Pancreatic islet transplantation is a curative treatment for diabetes but access to this therapy is limited by the availability of islets. Long-term cryopreservation of islets could partially address this limitation but current cryopreservation protocols, which use the toxic cryoprotectant dimethyl sulphoxide (DMSO), achieve suboptimal cryosurvival and are not clinically used. The aim of this project was to develop an improved and clinically-relevant method for cryopreservation of islets. The project examined the efficacy of the non-toxic cryoprotectant trehalose in combination with (1) membrane-permeabilising biopolymers PP-50 and PP-75 to increase intracellular uptake, and (2) antioxidants MitoQ and salidroside to reduce cryopreservation-induced oxidative stress.

Due to the complex architecture of pancreatic islets, human mononuclear cells were first used to optimise the use of permeabilising polymers. Current methods of viability assessment were critically examined and improved, followed by systematic screening of cryopreservation conditions. The efficacy of promising cryopreservation protocols were subsequently confirmed for mouse pancreatic islets using viability and functional assays.

PP-50 and PP-75 polymers were shown to be unsuitable for trehalose-based cryopreser- vation of mononuclear cells and islets due to potential toxicity and need for long incubation. Investigation of diffusion kinetics demonstrated that a minimal incubation time of 6 h at 37 °C is required to enable diffusion of solutes into the islet core. This finding implies that current clinical viability assessment protocols only survey the periphery of islets. A range of imaging modalities were used to confirm that islet core viability can be dramatically different from the islet periphery. Using these insights, a novel cryopreservation protocol was developed in which islets are pre-incubated for 6 h at 37 °C with trehalose and antioxidants, followed by addition of DMSO, resulting in significantly greater viability of cryopreserved islets.

In conclusion, this study identified important limitations of the current cryobiological viability assessment of mononuclear cells and pancreatic islets. Permeabilising biopolymers did not improve trehalose-based cryopreservation in primary cells. An improved method for cryopreservation of pancreatic islets was developed which combines DMSO and trehalose in a novel manner and can be further improved by addition of antioxidants.