of corneal stromal cells into keratocytes, fibroblasts and myofibroblasts.


RESULTS
Cell culture techniques were successfully used to establish a method for the isolation and culture of equine corneal keratocytes, fibroblasts and myofibroblasts. Immunohistochemical staining for alpha-smooth muscle and F-actin was used to definitively differentiate the three cell types.


CONCLUSIONS
Equine corneal stromal keratocytes, fibroblasts and myofibroblasts can be predictably isolated and cultured in vitro using this protocol.
College of Veterinary Medicine, University of Missouri, Columbia, MO, USA.
Dylan G
DG
Buss
Elizabeth A
EA
Giuliano
Ajay
A
Sharma
Rajiv R
RR
Mohan
D000199:Actins / Q000378:metabolism; D000818:Animals; D002469:Cell Separation / Q000662:veterinary; D002470:Cell Survival; D002478:Cells, Cultured; D003315:Cornea / Q000166:cytology* / Q000378:metabolism; D003319:Corneal Stroma / Q000166:cytology / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology; D006736:Horses / Q000033:anatomy & histology*; D016133:Polymerase Chain Reaction
D000199:Actins