Isolation , cultivation and immunofluorescence characterization of lamellar keratinocytes from equine hoof by using explants 1

RESUMO.[Isolamento, cultivo e caracterização por imunofluorescência de queratinócitos lamelares de casco equino utilizando explantes.] É notória a importância do casco na saúde dos equinos, mas o conhecimento em nível celular é pouco entendido. Estudos envolvendo o cultivo de queratinócitos equinos são escassos. Sabe-se que o desenvolvimento de cultivos de queratinócitos in vitro é uma condição para estudos sobre a biologia molecular, crescimento e diferenciação celular. Alguns métodos já estão estabelecidos, como para cultivo de queratinócitos de pele, mas poucas metodologias são encontradas para queratinócitos lamelares. O objetivo desse estudo foi padronizar o cultivo de queratinócitos provenientes de casco equino visando futuramente associar ao estudo da medicina regenerativa para assim estabelecer um modelo experimental in vitro e indicar o uso criterioso de terapias regenerativas para a laminite equina. Desta forma, o cultivo em monocamada e a caracterização de queratinócitos lamelares foram realizados. Para isso, o método de cultura primária utilizado foi através de explantes obtidos de três regiões do casco (dorso-medial, ABSTRACT.Pfeifer J.P.H., Santos V.H., Rosa G., Souza J.B., Watanabe M.J., Fonseca-Alves C.E., Deffune E. & Alves A.L.G. 2019. Isolation, cultivation and immunofluorescence characterization of lamellar keratinocytes from equine hoof by using explants. Pesquisa Veterinária Brasileira 39(4):292-298. Departamento de Cirurgia e Anestesiologia Veterinária, Faculdade de Medicina Veterinária e Zootecnia, Universidade Estadual Paulista “Julio de Mesquita Filho”, Distrito de Rubião Junior s/n, Botucatu, SP 18618-681, São Paulo, Brazil. E-mail: ana.liz@unesp.br The importance of the hoof to the horse health is clear, and the current knowledge regarding the cellular aspects of hoof keratinocytes is poor. Studies on equine keratinocyte culture are scarce. Developing keratinocyte cultures in vitro is a condition for studies on molecular biology, cell growth and differentiation. Some methods have already been established, such as those for skin keratinocyte culture. However, few methodologies are found for lamellar keratinocytes. The objective of this study was to standardize the equine hoof keratinocyte isolation and cultivation, and then characterize the cell immunophenotype. For this, the primary culture method used was through explants obtained from three regions of the equine hoof (medial dorsal, dorsal, and lateral dorsal). After the cell isolation and cultivation, the cell culture and its explants were stained with anti-pan cytokeratin (pan-CK) (AE1/AE3), vimentin (V9), p63 (4A4), and Ki-67 (MIB-1) antibodies. Cells were grown to third passage, were positive for pan-CK, p63 and Ki-67, and few cells had vimentin positive expression. As for the explants, the epidermal laminae were not stained for vimentin or Ki-67. However, some cells presented positive pan-CK and p63 expression. This study demonstrated the viability of lamellar explants of equine hooves as a form of isolating keratinocytes in primary cultures, as well as characterized the proliferation ability of such keratinocytes in monolayers.