Development of a canine skin analog

Abstract

Background. Skin covers an extensive area of the body and comprises three major layers, subcutis, dermis, and epidermis. This organ is one of the most tested for toxicity and safety evaluation during the process of drug research and development, in past usually performed in vivo using animals. On the last years a concern of sustainable and eco-friendly approach has been developing, with the expansion of non-animal alternatives (Klicks et al.; Mathes et al.).A histotypical cell cultured-derived tissue may be used to replace animal testing and are imperative to avoid armful, drawn-out tests to assess chemicals for their capacity to erode, bother or sensitize the skin. There are validated epidermal models for human (EPISKINTM and EpiDermTM) and rat (TER) skin (Flaten et al.; NIEHS; Netzlaff et al.). The aim of our study was the development of a histotypical canine skin equivalent, that can be used for the assessment of corrosion, irritation and sensibilization, avoiding in vivo animal testing. Methods. Canine keratinocyte progenitor cells were seeded in inserts and were allowed to grow until differentiation was reached, using an adapted version of the CELLnTEC commercial protocol, specific for human cells (CELLnTEC). For histological analysis, samples were fixed in 10% neutral-buffered formalin. Three-micrometer paraffin sections were routinely processed for biopsies and stained with hematoxylin and eosin. Corrosion, irritation and sensibilization protocols were adapted from human equivalent validated tests. Results. A multilayer (3-4 cell layers thick) of canine keratinocytes was developed in air-lift culture, originating a stratified epidermal-like tissue, confirmed by histological analysis. This epidermal-like tissue exhibited functional characteristics of normal epidermis. It showed adequate impermeabilization, after 0.1% Triton X-100 exposure for 4h and responded adequately to the positive (5% SDS and glacial acetic acid) and negative (PBS) controls used in “in vitro” corrosion and irritation assessment. Conclusions. As predicted, a canine skin analog was developed. This is a promising skin model for non-animal safety tests of veterinary pharmaceuticals or cosmetics, reducing in vivo testing, and can be commercialized as a service or a product.