A canine skin equivalent model has been validated for the assessment of a topical formulation effects. Skin equivalents were developed from freshly isolated cutaneous canine fibroblasts and keratinocytes, after enzymatic digestion of skin samples ( ) from different breeds. Fibroblasts were embedded into a collagen type I matrix, and keratinocytes were seeded onto its surface at air-liquid interface. Skin equivalents were supplemented with essential oils and polyunsaturated fatty acid formulation or with vehicle. Skin equivalents were histopathologically and ultrastructurally studied, and the three main lipid groups (free fatty acids, cholesterol, and ceramides) were analyzed. Results showed that the culture method developed resulted in significant improvements in cell retrieval and confluence. Treated samples presented a thicker epidermis with increased number of viable cell layers, a denser and compact stratum corneum, and a more continuous basal membrane. Regarding lipid profile, treated skin equivalents showed a significant increase in ceramide content ( ) when compared to untreated ( ) samples. Ultrastructural study evidenced a compact and well-organized stratum corneum in both treated and control skin equivalents. In conclusion, cell viability and ceramides increase, after lipid supplementation, are especially relevant for the treatment of skin barrier disruptions occurring in canine atopic dermatitis. 1. Introduction In recent years, significant progress has been made to produce engineered substitutes of human and animal skin [1]. Human skin equivalents (SE) reconstructed from both epidermal and dermal compartments are currently employed for safety and toxicity studies in both cosmetics and pharmaceutical compounds [2]. In veterinary medicine, tissue culture technology has been mainly applied to the development of in vitro models of canine skin pathologies such as epidermolysis bullosa [3]. Afterwards, Serra et al. [4] developed an SE from healthy canine skin suggesting the use of that organotypic skin culture as an alternative to the in vivo investigations for skin research. Nevertheless, to our knowledge, there are no studies evaluating the suitability of canine SE models for pharmacological compounds testing. A canine SE would be a useful tool for test topical or systemic treatments in veterinary medicine contributing to cutaneous drug research while sparing experimental animals. In this way, it would be possible to study the effects not only in skin cells but also in skin morphology and functionality. Atopic dermatitis (AD) is the most common
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