Leveraging physiology in human skin models - integrating adipose tissue and perfused vasculature

Even though anatomically relatively simple at first glance, skin is an incredibly complex organ with several vital functions such as a physical barrier and immune defense. Over the years, skin in vitro models have improved significantly in complexity through advances in the field of tissue engineering, especially with the emergence of Organ-on-Chips (OoCs) and 3D bioprinting. However, advancing skin tissue engineering technology to incorporate a perfused vasculature and a functional immune system is still a major challenge. Therefore, the primary objective of this thesis was to engineer the next generation of RhS (Reconstructed human Skin), modelling such distinct aspects of the human skin structure and function in a physiologically relevant model. The aim of Chapter 2 was to develop a true full thickness RhS with epidermis, dermis, and a subcutaneous adipose layer. Because adipose tissue is metabolically active, we hypothesized that the discrepancy in metabolic capability between native skin and RhS models could be restored by adding an adipose layer below the dermis. This layer was composed of adipose-derived stromal cells and differentiated adipocytes in a collagen-fibrin matrix. Chapter 3 delves into the isolation of primary human endothelial cells from skin biopsies and their separation into blood and lymphatic endothelial cells. These cells were used to generate perfused blood and lymphatic vessels in a multi-organ-chip (MOC) platform (TissUse HUMIMIC Chip2 and Chip3plus) with the objective to investigate the influence of culture length, varying flow rates and inflammatory conditions on cell morphology, biomarker secretion and phenotype. The deliberate choice was made to use a MOC platform, thereby allowing the future combination of different organ types and connecting them through vasculature. After lining the microfluidic channels with blood (BECs) and lymphatic endothelial cells (LECs) in the previous chapter, Chapter 4 aims to investigate a method combining bioprinting with OoC to bioengineer a perfused blood vasculature within the skin’s dermis. The chip potential was further explored by the addition of monocytes to the circulation and their ability to transmigrate into the vascularized fibroblast-populated dermis. The same methodology was utilized in Chapter 5, where – instead of blood vasculature – the focus was on lymphatic vasculature. Here, the aim was to investigate the influence of a perfused lymphatic vessel on a LN-on-chip model. This chapter emphasizes the necessity of a lymphatic vasculature in the LN on chip model, as indicated by a change in LEC phenotype, improved spatial organization, increased cytokine secretion and higher immune cell numbers. Chapter 6 combines expertise from the four previous chapters. Here, data of a pilot experiment with a vascularized skin-LN-on-chip model is presented as a proof of concept and an outlook for future experiments. To conclude, all results of this thesis are summarized, discussed, and contextualized for future perspectives in Chapter 7.