Cultivation and characterisation of primary and subcultured equine keratinocytes

Abstract

Summary We describe the establishment and characterisation of equine keratinocyte cultures with maintenance of a high proliferative capacity up to the second passage. Improved attachment and growth were obtained by seeding primary cells on equine feeder layers. Subcultured keratinocytes showed optimal growth when seeded on collagen type I. The proliferation rate of cells on this substrate exceeded that seen for cells seeded on equine feeder layers. By immunohistochemistry, epithelial origin and state of differentiation of the equine keratinocytes were determined. They expressed keratin and desmoplakin I/II, but lacked keratin 10. Electron microscopy revealed typical features of cultured keratinocytes. Purity of keratinocyte cultures was determined by vimentin staining. This is the first report on the establishment of equine keratinocytes derived from lip epithelium. It forms the basis to study equine keratinocyte biology and the pathogenesis of epidermal diseases. Since wound healing represents a severe problem in equine dermatology, our data may be essential for the establishment of new and improved therapy.

Characterization of extracellular matrix macromolecules in primary cultures of equine keratinocytes

“…We found all lamellar cells expressed vimentin in the cell cytoplasm regardless of the calcium concentration and did not display a fibroblastic cell morphology. Additionally, vimentin expression may be cell origin specific, as equine skin keratinocytes have been found to not express vimentin in culture [16] while lamellar keratinocytes in culture have been shown to express vimentin [14] in agreement with findings in this work.…”

“…Similar to another report [16], skin keratinocytes isolated from lip epithelium were found to proliferate and propagate in culture. In the present study, cells were found to adhere well to collagen type I during the initial isolation and this substrate was used for initial growth in addition to subculture.…”

“…Skin keratinocytes grew well in standard DMEM culture media with hormonal and serum supplementation and an approximate calcium concentration of 1.8 mM. This is in contrast to Dahm et al [16] who found media containing a calcium concentration of 0.6 mM optimal in their study, as with higher calcium they found early differentiation and loss of cell proliferation. Work presented here indicates that skin keratinocytes are able to proliferate at a higher calcium concentration, as long as suitable media and substrate conditions are present.…”

“…Calcium concentration present in culture medium is known to play a major role in cell proliferation and differentiation [17]. For equine skin keratinocytes, a calcium concentration of 0.6 mM was found to be optimal for cell proliferation and propagation [16]. Likewise, for lamellar keratinocytes, growth in calcium free DMEM media supplemented with 5% FBS plus additional calcium (0.6 mM) proved optimal for prolonged lamellar cell propagation.…”

“…Total cell lysates [16] and individual cell and matrix layers were prepared [29]. Samples were separated using 8-16% gradient SDS- PAGE mini gels (NuSep) followed by transfer to poly-vinyl-D-fluoride membrane (GE healthcare).…”