Characterization of canine epidermal organoid culturesby immunohistochemical analysis and quantitative PCR

Background – Keratinocyte organoids can be used as a tool to evaluate epidermal structure, function and dys-function.Objectives – To optimize the canine keratinocyte organoid system and produce organoids that are structurallyequivalent to in vivo canine epidermis, in order to enable studies that focus on epidermal diseases and diseasesresulting from an impaired epidermal barrier.Animals – Skin biopsies were obtained from five recently euthanized dogs of different breeds with no skinabnormalities.Methods and materials – Cells derived from microdissected interfollicular epidermis were seeded in basementmembrane extract and epidermal organoids were grown under different media conditions. Organoids were char-acterized to assess cell morphology and architecture in haematoxylin and eosin-stained slides and expression ofselected epidermal markers (keratin 5, keratin 10, loricrin and filaggrin) by immunohistochemical analysis andquantitative reverse transcription PCR.Results – The selected epidermal markers were expressed in the same epidermal layers in the organoids cul-tured in expansion medium and differentiation medium as in normal interfollicular epidermis, yet restriction to thedistinct layers was best achieved with expansion medium. Comparison of the mRNA expression levels of thesemarkers revealed that relative expression is similar in organoids cultured in expansion medium and normal canineepidermis, while it differs in organoids cultured in differentiation medium.Conclusion and clinical importance – Organoids cultured in expansion medium have an equivalent structureto the interfollicular epidermis and express key marker proteins in similar proportions. Epidermal organoids aretherefore a promising in vitro model to study epidermal structure, function and dysfunction