Nature inspired methods for long term banking of endocrine cells within encapsulation devices

Project Summary The objective of this study is to investigate and develop long term storage of cells using cryopreservation in the context of improving health of islets for islet transplantation as a Type Diabetes treatment The goal is to develop a high subzero cryopreservation method that cools cells inside protective immunoisolation devices to extend preservation time to weeks and months Our approach is based on the best strategies employed by freeze tolerant and hibernating animals in nature augmented with complementary strategies developed using recent scientific understanding and bioengineering principles Importantly our approach does not seek to solve all the problems needed for vitrification or classical cryopreservation but rather be the first to develop tissue preservation in a controlled partially frozen equilibrium state using high subzero temperatures ranging from to C combined with metabolic depression These are temperatures and strategies used in nature by species able to survive months in a state of suspended animation with the whole animal including every single organ being banked without injury Specific Aim SA is to select and optimize the cryostasis cocktail with components that are essential for cell survival post freezing Non metabolizable O methyl d glucose OMG and trehalose mix will be used to prevent cellular dehydration and intracellular ice formation during cryopreservation Unisol will be used as the base solution for testing the interaction of multiple doses of OMG trehalose with storage time as a function of temperature using TC cells Specific Aim SA is to achieve active suppression of metabolic rate and enhancement of stress tolerance TC cells will be stored in the cryostasis cocktail developed in SA as well with multiple dose testing of antioxidants AO anti apoptotic AA and metabolic suppressors MS Human islets will be cryopreserved and stored in the standard culture in DMSO and in the optimal dosage additives temperature and storage duration established through SA and SA Quality assessments of these islets include oxygen consumption rate OCR normalized to DNA percent recovery as measured by DNA to assess viability and glucose stimulated insulin secretion to measure function Specific Aim SA is to test encapsulated islets in the TheraCyte device using the most promising cryostasis cocktail established from SA and SA Islets will be cryopreserved and stored using the standard culture methods and the best conditions from SA and SA Once thawed islet quality assessments from SA will be performed immediately post thaw and after the hour culture recovery period Specific aim SA is to test cryopreserved encapsulated human islets in a diabetic nude mouse model Human islets within Theracyte immunoisolation devices that have been cryopreserved and stored in standard culture and the new cryostasis cocktail will be transplanted into diabetic nude mice Mice will be monitored for diabetes reversal by taking daily blood glucose and weekly intraperitoneal glucose tolerance tests over days Explanted devices will be assessed by device OCR DNA GSIS and histology to analyze islet health within the device Narrative Type Diabetes T D is a lifelong devastating metabolic disorder Current methods of treatment are supplemental insulin whole pancreas transplantation and islet transplantation These options are not ideal due to non physiological glucose control while using exogenous insulin the shortage of available donor organs and need for lifelong immunosuppression Cell encapsulation devices have the potential to address these limitations by enabling the transplantation of cells from sources of unlimited supply such as human stem cell derived islets without the need for immunosuppression Developing methods that not only preserve cells but allow for long term storage within devices is critically important for successful simplified and cost effective large scale implementation of such encapsulated cell therapies for the treatment of patients with diabetes