REAGGREGATION OF PANCREATIC ISLET CELLS: IMPLICATIONS FOR ISLET HEALTH, STORAGE AND NEW DRUG DISCOVERY

Abstract

Islets are clusters of cells in the pancreas that monitor and regulate blood glucose levels. In culture, single islet cells reaggregate into clusters, but the reaggregation process is random, resulting in large variation in the cluster numbers, sizes and cell composition. Our laboratory created a micromold, a specialized cell culture plate, to efficiently bioengineer small, uniformly-sized islet clusters that we called Kanslets. Currently, very little is known about the implications of islet cell clusters for the treatment of diabetes. The purpose of this dissertation was to optimize the experimental conditions necessary for islet storage (cryopreservation), drug discovery screening procedures, and islet health in culture utilizing Kanslets. The conventional methods of cryopreservation of islets use intact native islets, and the viability and function achieved after thawing with these methods is poor. In this dissertation work, we cryopreserved islets as single cells and, after thawing, reaggregated islet cells to form cryopreserved Kanslets using the micromold technology. We observed that the viability of cryopreserved Kanslets was superior to cryopreserved intact islets and cryopreserved Kanslets had the potential to lower blood glucose levels of diabetic rats for 10 months. Next, the effect of creating hybrid islets/spheroids using mixtures of cells was tested by combining cells from different human donors, or by combining mesenchymal stem cells (MSCs) and islet cells. In order to minimize the need for assay repetition during the drug screening procedures, multi-donor Kanslets, consisting of islet cells from different donors, were created. We observed that the multi-donor Kanslets were highly viable, but showed little response to drugs such as glibenclamide (insulin stimulator) and somatostatin (insulin inhibitor). To improve islet health in culture, islet cells were integrated with bone marrow MSCs and reaggregated into spheroids; these spheroids were highly viable even long-term in culture, but secreted less insulin irrespective of the proportion of MSCs and islet cells in the spheroids, when compared to the spheroids containing only islet cells. Overall, these findings greatly enhance the utilization of islets for islet transplantation, and also provide valuable insight for future work in reaggregating islet cells with MSCs. The work provides a platform for future researchers to further explore the implications of islet cell reaggregation for diabetes research or therapy.