The ultimate objective of the proposed research is to enable the long-term banking of curative doses of human pancreatic islets for the reversal of type 1 diabetes and the amelioration of type 2 diabetes and to understand storage injury near the glass transition temperature (TG) to enable a new type of long term storage to be developed. Banking should reduce or prevent the deterioration of islets prior to use, facilitate tolerance induction, enable delayed islet transplantation following transplantation of a kidney from the same donor weeks earlier, and have many other benefits. We have already demonstrated, in unpublished results, that it is possible to vitrify and rewarm human pancreatic islets with excellent islet number recovery and excellent retention of viability (based on vital staining), glucose-stimulated insulin release (stimulation index), glucose-stimulated oxygen consumption, and ability to reverse induced diabetes in mice with minimal islet doses, and we are working on scaling up from 13,000 IEQs per vitrified batch to full curative doses of islets (450,000-720,000). The next step is to establish that islets remain highly viable and functional following at least 3-4 weeks of storage and to determine the best storage conditions for islets. The best storage conditions are postulated to be quite unconventional due to the large volumes associated with vitrifying all islets from a single donor, the fact that the islets will be vitrified, and the desirability of rapid transfer into and out of long term storage without any risk of fracturing. In particular, we believe that islets should be stored near TG, and possibly even above TG, but storage in this temperature range has not been previously studied in any adequate way. Accordingly, Aim 1 is devoted to determining the effect of temperature on islet viability and functionality, Aim 2 is directed toward determining the effect of temperature on ice nucleation in both the islets and their vitrified medium and the relationship between ice nucleation and islet integrity, and Aim 3 is intended to determine the effect of storage time beyond 1 month. One month of storage should be sufficient for most islet banking needs, but two months would be very valuable for providing a safety margin and more flexibility to the clinician and the patient. At the same time, comparing 1 and 2 months of storage will open up information about rates of nucleation over time near TG that is presently entirely lacking, and will begin to answer questions about biological stability near TG that presently remain entirely obscure.
Public Health Relevance Statement: Project Narrative The ultimate objectives of this application are to enable improved treatment of type 1 diabetes (T1D) by establishing islet banking capability and to advance the science of cryobiology and cryopreservation in general by mapping the effects of time and temperature on islet integrity and on ice nucleation near the glass transition temperature (TG). Islet banking would allow islet transplants following transplantation of kidneys from the same donor at least 3-4 weeks earlier, reducing ultimate islet rejection. It would also provide more time for tolerance induction during islet storage and encourage collection of many more pancreases knowing that they can be used and not wasted, among other important benefits. To optimize islet banking, we propose to store islets near and even above TG based on a number of theoretical advantages of this temperature regime, including the prevention of fracturing and the ability to retrieve the islets from storage rapidly and without hazards. We will determine the effects of 1 and 2 months of storage at temperatures well above TG to temperatures far enough below TG to fully suppress ice nucleation and determine if the kinetics of biological deterioration are related to ice nucleation in either the vitrification solution or in the islets themselves, whether nucleation in islets differs from nucleation in solutions, and whether nucleation continues for more than a month at any temperature. Nucleation densities will be followed by changes in devitrification tendency, as measured by differential scanning calorimetry using previously established protocols and by photographic evidence of devitrification upon warming. We will also investigate fracturing tendency and document any fracturing photographically. The biological effects of all storage conditions, which includes vitrification followed by immediate rewarming (storage time of zero months), will be evaluated by rigorous methods in standard use at the City of Hope. Our laboratory has previously demonstrated excellent number recovery, viability, glucose-stimulated insulin release, glucose-stimulated oxygen consumption responses, and ability to cure induced T1D in mice with the subcapsular transplantation of minimal islet doses, and we are currently scaling up this process to the vitrification of full clinical islet doses. The final step is to establish ideal storage conditions for islets and to advance the science of cryobiology by evaluating circum-TG storage for the first time, potentially opening up a new form of biological storage for future exploitation for many other biological systems while answering a number of currently obscure but fundamental questions about biological rates of change near TG. These questions are particularly relevant to all large-volume biological vitrification systems, and especially to whole organs, which represent another area of currently intense scientific scrutiny.
Project Terms: Area; base; Biological; biological systems; Cities; Clinical; Collection; cryobiology; Cryopreservation; density; Deterioration; Development; Diabetes Mellitus; Differential Scanning Calorimetry; Dose; flexibility; Fracture; fracture risk; Future; Glass; Glucose; hazard; Human; Ice; improved; indexing; Injury; Insulin; Insulin-Dependent Diabetes Mellitus; islet; Islets of Langerhans; Islets of Langerhans Transplantation; Kidney; Kidney Transplantation; Kinetics; Laboratories; Measures; Methods; Mus; Non-Insulin-Dependent Diabetes Mellitus; novel; Organ; Oxygen Consumption; Pancreas; Patients; prevent; Prevention; Process; Protocols documentation; rate of change; Recovery; Research; response; Rewarming; Safety; scale up; Science; Stains; System; Temperature; Time; Transition Temperature; Transplantation
