The skin acts as the organism's first line of defense against physical, chemical, and biological agents. Currently, assessing the skin-sensitizing potential of new chemicals relies upon their application in animal models. However, due to their questionable biological similarity to humans, ethical issues, and current European legislation that requires animal models to be discontinued, new alternatives must be developed. To overcome these constraints, organotypic human skin models are a promising choice however, most of the available models lack an immune system and other native skin characteristics. Thus, the main objective of this thesis is to create and characterize an in vitro immunocompetent skin model comprising fibroblasts, keratinocytes, and Langerhans cells (LGs), capable of mimicking the interactions between different cells and simulating the systemic response of the human body. Furthermore, to overcome the limited number of LGs available, the use of mature dendritic cells was explored for the development of the immunocompetent skin model and their performance compared against LGs. For LGs isolation, magnetic sorting was the chosen method from the applied approaches since it resulted in a purer population of fully functional LGs capable of responding successfully to inflammatory stimuli and enhancing T-cell proliferation. Regarding mDCs, the THP-1 cell line was used to differentiate them. The acquired cells were found to be functional and capable of acting as LGs surrogates, as they respond to allergens and activate T cell proliferation in the same way the epidermal LGs do. Model characterization allowed us to confirm that the ratios of 1 LG to 50 KCs and 1 mDC to 5 KCs in the case of LGs and mDCs, respectively, were the most similar to the in vivo tissue organization and, as a result, had better epidermal stratification and differentiation. The presence of melanocytes in the LGs model was also confirmed, resulting in a more physiologically relevant and accurate representation of human skin. Furthermore, when exposed to a skin sensitizer, NiSO4, the immune cells within the models were able to migrate to the dermis, revealing that the cells were active and functional. In short, the two immunocompetent models established are capable of mimicking human skin organization, simulating crosstalk between different cells, and reproducing one of the key steps of the systemic response of the human body, making them an excellent in vitro research tool for studying the biology and functionality of dendritic cells under controlled conditions. Notwithstanding, these models can also be used to create future platforms for assessing the allergenicity and toxicity of novel chemicals.
- Langerhans cells
- In vitro immunocompetent skin model
- Mature dendritic cells
- THP-1
- Skin-sensitizer potential
- Mestrado em Engenharia Biomédica
The missing link: development of an immunocompetent in vitro skin model
Vieira, S. A. (Student). 7 Nov 2023
Student thesis: Master's Thesis