Culture and characterization of equine terminal arch endothelial cells andhoof 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 arter ies of the disarticulated lower portion of the forelimbs from 4 horses that had been heparinized prior to euthanasia. Phosphate-buffered saline soluti on was used to remove and discard RBC from blood vessels, and collagenase w as used to loosen and flush EC from the vasculature. Hoof keratinocytes wer e obtained from 4 recently euthanatized horses by use of dispase/trypsin di ssociation of the coronary band epidermis. Use of an extracellular matrix g el as a culture flask attachment factor was important to the success of hoo f keratinocyte cultures. Results-EC from the palmar digital arteries were successfully cultured and characterized by in vitro morphology, uptake of a fluorescence-labeled acet ylated-low density lipoprotein, and lack of expression of von Willebrand fa ctor and smooth muscle actin. Hoof keratinocytes were characterized by morp hology in culture and expression of keratin proteins, as determined by immu nochemical reaction. Keratinocyte cultures were also positive for vimentin expression. Conclusions-Culture techniques to isolate and characterize hoof cells shoul d aid investigators in their study of equine hoof pathobiologic features, e specially as it relates to laminitis.