Culture and characterization of equine terminal arch endothelial cells and hoof keratinocytes.

OBJECTIVES: To develop methods to isolate, culture, and characterize equine hoof endothelial cells (EC) and keratinocytes. SAMPLE POPULATION: Cells harvested from the forelimbs of 8 horses. PROCEDURE: EC were obtained via catheters placed in the palmar digital arteries of the disarticulated lower portion of the forelimbs from 4 horses that had been heparinized prior to euthanasia. Phosphate-buffered saline solution was used to remove and discard RBC from blood vessels, and collagenase was used to loosen and flush EC from the vasculature. Hoof keratinocytes were obtained from 4 recently euthanatized horses by use of dispase/trypsin dissociation of the coronary band epidermis. Use of an extracellular matrix gel as a culture flask attachment factor was important to the success of hoof keratinocyte cultures. RESULTS: EC from the palmar digital arteries were successfully cultured and characterized by in vitro morphology, uptake of a fluorescence-labeled acetylated-low density lipoprotein, and lack of expression of von Willebrand factor and smooth muscle actin. Hoof keratinocytes were characterized by morphology in culture and expression of keratin proteins, as determined by immunochemical reaction. Keratinocyte cultures were also positive for vimentin expression. CONCLUSIONS: Culture techniques to isolate and characterize hoof cells should aid investigators in their study of equine hoof pathobiologic features, especially as it relates to laminitis.