Development of Novel Storage Method for Highly Viable and Well-Characterized Vitrified Human Islets

Project Summary Abstract 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 typediabetes and the amelioration of typediabetes and to understand storage injury near the glass transition temperatureTGto enable a new type of long term storage to be developedBanking should reduce or prevent the deterioration of islets prior to usefacilitate tolerance inductionenable delayed islet transplantation following transplantation of a kidney from the same donor weeks earlierand have many other benefitsWe have already demonstratedin unpublished resultsthat it is possible to vitrify and rewarm human pancreatic islets with excellent islet number recovery and excellent retention of viabilitybased on vital stainingglucose stimulated insulin releasestimulation indexglucose stimulated oxygen consumptionand ability to reverse induced diabetes in mice with minimal islet dosesand we are working on scaling up fromIEQs per vitrified batch to full curative doses of isletsThe next step is to establish that islets remain highly viable and functional following at leastweeks of storage and to determine the best storage conditions for isletsThe best storage conditions are postulated to be quite unconventional due to the large volumes associated with vitrifying all islets from a single donorthe fact that the islets will be vitrifiedand the desirability of rapid transfer into and out of long term storage without any risk of fracturingIn particularwe believe that islets should be stored near TGand possibly even above TGbut storage in this temperature range has not been previously studied in any adequate wayAccordinglyAimis devoted to determining the effect of temperature on islet viability and functionalityAimis 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 integrityand Aimis intended to determine the effect of storage time beyondmonthOne month of storage should be sufficient for most islet banking needsbut two months would be very valuable for providing a safety margin and more flexibility to the clinician and the patientAt the same timecomparingandmonths of storage will open up information about rates of nucleation over time near TG that is presently entirely lackingand will begin to answer questions about biological stability near TG that presently remain entirely obscure Project Narrative The ultimate objectives of this application are to enable improved treatment of typediabetesT Dby 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 temperatureTGIslet banking would allow islet transplants following transplantation of kidneys from the same donor at leastweeks earlierreducing ultimate islet rejectionIt 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 wastedamong other important benefitsTo optimize islet bankingwe propose to store islets near and even above TG based on a number of theoretical advantages of this temperature regimeincluding the prevention of fracturing and the ability to retrieve the islets from storage rapidly and without hazardsWe will determine the effects ofandmonths 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 themselveswhether nucleation in islets differs from nucleation in solutionsand whether nucleation continues for more than a month at any temperatureNucleation densities will be followed by changes in devitrification tendencyas measured by differential scanning calorimetry using previously established protocols and by photographic evidence of devitrification upon warmingWe will also investigate fracturing tendency and document any fracturing photographicallyThe biological effects of all storage conditionswhich includes vitrification followed by immediate rewarmingstorage time of zero monthswill be evaluated by rigorous methods in standard use at the City of HopeOur laboratory has previously demonstrated excellent number recoveryviabilityglucose stimulated insulin releaseglucose stimulated oxygen consumption responsesand ability to cure induced T D in mice with the subcapsular transplantation of minimal islet dosesand we are currently scaling up this process to the vitrification of full clinical islet dosesThe 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 timepotentially 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 TGThese questions are particularly relevant to all large volume biological vitrification systemsand especially to whole organswhich represent another area of currently intense scientific scrutiny