Methodological approaches in aggregate formation and microscopic analysis to assess pseudoislet morphology and cellular interactions.

Microscopy has revolutionised our view on biology and has been vital for many discoveries since its invention around 200 years ago. Recent developments in cell biology have led to a strong interest in generating spheroids and organoids that better represent tissue. However, the current challenge faced by many researchers is the culture and analysis of these three-dimensional (3D) cell cultures. With the technological improvements in reconstructing volumetric datasets by optical sections, it is possible to quantify cells, their spatial arrangement, and the protein distribution without destroying the physical organization. We assessed three different microwell culture plates and four analysis tools for 3D imaging data for their applicability for the analysis of 3D cultures. A key advantage of microwell plates is their potential to perform high-throughput experiments in which cell cultures are generated and analysed in one single system. However, it was shown that this potential could be impacted by the material composition and microwell structure. For example, antibody staining was not possible in a hydrogel microwell, and truncated pyramid-structured microwells had increased background fluorescence due to their structure. Regarding analysis tools, four different software, namely CellProfiler, Fiji/ImageJ, Nikon GA3 and Imaris, were compared for their accuracy and applicability in analysing datasets from 3D cultures. The results showed that the open-access software, CellProfiler and Fiji, could quantify nuclei and cells, yet with varying results compared to manual counting, and may require post-processing optimisation. On the other hand, the GA3 and Imaris software packages showed excellent versatility in usage and accuracy in the quantification of nuclei and cells, and could classify cell localisation. Together these results provide critical considerations for microscopic imaging and analysis of 3D cell cultures.

The Role of Pancreatic Alpha Cells and Endothelial Cells in the Reduction of Oxidative Stress in Pseudoislets.

Pancreatic beta cells have inadequate levels of antioxidant enzymes, and the damage induced by oxidative stress poses a challenge for their use in a therapy for patients with type 1 diabetes. It is known that the interaction of the pancreatic endocrine cells with support cells can improve their survival and lead to less vulnerability to oxidative stress. Here we investigated alpha (alpha TC-1), beta (INS1E) and endothelial (HUVEC) cells assembled into aggregates known as pseudoislets as a model of the pancreatic islets of Langerhans. We hypothesised that the coculture of alpha, beta and endothelial cells would be protective against oxidative stress. First, we showed that adding endothelial cells decreased the percentage of oxidative stress-positive cells. We then asked if the number of endothelial cells or the size (number of cells) of the pseudoislet could increase the protection against oxidative stress. However, no additional benefit was observed by those changes. On the other hand, we identified a potential supportive effect of the alpha cells in reducing oxidative stress in beta and endothelial cells. We were able to link this to the incretin glucagon-like peptide-1 (GLP-1) by showing that the absence of alpha cells in the pseudoislet caused increased oxidative stress, but the addition of GLP-1 could restore this. Together, these results provide important insights into the roles of alpha and endothelial cells in protecting against oxidative stress.

The Role of Alpha Cells in the Self-Assembly of Bioengineered Islets.

Vascularization is undoubtedly one of the greatest challenges in tissue engineering. Its importance is particularly evident when considering the transplantation of (bioengineered) pancreatic islets of Langerhans, which are highly sensitive to the delivery of oxygen and nutrients for their survival and function. Here we studied pseudoislets of Langerhans, which are three-dimensional spheroids composed of ß (INS1E), α (alpha TC-1), and endothelial (HUVEC) cells, and were interested in how the location and prevalence of the different cell types affected the presence of endothelial cells in the pseudoislet. We hypothesized that alpha (α) cells play an essential role in islet self-assembly and the incorporation of endothelial cells into the pseudoislet, and are thus important to consider in tissue engineering or regenerative medicine strategies, which typically focuses on the insulin-producing beta (ß) cells alone. We first determined the effect of changing the relative ratios of the cells and found the cell distribution converged on a steady state of ∼21% α cells, 74% ß cells, and 5% endothelial cells after 10 days of culture regardless of their respective ratios at seeding. We also found that the incorporation of endothelial cells was related to the pseudoislet size, with more endothelial cells found in the core of larger pseudoislets following a concomitant increase of α cells and a decrease in ß cells. Finally, we observed that both endothelial and ß cells were found adjacent to α cells significantly more frequently than to each other. In conclusion, this study demonstrates that the self-assembly of a pseudoislet is an intrinsically cell-regulated process. The endothelial cells had preferential proximity to the α cells, and this persisted even when challenged with changing the cell ratios and numbers. This study gives insight into the rules governing the self-organization of pseudoislets and suggests an important role for α cells to promote the incorporation of endothelial cells.