Epidermal-like architecture obtained from equine keratinocytes in three-dimensional cultures

Abstract

Despite the high prevalence of skin conditions in the horse, there is a dearth of literature on the culture and biology of equine skin cells, and this is partially attributable to the lack of suitable in vitro skin models. The objective of this study was to develop a three-dimensional (3D) culture system that would support the proliferation and differentiation of equine keratinocytes, similar to that observed in natural epidermis. Cell monolayers were obtained from explants of equine skin and serially passaged as highly pure keratinocyte populations (> 95% of cells), based on their expression of cytokeratins, including CK-5 and CK-14, which are associated in vivo with proliferating keratinocyte populations. Explant-derived keratinocytes were seeded into Alvetex™ 3D tissue scaffolds for 30 days under conditions that promote cell differentiation. Ultrastructural, immunohistochemical and biochemical analyses revealed that keratinocytes within scaffolds were able to proliferate and attain tissue polarity, including differentiation into basal and suprabasal layers. The basal layer contained distinct cuboidal cells with large nuclei and stained for proliferative markers such as CK-5 and CK-14. In contrast, the suprabasal layers consisted of cells with distinct polyhedral morphology, abundant cytoplasmic processes and desmosomes indicative of stratum spinosum and distinct flattened cornified cells that expressed involucrin, a marker of terminal differentiation. Thus, keratinocytes derived from primary equine skin explants were able to attain epidermal-like architecture in culture. This novel system could provide a very useful tool for modelling skin diseases, drug testing/toxicity studies and, potentially, equine regenerative medicine. Copyright © 2016 John Wiley & Sons, Ltd.

Development and characterization of an equine skin‐equivalent model

“…The normal thickness of the epidermis and the cornification process were also achieved after the culture period. The unique model of equine epidermis‐like structure developed until now had failed to form a stratified epidermis with well‐defined layers and corneocytes in the upper layers and a continuous basement membrane . This result is perhaps due to the reduced subpopulation of epidermal stem cells obtained after the isolation procedure, as previously described, and also the absence of a dermal compartment, based on the fact that epidermal differentiation and basement membrane formation are linked to the underlying connective tissue .…”

“…In all of these studies, however, primary cultures of keratinocytes could not be passaged more than four times. Finally, a study established equine keratinocyte cultures from skin explants . In this case, although cells proliferated for up to six passages and expressed a terminal marker of cornification in suprabasal cell layers in a three‐dimensional culture system, the authors failed to observe a complete epidermis with four defined strata.…”

“…Primary keratinocytes (1 × 10 5 cells/cm 2 ) were then plated onto collagen‐coated flasks and grown in a humidified atmosphere at 37°C with 5% CO 2 . The culture medium consisted of DMEM containing 5% FBS, 0.01 μg/mL epidermal growth factor (Austral Biologicals, San Ramon, CA, USA) and β‐mercaptoethanol (0.1 mmol/L; Invitrogen), as previously described . The medium was changed every 2–3 days and subcultured when 90% confluence was reached.…”

“…Two studies described media, techniques and procedures in the culturing and isolation of primary equine keratinocytes, but cells proliferated at low rates or were not passaged more than four times . Two recent studies, however, reported an equine keratinocyte culture grown for at least six passages and a three‐dimensional culture system that enabled keratinocyte differentiation into an epidermis‐like structure . Thus, efficient isolation and culture techniques have the potential to provide long‐term cultures of equine keratinocytes that may be used to develop ESE, incorporating both dermal and epidermal compartments.…”

“…18,19 Two recent studies, however, reported an equine keratinocyte culture grown for at least six passages 20 and a three-dimensional culture system that enabled keratinocyte differentiation into an epidermis-like structure. 21 Thus, efficient isolation and culture techniques have the potential to provide long-term cultures of equine keratinocytes that may be used to develop ESE, incorporating both dermal and epidermal compartments. The objective of this study was the development of an equine skin model that could provide a useful tool to investigate the behaviour of equine skin cells in physiological and pathological conditions and could be used to establish tissue-engineered skin therapy in horses.…”