Fredrik Carl Wieland

Complex tissues in vitro have applications in modelling development and disease, screening and discovering drugs, and as medical therapies. Three-dimensional (3D) aggregate cell clusters more closely mimic the in vivo environment, particularly the cell–cell and cell–matrix interactions, and can aid in clinical translation, as pre-clinical studies use non-human animal models with varying human mimicry.

To this end, in Chapter 2, we reviewed the intricate multicellular interactions within the pancreatic islet of Langerhans and their translation into in vitro co-cultures known as pseudoislets. We focussed on how a deeper understanding of islet structure, cell–cell interactions, ECM proteins, and paracrine signalling can together create an optimal microenvironment for islet cells. Chapter 2 highlighted the delicate relationship between gap junctions and cell function, and that endothelial cells contribute crucial ECM proteins that influence β cell glucose responsiveness, underscoring the importance of a heterogeneous cell mixture. Explanations were made on how islet structure affects glucose responsiveness, emphasising the importance of 3D cell culture and co-culture with α and endothelial cells.

Studying complex cell aggregates necessitates several complex approaches to quantify cellular self-assembly and organization. Chapter 3 outlines the methodology developed in this thesis to optimize a microscopy workflow for achieving single-cell resolution within pseudoislets cultured in microplates. The lessons learned are that careful consideration is crucial when selecting 3D culture and analysis software, as each setup may yield different results. While Nikon GAP and Imaris analysis software outperformed Fiji and CellProfiler in our study, they may not be as accessible. We recommend clearly defining analysis requirements and conducting testing to determine the most suitable culture and software solution.

Chapter 4 aimed to explore the organization of pseudoislets and understand the relationship between pancreatic endocrine cells and endothelial cells. By varying the relative ratios and the total number of cells, the study investigated the impact on pseudoislet composition and organization. Spatial organization during self-assembly was quantified, revealing insights into the rules governing pseudoislet self-organization. Surprisingly, the initial seeding ratio did not influence the final cell distribution after ten days in culture. However, pseudoislet size affected endothelial cell incorporation and α cell numbers slightly increased with pseudoislet size. The increase of α cells suggests that they play a crucial role in endothelial cell function, potentially aiding pseudoislet endothelialisation. Specifically, endothelial cells were found adjacent to α cells significantly more frequently than to β cells. This finding is of importance for regenerative medicine, particularly, where rapid vasculature formation around transplanted β cells is crucial for viability and function.

The lessons learned from Chapter 4 were continued into Chapter 5, which delves into understanding the impact of interactions between α and endothelial cells on the levels of oxidative stress experienced by the pseudoislet. Oxidative stress is known for its detrimental effect on β cell viability due to their low levels of antioxidant enzymes. The composition of the pseudoislet, namely including α and endothelial cells, was found to reduce oxidative stress, highlighting their supportive role. Exploring cell interactions revealed that the reduction in oxidative stress was attributed to antioxidant enzymes protecting endothelial and β cells via the incretin glucagon-like peptide-1 (GLP-1). Moreover, the presence of α cells significantly reduced the number of cells experiencing oxidative stress, and adding GLP-1 effectively rescued the pseudoislets lacking α cells. These findings underscore the critical role of α cells in mitigating oxidative stress within the pseudoislet. Also, the study has implications for developing cell-based therapies for type 1 diabetes, indicating that including both α and endothelial cells could enhance the successful transplantation of de novo cell-derived islet cells into patients.

This thesis focused on exploring approaches to capture and comprehend aggregated cells' complexity, enhance understanding of self-assembly intricacies, and shed light on the importance of support cells in pancreatic (pseudo)islets. The future of in vitro models holds promise, with advancements like self-assembly, 3D bioprinting, and gene editing poised to advance their fidelity, particularly for applications in regenerative medicine.