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Développement, validation et caractérisation d’un modèle ex vivo de peau humaine perfusé : FlowSkin

Emma Raude 1
1 LAAS-ELIA - Équipe Ingénierie pour les sciences du vivant
LAAS - Laboratoire d'analyse et d'architecture des systèmes
Abstract : Organotypic models as human skin explants are the most complex and among the most representative of in vivo skin existing today to test the efficacy or the safety of molecules of therapeutic interest during preclinical studies. However, the loss of vascularization and lymphatic system in these models remains a major limitation in tissue homeostasis that impedes the prediction of skin responses to a treatment. In addition, exchanges of nutrients and oxygen being limited to diffusion, models lifetime is limited. Different strategies have been implemented to study and improve mass transport mechanism in such models. Microfluidics offers a great potential to control diffusion and convection mechanisms that permit molecular exchanges in skin models.The objective of this project is to develop, characterize and validate an ex vivo perfused human skin model. The purpose of this intra-tissue infusion is to promote the exchanges of nutrients, oxygen or drugs, but also to improve metabolic waste elimination.The first objective of my work consisted in implementing an intra-tissue flow in a human skin explant, and in setting up a process to maintain the perfused model in culture for several days. To this end, a porous device was implanted in the dermis of the ex vivo human skin model NativeSkin, developed by the company Genoskin. The implantable device is then connected to a microfluidic system allowing the infusion of compounds within the tissue.The second objective was to develop analysis methods of the diffusion of compounds in skin explants. Four methods have been developed: macroscopic and qualitative evaluation of the diffusion using a dye, the study of the diffusion in real time by X-ray radiography, the study of the diffusion in three dimensions by X-ray tomography, and finally the analysis of the diffusion of fluorescent dextrans of different molecular weights, on histological sections. A numerical model allowing to simulate the diffusion in the skin model has also been developed using COMSOL software, allowing to predict the diffusion profile of a compound.The third and last objective of my work was to determine perfusion parameters allowing efficient molecular exchanges of compounds in the skin explant, but without damaging the tissue. A first series of experiments (8 donors) was carried out on models perfused with a constant flow-rate (2.5 µL/min) with culture medium, for 10 days. The results showed that at the end of the culture, skin models did not show any alteration in cell viability or tissue integrity, with maintenance of cell proliferation and metabolism. However, diffusion characterization in the model demonstrated a lack of reproducibility in the experiments, with significant inter and intra-donor variability. In addition, the infusion of different molecular weights dextrans has demonstrated that the mass transport of high molecular weight compounds was limited through the implantable device. We demonstrated that the control of the fluid pressure is critical and that imposing a pulsatile injection with slight overpressures improves the efficiency and reproducibility of the molecular species delivery and collection in the explant.These results have shown the potential of the developed FlowSkin model as a new tool to study the efficacy or toxicity of intravenously administered drugs directly onto human skin. In addition, the combination of FlowSkin with perfusion of oxygen carriers offers unique opportunities to extend the lifetime and further improve the relevance of such ex vivo skin model.
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Contributor : Abes Star :  Contact
Submitted on : Tuesday, March 30, 2021 - 12:38:08 PM
Last modification on : Friday, April 2, 2021 - 10:06:41 AM

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Emma Raude. Développement, validation et caractérisation d’un modèle ex vivo de peau humaine perfusé : FlowSkin. Médecine humaine et pathologie. INSA de Toulouse, 2020. Français. ⟨NNT : 2020ISAT0015⟩. ⟨tel-03185460⟩

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