Modelling stromal-competent human lymph nodes in vitro

With the rapid progress in the global need for humane research, the immune system remains cemented as a critical factor that requires addressing and incorporation into animal-alternative models. While the LN itself acts essentially as the control centre of human immunology, and advancements in lymphoid organ models have shown promising developments, incorporation of the influential LNSCs has yet to be properly achieved. Therefore, this thesis aims to build a 3D LN model with a stromal cell backbone in both static and dynamic settings to mimic physiological LN functioning. Chapter 2 provides a comprehensive overview of all single- and multi-OoC models that have incorporated the human immune system. This explores the level of immunocompetency for each OoC model with the use of innate and adaptive immune cells, while also discussing the current limitations, challenges and degrees of physiological improvements. Before diving into the development of a LN model with an FRC component, Chapter 3 outlines our initial efforts in examining whether it was possible to culture human LN-isolated FRCs ex vivo, and whether these FRCs could represent characteristics of human LNSCs subsets. In Chapter 4, we investigate the possibility of constructing a human LN model using cultured FRCs to act as the foundation for DC integration. However, the LN contains a variety of immune cells, such as a plethora of lymphocyte and myeloid cell subsets. Therefore, in order to advance the two cell FRC-DC model into one reflecting a LN environment to study immune cell functionality, Chapter 5 reports the incorporation of native LN-derived immune cells into a LN model, enriched with FRCs. As mentioned, the LN is a dynamic environment characterised by immune cell trafficking, chemotactic gradients and cell-cell interactions. Static models fail to mimic these elements, which are pivotal in facilitating efficient and potent immune responses. Therefore, the last experimental study of this thesis, described in Chapter 6, aimed to bring the LN model into an OoC device and establish the lymphatic vasculature through the LN model using autologous LECs. Finally, Chapter 7 collectively discusses the key findings of each chapter to conclude this thesis.