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Aims/hypothesis: There are potential advantages to the low-temperature (-196 °C) banking of isolated islets, including the maintenance of viable islets for future research. We therefore assessed the in vitro and in vivo function of islets cryopreserved for nearly 20 years., Methods: Human islets were cryopreserved from 1991 to 2001 and thawed between 2012 and 2014. These were characterised by immunostaining, patch-clamp electrophysiology, insulin secretion, transcriptome analysis and transplantation into a streptozotocin (STZ)-induced mouse model of diabetes., Results: The cryopreservation time was 17.6 ± 0.4 years (n = 43). The thawed islets stained positive with dithizone, contained insulin-positive and glucagon-positive cells, and displayed levels of apoptosis and transcriptome profiles similar to those of freshly isolated islets, although their insulin content was lower. The cryopreserved beta cells possessed ion channels and exocytotic responses identical to those of freshly isolated beta cells. Cells from a subset of five donors demonstrated similar perifusion insulin secretion profiles pre- and post-cryopreservation. The transplantation of cryopreserved islets into the diabetic mice improved their glucose tolerance but did not completely normalise their blood glucose levels. Circulating human insulin and insulin-positive grafts were detectable at 10 weeks post-transplantation., Conclusions/interpretation: We have demonstrated the potential for long-term banking of human islets for research, which could enable the use of tissue from a large number of donors with future technologies to gain new insight into diabetes.

Background: Success of human pancreatic islet isolation depends largely on the techniques used during pancreas procurement and the quality of the gland. Warm ischemia of the pancreas of any duration during organ procurement may be detrimental to subsequent islet yield and functional viability of the islets. The aim of this study was to correlate pancreas procurement technique with the core temperature within the pancreas during in situ procurement to the recovery and in vitro function of isolated human islets., Methods: Alternative pancreata were recovered from human cadaveric organ donors with needle-point myocardial temperature probes placed within the body of the pancreas. After vascular flushing with University of Wisconsin organ preservation solution, the first group of human pancreata were removed after standard liver and kidney procurement followed by in situ dissection of the pancreas. The second group of pancreata were removed using a similar protocol, except that the lesser sac was opened (and the spleen was mobilized to the midline) rapidly at the time of aortic cross-clamp. An additional 3-4 L of ice slush solution was placed behind and in front of the pancreas and replenished throughout the procurement process for the liver and kidneys. Immediately after recovery, in both groups, the pancreas was placed in cold University of Wisconsin solution and transported to the islet isolation laboratory for processing. Islets were isolated using a standard protocol of Liberase perfusion via the duct, gentle mechanical dissociation, and Ficoll purification., Results: The absence of in situ ice-chilling of the entire pancreas during liver and kidney procurement caused the core pancreas temperature to rise to a peak of 18.2 degrees C; but when the pancreas was surrounded and replenished with iced saline slush, the core pancreas temperature was maintained at approximately 4 degrees C. The lower pancreas temperature correlated with a significantly higher recovery of islets in the group of pancreata surrounded with iced saline (223+/-35x10(3) IE vs. 103+/-26x10(3) IE; mean +/- SEM). Functional ability of the islets was also significantly improved in glucose static incubation, with a stimulation index of 4.4+/-0.3 in the iced-saline pancreas group versus 3.0+/-0.4 in the standard procurement group (P<0.05, paired t test)., Conclusions: Procurement technique and the maintenance of a low temperature of the pancreas are critical to subsequent islet recovery and function. Maintaining a low pancreas temperature during procurement through the addition and replenishment of iced saline slush surrounding the anterior and posterior aspects of the pancreas greatly improves islet yield and functional viability of the isolated islets and is essential for success in clinical islet transplantation.

Effective intraductal delivery of the enzyme collagenase into the pancreas is crucial to the subsequent ability to isolate viable islets. Most clinical islet transplant centers load the enzyme into the pancreas by retrograde injection using a syringe following cannulation of the pancreatic duct. An alternative approach is to perfuse the pancreas via the pancreatic duct with collagenase solution using a recirculating perfusion device system. This provides control over perfusion pressures and collagenase temperature. This study reports on our evaluation of the delivery of Liberase-HI into the pancreas of 14 consecutive adult multiorgan cadaveric donors. Alternate glands were procured and processed using an identical protocol with the exception of collagenase delivery. The first group of pancreases was loaded using the perfusion technique where cold (4 degrees C) Liberase-HI was perfused at 80 mmHg for 5 min after which the pressure was increased to 180 mmHg. The collagenase solution was then slowly warmed to 35 degrees C, transferred to the dissociation chamber and mechanically dissociated, and then purified using discontinuous gradients of Ficoll. Pancreases in the second group were loaded with collagenase (28-32 degrees C) using the syringe technique before mechanical dissociation and purification. There were no significant differences in pancreas cold ischemia, donor age, body mass index, maximum blood glucose, or serum amylase of the donors between the two groups. Mean collagenase digestion time in the digestion chamber was not different between the two groups; however, the amount of undigested tissue remaining after dissociation was significantly higher in the syringe-loaded group (15.3 +/- 2.6 g vs. 4.6 +/- 2.1 g, mean +/- SEM, p < 0.05). Postdigestion recovery of islets was 471 +/- 83 x 10(3) IE in the perfusion group compared with 391 +/- 57 x 10(3) IE for the syringe-loaded group. Postpurification recovery was higher in the perfused group (379 +/- 45 vs. 251 +/- 28 x 10(3) IE, p < 0.05, two-tailed paired t-test). No difference in in vitro islet viability was observed between the two groups following glucose perifusion with the calculated stimulation index of 4.6 +/- 0.6 for the perfusion group and 4.2 +/- 0.7 for the syringe-loaded group. Controlled perfusion via the pancreatic duct allows the effective delivery of the enzyme achieving maximal distension to all regions of the pancreas leading to an increased recovery of the islets with no detrimental effect on subsequent in vitro islet function.

We compared the efficacy of fresh and frozen/thawed islets by determining the minimum number required to consistently reverse diabetes in mice. Defined numbers of islets, isolated from Balb/c (H-2d) and CBA/J (H-2k) mice, were transplanted into streptozotocin-induced diabetic Balb/c mice. Frozen/thawed grafts were cooled slowly to -40 degrees C, stored at -196 degrees C, and thawed rapidly. At 100 days after transplantation, isografts were recovered for measurement of insulin content. Mean (+/-SD) recovery of cryopreserved islets after thawing was 80 +/- 3% (range 67-89%). For both fresh and frozen/thawed isografts and allografts, 200 islets were required to establish normoglycemia. The degree of metabolic function provided by equivalent quantities of fresh and frozen/thawed grafts was similar; and all normoglycemic isograft recipients remained so until graft nephrectomy. The insulin content of fresh and frozen/thawed isografts containing 200 and 300 islets were 151 +/- 25 and 126 +/- 8 mU and 259 +/- 36 and 278 +/- 20 mU, respectively. Among allograft recipients, median survival ranged from 15 to 20 days, and was not influenced by cryopreservation or graft size. The results of this study demonstrate a high rate of recovery of viable islets following cryopreservation. The function of equivalent quantities of fresh and cryopreserved islet isografts and allografts in nonimmunosuppressed recipients is similar.

Intraductal distention of the pancreas with collagenase followed by stationary warm incubation improves the recovery of islets of Langerhans in the rat, but controlled studies are needed for valid comparison with standard isolation methods. We have modified Gotoh's technique of stationary digestion for high-yield isolation in the rat (Stationary). The method is subjected herein to rigorous blinded comparison with the standard chopped tissue (Chopped) technique, based on Lacy et al., as performed in our laboratory for over 10 yr. Islet recovery was determined by a single observe 'blinded' to the method of isolation used, and only intact islets of diameter > or = 100 microns were included. Stationary gave 719 +/- 114 islets per pancreas (mean +/- SD, n = 21 isolations) vs. 487.5 +/- 69 for Chopped (n = 36 isolations), a 47.5% increment in yield (p < 0.0001). In vitro islet perifusion showed no statistical difference in stimulation index (SI) or stimulated area under the curve (SAUC) between the two methods, but Stationary showed a trend towards improved phase II insulin release. In vivo function was assessed by isogeneic transplantation of 2,000 islets beneath the renal capsule of streptozotocin diabetic recipients (65 mg/kg Sigma); Stationary recipients (n = 7) became normoglycemic (< or = 8 mmol/L) by 3.3 +/- 4.8 days vs. 1.6 +/- 1.5 days for Chopped recipients (p = 0.4 ns, mean +/- SEM). IVGTT performed at 1 mo posttransplant gave K-values for Stationary of 2.64 +/- 0.8 vs. 2.62 +/- 0.8 for Chopped (mean +/- SD, p = 0.9 ns, n = 6, unpaired t-test), which were not distinguishable from normal control rats (2.59 +/- 0.8) (p = 0.9 ns, n = 10). Graft function remained stable until graft bearing nephrectomy induced hyperglycemia uniformly within 1 day. Graft histology showed a healthy well-preserved structure on light microscopy, with well-granulated beta cells on EM. Economic costs of rat, collagenase, and Ficoll were 26% ($50.82) lower per recipient for Stationary. We conclude that modified stationary digestion significantly improves islet recovery with excellent in vitro and in vivo function, and is cost effective.

Previous studies in rodent and canine animal models have suggested a detrimental impact on islet recovery and function when pancreas excision is preceded by in situ vascular flushing with cold preservation solutions. We studied the efficacy of islet isolation from 19 consecutive cadaver pancreases procured alternately by initial pancreatectomy before in situ flush (group 1, our standard procurement technique, n = 9) or pancreas removal following in situ vascular flushing with cold University of Wisconsin solution (group 2, n = 10). Once procured, pancreases were weighed, the main pancreatic duct was cannulated, and 150 ml of collagenase solution was injected. The pancreases were transported to the isolation laboratory and processed within 2 hr. Islets were counted and sized after dithizone staining, and the islet equivalents were calculated. Aliquots of isolated islets were cryopreserved using established techniques. Islet function of both freshly isolated and frozen-thawed islets was assessed using a glucose-stimulated perifusion system. Significantly more pancreas was harvested after University of Wisconsin flush (90.6 +/- 6.9 g for group 1 versus 66.7 +/- 4 for group 2, P < 0.05). The quantity of islets per gram of processed pancreas released during enzymatic digestion from each of the experimental groups did not differ significantly (4.5 +/- 0.6 x 10(3) islet equivalents per gram for primary pancreatectomy versus 4.0 +/- 0.4 x 10(3) University of Wisconsin flush). Similarly, following Ficoll purification, the overall yields of islets did not differ significantly. Total islet yield in the primary pancreatectomy group was 181 +/- 25 x 10(3) islet equivalents (2.7 +/- 0.3 x 10(3) IE/g) versus 217 +/- 41 x 10(3) for the University of Wisconsin flush group (2.9 +/- 0.8 x 10(3) islet equivalents/g; P not significant). No differences were observed in in vitro viability. Perifusion stimulation indexes (peak/basal insulin release) were 5.9 +/- 1.3 for group 1 and 7.1 +/- 1.5 for group 2. These results conflict with published results in animal models and indicate that large numbers of viable islets can be recovered from cadaver pancreas utilizing either procurement technique. The decreased operating time, simplicity, and safety favor the use of total pancreatectomy after limited in situ vascular flushing as the method of choice for pancreas procurement for subsequent islet isolation.

Immunomodulation of rodent islets can significantly prolong allograft survival. We utilized a murine model of primary islet transplantation to study the relationship between allograft survival and the quantitative pretransplant expression of class I and II MHC antigens in freshly isolated CBA/J islets, and islets subjected to 37 degrees C tissue culture, brief culture at 7 degrees C, or exposure of the donor to lipopolysaccharide. Seven-day culture resulted in decreased class II expression, a tendency to decreased class I expression, and a significant prolongation of allograft survival. Brief culture at 7 degrees C resulted in increased class I expression, a trend to decreased class II expression, and no significant change in allograft survival. Donor pretreatment with LPS resulted in increased class I expression without significant change in class II expression and was correlated with prolongation of allograft survival. These studies demonstrate that an upregulation of MHC class I by in vitro or in vivo islet pretreatment is not associated with an acceleration of islet rejection. Reduction of class II was associated with delayed rejection. These results do not support a major role of the indirect pathway of antigen presentation in islet rejection in vivo. Certain protocols that alter the usual expression of class I and II on pancreatic islets are associated with alteration in the initiation and/or propagation of the normal cell-mediated rejection process, suggesting that the concept of pretransplant treatment should continue to be pursued.

The authors used the principal features of a collagenase perfusion technique and an automated dissociation technique to determine if islets could be isolated from the large mammal pancreas and to compare the effects of the two methods on isolated islets. The pancreases of 16 dogs were cannulated and perfused with collagenase at 4 degrees C, then warmed to 37 degrees C. Group 1 (eight) pancreases were perfused at 37 degrees C until digested, then dissociated manually by teasing and trituration. Group 2 (eight) pancreases were transferred to a closed chamber for continued collagenase digestion and dissociation at 37 degrees C. Islets were purified using identical Ficoll gradients. Aliquots were stained with dithizone and evaluated for number, size and purity. Total islet volume was calculated. Group 2 pancreases were thoroughly digested leaving only a few residual ducts, but undigested fragments persisted in group 1 pancreases. Islet size was similar in both groups. There was a greater islet volume before and after Ficoll purification in group 2, but the difference was not significant. Purity was greater than 90% in both groups. Perifusion with 28 mM glucose elicited a biphasic insulin release from islets in both groups. The data show that the combined protocol enables mass isolation of purified islets from the canine pancreas. Compared with the manual technique, the automated protocol for pancreas dissociation tends to improve the yield of islets without compromising islet size and viability. It provides the advantages of a closed system with increased control over the extent of collagenase digestion.

Human islets of Langerhans were isolated with the principles of collagenase perfusion via the pancreatic duct and gentle dissociation of tissue. The number of islets released was 161 x 10(3), distributed as 76 x 10(3) large (greater than 100-micron) and 85 x 10(3) small (less than 100-micron) islets. Recovery after Ficoll-gradient purification was 61% for the large islets and 42% for the small islets. The final islet volume was 240 microliter, with purity of 70-90% (large islets) and 20-40% (small islets). Perifusion with glucose elicited a biphasic release of insulin, with the response rising sixfold from basal secretion. Implantation of pure islets under the kidney capsule of normal or streptozocin-induced diabetic nude mice resulted in human C-peptide secretion and partial or complete reversal of hyperglycemia, confirmed by histological recovery. The data show that these methods provide large quantities of viable purified human islets.

Reliable high-recovery human islet storage would facilitate tissue matching, organ sharing, and immune manipulation of donor islets and prospective diabetic recipients. Collagenase-isolated, Ficoll-purified pancreatic islets (median 21,000, 15% of total islet yield) from eight cadaver pancreases were cultured in vitro for 24 h, equilibrated in three steps with dimethyl sulfoxide (DMSO) to a 2-M concentration, supercooled, nucleated, and cooled at 0.25 degree C/min to -40 degrees C before storage at -196 degrees C for 44.25 +/- 8.75 days. Rewarming at 200 degrees C/min and removal of DMSO with 0.75 M sucrose preceded 48 h of culture and retesting. Recovery postthaw by microscope count on duplicate aliquots was 94.2 +/- 3.5% of prefreeze counts and by triplicate assay of extractable insulin was 90.0 +/- 22.3% on day 0 and 74.1 +/- 12.6% after a 48-h culture. Nonfrozen islets increased basal insulin secretion 7.7 +/- 2.8 times after stimulation with 300 mg/dl glucose in perifusion, whereas islets frozen-thawed and cultured 48 h increased 6.2 +/- 0.8 times (NS). Peak stimulated insulin release was 0.92 +/- 0.14 microU.islet-1.min-1 before storage and 0.73 +/- 0.14 microU.islet-1.min-1 (79% of control, NS) after freeze-thaw and a 48-h culture. Total insulin secretion (area under curve) was 66% of prefreeze values at 48 h. Immunocytochemical stains revealed preservation of islet morphology postthaw. Electron microscopy showed intact cellular and nuclear membranes and intracellular organelles. Frozen-thawed islets harvested 14 days after renal subcapsular xenografting in nude mice were revascularized and well granulated. Cryopreservation can achieve prolonged storage of large numbers of human islets with high recovery numerically and functionally, making this a feasible approach for future trials of human islet transplantation.

Long-term islet storage would facilitate many aspects of islet research and clinical islet transplantation. Collagenase-isolated, Ficoll-purified islets from eight cadaveric pancreases were stored in liquid nitrogen for 44 +/- 9 days after dimethyl sulfoxide equilibration and slow cooling. Rapid rewarming and 48 h of culture preceded repeat evaluation of recovery by islet counts, insulin extraction, and glucose-stimulated perifusion. Islet recovery was 94 +/- 4% by count and 90 +/- 22% by insulin extraction immediately after thawing. After an additional 48 h in culture, recovery was 74 +/- 12% by insulin extraction and 79% by quantitative perifusion culture. Perifusion demonstrated normal baseline and first-phase insulin secretion with decreased second-phase insulin secretion after cryopreservation. Insulin-stained sections and electron microscopy revealed preserved islet morphology and ultrastructure. Granulated islets with preserved morphology were recovered 14 days after renal subcapsular xenografting into nude mice. This study demonstrates high recovery and good functional activity of human islets after prolonged cryopreservation.