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
Is data on this page outdated, violates copyrights or anything else? Report the problem now and we will take corresponding actions after reviewing your request.