DEVELOPMENT AND SCALE-UP OF 3D ENDOGENOUS HUMAN SKIN EQUIVALENT MODELS AS PLATFORM FOR COSMETICS TESTING

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The current necessity of development new biological in vitro models that mimic the characteristics and the complexity of human tissue arises from the need to find a valid alternative to animal models to test and validate new products, and to screen substances and procedures for tissue repair and regeneration. In particular, in the cosmetics field, big multinational companies have developed and devised methods for the realization of testing platforms in large scale, since the European Regulations (Decree 76/768/EEC and the EU Cosmetic Regulation 1223/2009) ban the putting on the market cosmetic products whose ingredients, or parts thereof, have been tested on animal models. in this scenario, researchers have been spent many efforts to develop innovative tissue engineering strategies to create 3D skin equivalent models that faithfully recapitulate the characteristics of human skin in terms of organization, complexity, architecture and responsivity to specific exogenous stimuli. The optimization of the process to produce these skin equivalent represents a crucial step to obtain i) tissue in large scale in order to allow the screening of a large number of molecules/exogenous factor ii) a high-fidelity replica of the native counterpart in order to evaluate the effect of molecules/exogenous factor on the mechanical properties and ECM composition organization and hydration. For this purpose, in my PhD work, after a deep study of the literature, it was developed a method of production of 3D skin equivalent models in large scale with great reproducibility. In the first part of the thesis, there is a description of the principal systems that composed the human skin and a summary about the main arguments of European Regulation related to cosmetics testing, the development of alternatives animal tests and its principal applications. In the second chapter, we exploited a bottom-up tissue engineering approach to build up the skin tissues. Such approach allowed to obtain skin tissues composed of endogenous extracellular matrix (ECM), produced by human dermal fibroblasts and by stratified epithelial cells that constitute a fully differentiated epithelium resembling the human epidermis. In the third chapter, we performed a morphological characterization of our 3D skin tissue by histological, biochemical and mechanical analysis in order to better describe the main features of this human skin equivalent models. Furthermore, to validate the skin produced as testing platform, we induced different kinds of damages (UVA, H2O2) and after evaluating the effect on the tissue, with the aim to study the effectiveness of molecules having antioxidant and photo-protective action. Finally, in the last part of thesis, we described firstly, a comparison between 3D skin models designed by us with the commercially available gold standard models produced by best international companies, and then a scale-up strategy in order to improve the production process of 3D-skin equivalent models with the prospective of realization of a start-up. In this last part, we described phases and all critical steps of human skin equivalent production passing from the realization of skin tissue in small scale to the large scale and the development of a working plan of all activities to better control step by step the quality and the effectiveness of final product. All results reported in my thesis strongly suggested a possible use of the developed skin tissues as a valid alternative to the use of animal models for the testing of new cosmetic compounds.