FACULTY OF HEALTH AND SOCIETY | Dissertation defence
Dissertation defence – Sylwia Hasterok

Physiologically relevant human skin models are important for investigating skin biology, inflammation, and disease under controlled conditions. However, conventional in vitro models often lack the structural complexity of native skin and fail to adequately reproduce interactions between skin-resident cells and immune components. Therefore, addressing these limitations remains essential for developing models that better reflect human skin biology in health and disease. This thesis aimed to establish a methodological framework of tools and techniques for developing physiologically relevant human skin in vitro models, providing a foundation for the future integration of immune components.

The work comprised five studies, including a literature review and four experimental studies. The literature review examined existing approaches to 3D model reconstruction and informed the selection of a strategy for in vitro skin model development. The experimental studies addressed complementary methodological aspects of in vitro skin model development and future immune cell integration. Three-dimensional skin equivalents containing fibroblasts, keratinocytes, and either melanocytes or melanoma cells were established to represent healthy and melanoma-associated skin conditions. The models reproduced selected structural features of human skin, although differences from native tissue remained. Functional evaluation revealed differential responses in IDO-1 expression and tryptophan–kynurenine metabolism following IFN-γ or UVB exposure, supporting the models' utility for investigating melanomaassociated metabolic responses. In complementary studies, an optimised differentiation approach generated distinct Langerhans cell subsets from human peripheral blood monocytes, reflecting aspects of their in vivo heterogeneity.

Furthermore, a high-sensitivity imaging technology combining upconverting nanoparticles and segmentation-based analysis enabled the visualisation and quantitative assessment of low-abundance cells, such as immune cells, in multicellular systems. This approach provides a basis for the future detection and characterisation of scarce cells, such as Langerhans cells, in more complex skin models.

Overall, this thesis advances the development of physiologically relevant human skin in vitro models by establishing and evaluating 3D skin equivalents and developing complementary methods for immune-cell generation and detection.

Together, these methodological advances provide a foundation for future model refinement and immune integration, supporting the development of more representative experimental systems for investigating skin biology and melanoma-associated processes.