Optimisation of microscopic techniques to assess isolated islet characteristics

Abstract

Islets of Langerhans or pancreatic islets constitute ~2% of the mass of the human pancreas and present on isolation as spheroids of 100-200 µm diameter. The 3D cellular organisation of islets is specific to each species, and important for islet viability and functionality. Isolated donor islets are used in transplantation for ameliorating Type I diabetes in humans, however current techniques to assess islet viability are highly specialised and not easily accessible in a non-clinical set up. The research in this thesis aimed to create and optimise methodologies for multiple microscopic techniques and analysis for isolated pancreatic islets. I found that live imaging of 3D intact pancreatic islets has multiple challenges, one of the most important being techniques to efficiently immobilise these organoid structures while retaining high-quality imaging and flexibility in the experimental set-up. I developed a tailor-made hydrogel for pancreatic islets and validated its use in live intact islets. The hydrogel was combined with experimental and commercially available chemical dyes and enabled optimisation of the analysis. Limitations in the labelling and imaging are discussed. Alternative dyes were tested to label different structures as steps towards automated viability assessment of isolated islets. New applications for an experimental dye to label alpha and beta cells were tested in human islets. In pursuit of a better understanding of the insulin metabolic pathways for its synthesis, maturation and release, a fluorescence timer tag was designed and validated for its use in beta cell lines and pancreatic islets. This validation was a multiple optimisation processes consisting of immunostaining and histology, imaging analysis and characterisation in live beta cells. The thesis offers insight into the complexities, opportunities and limitations offered by microscopic techniques in islet assessment with the aim of enabling assessment of islet health before transplantation and for research purposes