Two-layer method in short-term pancreas preservation for successful islet isolation

BACKGROUND: We sought to determine whether the two-layer method (TLM) offers advantages over UW storage solution for locally procured pancreata with cold ischemia time of <8 hours for successful islet isolation. METHODS: From October 2003 through February 2005, 22 human pancreata were procured locally from cadaveric donors and preserved using UW solution (n = 11) or TLM (n = 11). RESULTS: Donor characteristics were similar in the two groups, with no statistical difference. Cold ischemia time was 4.5 +/- 0.6 (2.5 to 8) hours in the UW and 5.1 +/- 0.5 (3 to 8) hours in TLM group (P > .05). Organs preserved with TLM were exposed to PFC for 4 +/- 0.5 (2 to 7.5) hours. After TLM preservation, 8 of 11 (72%) pancreata yielded >300,000 IEQ pancreatic islets, which met all criteria for clinical transplantation; after UW cold storage, only 3 of 11 isolations were equally successful (27%) (P < .05). Mean IEQ was higher in the TLM than in the UW group: 349,000 +/- 37,000 vs 277,800 +/- 34,000; IEQ/g was also higher at 5100 +/- 760 vs 3000 +/- 570, respectively (P < .05). Islet quality, characterized by purity, viability, and insulin SI, did not differ statistically in the two groups: 67 +/- 4 vs 74 +/- 4%, 87 +/- 2 vs 83 +/- 4%, and 4 +/- 0.7 vs 4.8 +/- 1, respectively (P > .05). CONCLUSIONS: The Two Layer Method for locally procured human pancreata with cold ischemia time lower than 8 hours offers significant advantage over UW cold storage increasing the pancreatic islet isolation yield and the isolation success rate.     

Influence of preservation solution on human islet isolation outcome

BACKGROUND: The influence of the preservation solution used for in situ perfusion of the donor and pancreas storage on islet isolation has received little attention. METHODS: In this prospective controlled trial, we compared the outcome of human islet isolation from pancreata perfused with University of Wisconsin (UW) solution or Celsior, an alternative colloid-free extracellular solution. RESULTS: At the 1-year interim analysis, the viability and insulin secretion of islets isolated from donors perfused with UW (n=19) or Celsior (n=5) were identical. However, total islet recovery (IEQ) and isolation yield (IEQ/g) were 1.8-fold and 2.1-fold inferior in the Celsior group (P<0.05 vs. UW). Overall, 13 (68%) of islet preparations were effectively transplanted from the UW group vs. none from the Celsior group (P=0.01). The clinical study was discontinued and the causes of these differences were further explored in the pig (n=14). In contrast to UW, Celsior induced cell swelling and pancreas edema after only four hours of cold storage. These abnormalities were delayed when the donor was perfused with Solution de Conservation d'Organes et de Tissus (SCOT), an extracellular solution containing polyethylene glycol. CONCLUSIONS: Our results suggest that colloid-free preservation solutions might be suboptimal for pancreas perfusion and cold storage prior to islet isolation and transplantation. Because pancreata are now frequently recovered for islet transplantation, preliminary experimental and clinical data about islet isolation should be obtained prior to the routine implementation of new preservation solutions for abdominal perfusion during multiorgan recovery.     

Adaption of neutral protease activity for islet isolation from the long-term two-layer method-stored pig pancreas

BACKGROUND: Islet release from the pancreas is mediated by both collagenase and neutral protease (NP), a critical effector of islet integrity. To prove the hypothesis that adjustment of NP reduces islet damage after prolonged ischemia, adult pig pancreata were digested after 7-hour preservation by the two-layer method (TLM) using a 2-component enzyme blend consisting of collagenase NB-8 and NP. METHODS: After intraductal University of Wisconsin (UW) flush resected pancreata were distended with 4.4 PZ-U/g of UW-dissolved Serva collagenase either before (TLM-preloaded, n = 7) or after (TLM-postloaded, n = 10) cold storage, or for immediate processing (n = 6). NP was adjusted after preliminary experiments to respectively 1.1, 0.2, or 0.8 DMC-U/g for unstored, TLM-preloaded, or postloaded organs. RESULTS: Purified islet yield decreased from 3670 +/- 730 islet equivalents (IEQ)/g in unstored pancreata to 1800 +/- 180 and 2080 +/- 290 IEQ/g in TLM-preloaded or postloaded organs, respectively (P < .05). Although purity was always >90%, IEQ recovery was significantly decreased in TLM-preloaded pancreata. Quality control revealed consistently high viability as determined using trypan-blue exclusion (>95%) or formazan production. Compared with unstored organs (2.47 +/- 0.36; P < .05), glucose stimulation index was reduced in TLM-preloaded (1.48 +/- 0.15) and TLM-postloaded pancreata (1.81 +/- 0.20). Normoglycemia in diabetic nude mice transplanted with islets from TLM-preloaded pancreata was transient in contrast to sustained function in the other groups. CONCLUSIONS: Significant amounts of viable pig islets can be isolated after prolonged TLM preservation by reducing NP activity. Nevertheless, early enzyme administration prior to long-term storage deteriorates islet graft function.     

Islet Isolation From Human Pancreas With Extended Cold Ischemia Time

The general consensus among transplant centers is that a cold ischemia time (CIT) beyond 8 hours results in reduced yields and quality of human islets. We sought to optimize the isolation process and enzymes for pancreata with extended CIT. We processed 16 extended CIT pancreata (13.2 ± 0.7 hours). Donors averaged 50.8 ± 2.6 (standard error of the mean) years old with a body mass index of 28.6 ± 1.5. Glands were shipped in cold organ preservation solution without oxygenated perfluorocarbon. Isolations were performed under a protocol optimized for digestion with the new cGMP collagenase from Roche. Purification used continuous Euroficoll/University of Wisconsin gradients. Islets were cultured in two types of Prodo cGMP islet culture media and/or in Miami 1A media. Glucose-stimulated insulin secretion assays were performed after 8 to 16 days of culture. Prepurification yield averaged 415 ± 41 KIEQ postpurification, 359 ± 29 KIEQ (purification loss 13.5%); and postculture 317 ± 27 KIEQ (culture loss 11.7%). Our process liberated an average of 4278 IEQ/g of pancreas (97 ± 5 g). Most islets were recovered in the purest fraction (purity 79.7% ± 1.9%). Culture loss in our enhanced culture media was 11.7%. After 2 to 3 days in culture, viability was 92% ± 1%. Islets exhibited compactness and dithizone staining. Glucose-stimulated insulin secretion assays performed after 3 to 23 days in our PIM(R) media resulted in a stimulation index of 6.8 ± 1.7 (G50 to G350). We concluded that our human islet isolation process permitted the recovery of large numbers of high-quality human islets from extended CIT pancreata and that our cGMP islet culture media was superior to the current standard CMRL-based media.     

A meta-analysis for comparison of the two-layer and university of Wisconsin pancreas preservation methods in islet transplantation

Conflicting results have been reported on the effectiveness of the two-layer method (TLM) compared with the University of Wisconsin (UW) method for preserving pancreata. The objective of this study was to compile the evidence for or against any difference in human islet yield and viability between these two. PubMed (January 2000 to May 2008) and Cochran Library searches were performed and 17 studies were included for the meta-analysis. Data on donor characteristics, preservation time, and outcomes were abstracted. Studies were subgrouped based on how TLM was used (UW + TLM or TLM alone), on mean cold ischemic time (CIT) (>20 h or <20 h), and on whether special chemical was used (yes or no). Meta-analysis of all studies and subgroups was performed and the pooled standardized mean differences (SMD) with 95% confidence intervals (CI) were reported. Overall, the use of TLM significantly increased islet yield [SMD, 0.74 (0.44-1.04)] and viability [SMD, 0.63 (0.14-1.12)]. The beneficial effects of TLM on islet yield were more evident when TLM was used following UW storage or when prolonged CIT was used. TLM used alone, shorter CIT, and no chemical use all resulted in similar islet viability between TLM and UW groups. Beneficial effects of TLM on islet viability were demonstrated only when TLM was used following UW storage, or with prolonged CIT, or with chemical use. In conclusion, the TLM was beneficial for prolonged pancreas preservation before human islet isolation; however, benefit of the TLM for short-term preservation was not clear.     

Impact of Hypothermic Preservation on Tissue Yield and Viability in Pig Pancreata

Introduction

Chronic shortage of quality human cadaveric pancreata limits islet transplantation. Porcine islet xenotransplantation is being explored to increase the donor pool. For clinical-ready islets, centralized animal husbandry, Current Good Manufacturing Practice–regulated processing facilities, and organ transportation support are required. Amount of cold ischemia time (CIT) before isolation significantly affects transplantation. The goal of this study was to determine the maximum safe CIT of whole pancreata before islet isolation.

Materials and Methods

Pancreata were rapidly removed from Yorkshire pigs (age, 14–22 days) and stored in modified University of Wisconsin solution or in EuroCollins solution at 4^°C. Pancreata were processed with <1 hour CIT (control) or stored for 4 or 12 hours before isolation. Islet yield and percent purity and viability were determined after 7 days of in vitro tissue culture and maturation. Samples from nonprocessed pancreata were collected and snap-frozen in liquid nitrogen at 0, 3, 6, 9, 12, 15, and 24 hours of preservation, then analyzed for adenosine diphosphate/adenosine triphosphate ratio as a measure of tissue energetics.

Results

Up to 12 hours in cold storage had no significant impact on overall islet yield after 7 days of in vitro culture compared with controls; islet yield at the end of the maturation process was 28,700 ± 500 islet equivalents per pancreas (mean ± SEM control yield, 30,300 ± 900 islet equivalents per pancreas); islet purity was 75 ± 5% compared with 74 ± 5% in controls. Islet viability was significantly reduced at 12 hours compared with controls (80 ± 6% vs 96 ± 5%; P < .05). The tissue adenosine diphosphate/adenosine triphosphate ratio was maintained within the first 6 hours (1.6 ± 0.1 to 1.8 ± 0.2; P = NS) but was markedly increased during the 24-hour study (3.3 ± 0.1 at 24 hours), indicating a progressive loss of adenosine triphosphate tissue stores.

Conclusions

Young pig pancreata can be hypothermically stored for up to 12 hours without affecting islet yield and purity; however, islet viability is reduced. These data highlight the need for uniform shipping parameters to standardize islet quality, ideally with CIT <6 hours.     

Twenty-four hour hypothermic machine perfusion preservation of porcine pancreas facilitates processing for islet isolation

Procurement of donor pancreata for islet isolation and transplantation is not yet widely practiced due to concerns about the impact of postmortem ischemia on functional islet yields. Perfusion/preservation technology may help to circumvent ischemic injury as applied in this study of porcine pancreata prior to islet isolation. Pancreata harvested from adult pigs were assigned to 1 of 3 preservation treatment groups: G1, fresh controls, processed immediately with minimum cold ischemia (<1 hour); G2, static cold storage, flushed with cold UW-Viaspan and stored at 2 degrees -4 degrees C for 24 hours; and G3, hypothermic machine perfusion (HMP) on a pulsatile LifePort machine Organ Recovery Systems, Inc., Des Plaines, Ill, United States with KPS1 solution at 4-7 degrees C and low pressure (10 mm Hg) for 24 hours. Islet isolation was then accomplished using conventional methods. Product release criteria were used to assess islet yield and function. Islet yield was markedly different between the treatment groups. There was a statistically significant increased yield in the HMP group over static cold storage in UW-Viaspan (P < .05). Functionally, the islets from each experimental group were equivalent and not significantly different from fresh controls (G1). Dithizone staining of islets showed consistently more uniform digestion of pancreata from G3 compared with G1 and G2, with greater separation of the tissue and fewer entrapped islets. HMP for 24 hours was well tolerated, leading to moderate edema but no loss of function of the harvested islets. The edema appeared to aid in enzymatic digestion, producing a greater yield and purity of islets compared with pancreata subjected to 24 hours of static cold storage.     

Analysis on donor and isolation-related factors of successful isolation of human islet of Langerhans from human cadaveric donors

We analyzed the preexisting donor factors and isolation variables that affected isolation of human islets of Langerhans. Sixty-nine pancreata from cadaveric donors were analyzed for donor factors of age, gender, body mass index, cause of death as well as graft factors of cold ischemia time, pancreas status, distensibility during intraductal collagenase distension and time of collagenase expansion and digestion. Islet isolations that recovered >100,000 IEQ (n = 53) were compared to those generating less than 100,000 IEQ (n = 16) to analyze the factors affecting islet yield during donor harvest and isolation procedures. The mean islet recovery was 216.0 x 10(3) (IEQ) or 2840 (IEQ) per gram of pancreas. Mean purity was 54%. The success rate of islet isolation was 76%. Mean age was 31 years, and mean cold ischemia time was 6.9 hours. In univariate analysis, the status of the pancreas was the only significant factor for successful isolation, and gender, time of collagenase expansion and digestion were marginal factors. In stepwise multivariate logistic regression analysis of donor and isolation-related factors, donor gender, pancreas status and digestion time were significant factors. During the same period we performed three cases of clinical islet allotransplantation and one autotransplantation. This study confirmed that the same donor factors and variables in the isolation process can affect the ability to obtain successful human islet isolation. Enough experience and pertinent review of donor and isolation factors can make islet isolation consistent, supporting clinical islet transplantation without unnecessary cost.     

Procurement of the human pancreas for pancreatic islet transplantation

BACKGROUND: The full potential of pancreatic islet transplantation (PIT) has not been realized because of the difficulties associated with islet isolation, particularly when associated with a remote islet isolation center. Herein, we describe the principles of pancreatic procurement for PIT, which have allowed us to achieve a successful pancreatic islet isolation rate 67% of the time when using a remote islet isolation center. METHODS: Between January 16, 2002 and June 30, 2003, 39 pancreata were procured and processed for PIT at a distant islet isolation center. All pancreata were procured by a single surgeon, and special attention was given to careful dissection of the pancreas, maintenance of arterial inflow and the pressure differential between arterial and venous systems during perfusion, rapid organ cooling, and rapid removal of the pancreas from the body. RESULTS: Twenty-six of 39 (67%) procured pancreata yielded more tha 5,000 islet equivalents (IEQ)/kg recipient weight and were transplanted. Median IEQs per isolation was 413,867, whereas median purity and viability were 65% and 100%, respectively. The median time for pancreatic excision was 34 minutes, whereas cold ischemia time was 6 hours and 40 minutes. DISCUSSION: The principles we have adopted for pancreatic procurement for PIT have resulted in a 67% islet isolation success rate despite the maintenance of more than 5,000 IEQ/kg and the use of a remote islet isolation center.     

Efficacy of the oxygen-charged static two-layer method for short-term pancreas preservation and islet isolation from nonhuman primate and human pancreata

Previous reports indicate that the two-layer method (TLM) of human pancreas preservation is superior to University of Wisconsin solution (UW) when pancreata are preserved for extended periods (i.e., >24 h) prior to islet isolation. In this study, the efficacy of using the TLM for preserving pancreata for short periods (i.e., <13 h) was evaluated using both nonhuman primate and human pancreata preserved with a TLM kit precharged with oxygen. An oxygen precharged TLM (static TLM) was established and compared with the original TLM with continuous oxygen supply. For the static TLM, the perfluorochemical was fully oxygenated and the oxygen supply removed prior to pancreas preservation. In the primate model, pancreata were preserved by the static TLM, the original TLM, and UW for 5 h prior to islet isolation. In the human model, pancreata were preserved with the static TLM or the original TLM or UW for 4-13 h. Both primate and human pancreata were processed by intraductal collagenase injection and digestion followed by continuous density gradient purification to isolate islets. Islets were assessed for islet yield, purity, viability, and in vitro functionality. In the primate model, islet yield, viability, and in vitro functionality were significantly improved by both the static TLM and the original TLM with similar results. Postculture islet yields were 23,877 +/- 3619 IE/g in the static TLM, 21,895 +/- 3742 IE/g in the original TLM, and 6773 +/- 735 IE/g in UW. In the human model, both the static TLM and the original TLM significantly increased islet yield compared with UW with postculture islet yields of 2659 +/- 549 IE/g in the static TLM, 2244 +/- 557 IE/g in the original TLM, and 1293 +/- 451 IE/g in UW. Nonhuman primate and human pancreata stored in the static TLM, immediately upon procurement, yield isolated islets of a substantially higher quantity than when pancreata are stored in UW. Thus, the use of the static TLM should replace the use of UW for storage of pancreata during transport prior to islet isolation.     

Human islet transplantation from pancreases with prolonged cold ischemia using additional preservation by the two-layer (UW solution/perfluorochemical) cold-storage method

BACKGROUND: A two-layer (University of Wisconsin solution/perfluorochemical [UW/PFC]) cold-storage method delivers sufficient oxygen to the pancreas during preservation and restores the ischemically damaged pancreas. In this study, we determined whether the additional preservation by the two-layer method could improve islet recovery from human pancreases with prolonged cold storage in UW. METHODS: Human pancreases were procured from cadaveric organ donors and preserved by the two-layer method (UW/PFC) for 2.9+/-0.7 hours (mean+/-SEM) at 4 degrees C after 11.8+/-1.5 hours of cold storage in UW (UW/PFC group, n=7), or by cold UW alone for 11.3+/-0.3 hours (UW group, n=14). The selected pancreases met the criteria of having at least 10 hours of cold storage in UW. All were processed by using a standard protocol of Liberase perfusion with Pefabloc by way of the duct, gentle mechanical dissociation, and Ficoll gradient purification. Transplanted islets were selected with the criteria of the Edmonton protocol (>5,000 islet equivalents [IE]/kg recipient body weight). RESULTS: The islet recovery was significantly increased in the UW/PFC group compared with the UW group (349.2+/-44.1 x 10 and 214.0+/-31.0 x 10 IE, respectively; <0.05). This resulted in islet yields of 4.6+/-1.0 x 10 IE/g of pancreas in the UW/PFC group compared with 2.0+/-0.3 x 10 IE/g of pancreas in the UW group ( <0.05). Five of 7 cases (71%) in the UW/PFC group and 5 of 14 cases (36%) in the UW group were transplanted. The islet grafts in the UW/PFC group improved the ability of glycemic control and decreased exogenous insulin administration in all recipients. CONCLUSIONS: Improvements in methods to preserve and recover ischemically damaged human pancreases before islet isolation and transplant could be extremely beneficial to the field of clinical islet transplantation. This preliminary study shows that additional short preservation by the two-layer (UW/PFC) cold-storage method can significantly improve islet recovery and increase opportunities of islet transplantation from human pancreases after prolonged cold ischemia.     

Influence of pancreas preservation on human islet isolation outcomes: impact of the two-layer method

BACKGROUND: Human pancreas preservation for islet transplantation holds additional challenges and considerations compared with whole pancreas transplantation. The purpose of this study was to clarify the limitations of the University of Wisconsin (UW) solution and the potentials of the two-layer method (TLM) for pancreas preservation before human islet isolation. METHODS: We retrospectively evaluated human islet isolation records between January 2001 and February 2003. One hundred forty-two human pancreata were procured from cadaveric donors and preserved by means of the UW solution (n=112) or TLM (n=30). Human islet isolations were performed using a standard protocol and assessed by islet recovery and in vitro function of islets. RESULTS: Eight to ten hours of cold ischemia in the UW solution is a critical point for successful islet isolations. It is difficult to recover a sufficient number of viable islets for transplantation from human pancreata with more than 10 hours of cold storage in the UW solution. The overall islet recovery in the TLM group was significantly higher than in the UW group. With 10 to 16 hours of cold storage, the success rates of islet isolations remained at 62% in the TLM group but decreased to 22% in the UW group. Transplanted islets in the TLM group worked well in the recipients. CONCLUSIONS: There are time limitations for using the UW solution for pancreas preservation before human islet isolation. The TLM is a potential method to prolong the optimal cold storage time for successful islet isolations.     

Short-term storage of the ischemically damaged human pancreas by the two-layer method prior to islet isolation

A two-layer cold storage method (TLM) allows sufficient oxygen delivery to pancreata during preservation and resuscitates the viability of ischemically damaged pancreata in the canine pancreas transplant model. In this study, we applied a short-term preservation of the TLM to human pancreata after prolonged cold ischemia prior to islet isolation, and investigated the mechanisms of resuscitation of the ischemically damaged human pancreas by the TLM. Human pancreata were procured from cadaveric donors and preserved by the TLM for 3.2 +/- 0.5 h after 11.1 +/- 0.9 h of cold storage in UW (TLM group), or by cold UW alone for 11.0 +/- 0.3 h (UW group). Islet isolations of all pancreata were performed using the Edmonton protocol. Islet recovery and in vitro functional viability of isolated islets were significantly increased in the TLM group compared with the UW group. According to the criteria of the Edmonton protocol, 10/14 cases (71%) in the TLM group were transplanted to patients with type I diabetes mellitus compared with only 5/21 cases (24%) in the UW group. In the metabolic assessment of human pancreata, levels of energetic parameters (ATP, total adenylates, and energy charge) were significantly increased, and malondialdehyde (MDA) levels were significantly decreased after the TLM preservation. There was no observable change in the incidence or degree of mitochondrial injury after the TLM preservation. Additional short-term storage by the TLM resuscitates the ischemically damaged human pancreas by regenerating the energetic status and prevents further damage by oxidative stress, ultimately leading to improvements of islet recovery and in vitro function. Use of the TLM following prolonged storage in UW provides an excellent adjunctive protocol for treating human pancreata for the rigors of the islet isolation process.     

Can the density of native pancreatic tissue slices predict human islet isolation and purification outcome?

INTRODUCTION: With currently available technology, the outcomes of human islet isolation and purification are still inconsistent, in part due to a lack of control of the pancreas donor and the procurement conditions. Using a single donor pancreas, the critical islet mass for establishing insulin independence of approximately 5000 engrafted islet equivalents (IEQ)/kg of recipient weight can only be retrieved from about one third of isolations. The purpose of this study was to analyze whether successful islet isolation and purification outcomes might be predicted from the density of native pancreatic tissue. METHODS: Tissue slices (TS) were obtained from the neck of 9 nondistended human donor pancreata. The density of the TS was determined using gravity sedimentation in continuous density gradients under either iso-osmolar or hyperosmolar conditions. Correlation coefficients were calculated with regard to the density of isolated exocrine and endocrine tissue, donor age, body mass index (BMI), cold ischemia time (CIT), IEQ prepurification and postpurification, IEQ recovery, and purity. RESULTS: (1) There was no change in density over time for TS in 300 mOsm/kg (mean, 1.079 +/- 0.0019 g/cm(3)) (2) In 500 mOsm/kg, there was a significant increase in density from 1.086 +/- 0.0021 g/cm(3) to 1.092 +/- 0.0021 g/cm(3) over time. (3) Density of isolated exocrine and endocrine became more distinct with lower density of TS (r = -0.776; P < .05). (4) Donor age, BMI, recovery of IEQ from gradients, and number of IEQ after purification did not correlate significantly with TS density. (5) In contrast, a significant inverse correlation existed betwen TS and CIT (r = -0.829; P < .05), and between TS versus IEQ number prior to purification (r = -0.867; P < .05). CONCLUSION: No homogeneous distribution of pancreas tissue density was seen among 9 consecutive human organs. Taken together, the density of native pancreas TS is not a suitable sole predictor for successful islet isolation and purification.     

Improved Islet Yields From Macaca Nemestrina and Marginal Human Pancreata After Two-Layer Method Preservation and Endogenous Trypsin Inhibition

We tested whether two-layer method (TLM) pancreas preservation and trypsin inhibition (Pefabloc) during processing allows longer preservation while retaining or improving viable islet recovery. Non-marginal primate (Macaca nemestrina) and marginal human (ischemic or preservation-injured) pancreata were processed with a research-oriented pan technique (Seattle method). Organs were processed upon arrival (+/- Pefabloc), or after TLM or University of Wisconsin solution (UW) preservation (+ Pefabloc). Islet yield, viability, and function were assessed. Pefabloc increased M. nemestrina islet yields from 9696 +/- 1749 IE/g to 15 822 +/- 1332 IE/g (p < 0.01). Two-layer method preservation (< 6 h) further increased yields, to 23 769 +/- 2773 IE/g (vs. + Pefabloc; p < 0.01). Similarly, Pefabloc increased marginal human islet yields from 2473 +/- 472 IE/g to 4723 +/- 1006 IE/g (p < 0.04). This increase was maintained after lengthy TLM preservation (> 30 h; 4801 +/- 1066 IE/g). We also tested the applicability of TLM preservation (23.5 +/- 3.2 h) to the processing of marginal human pancreata by the Edmonton/Immune Tolerance Network clinical protocol. Islet yield and function approached published results of pancreata processed 4.8 +/- 0.8 h after organ recovery (p = 0.06). Pefabloc, and TLM vs. UW preservation, prolonged the tolerable interval between organ recovery and islet isolation. Islet yield, viability, and functionality improved from both marginal and nonmarginal pancreata.     

Resuscitation of the ischemically damaged human pancreas by the two-layer method prior to islet isolation

A two-layer cold storage method (TLM) allows sufficient oxygen delivery to pancreata during preservation and resuscitates the viability of ischemically damaged pancreata. This study determined the effect of additional preservation of ischemically damaged human pancreata by the TLM before islet isolation. Human pancreata were procured from cadaveric organ donors and preserved by the TLM for 3.2 +/- 0.5 hours (mean +/- SEM) at 4 degrees C after 11.1 +/- 0.9 hours of cold storage in University of Wisconsin solution (UW) (TLM group), or by cold UW alone for 11.0 +/- 0.3 hours (UW group). Islet isolations of all pancreata were performed using the Edmonton protocol. Islet recovery and in vitro function of isolated islets were significantly increased in the TLM group compared with the UW group. In the metabolic assessment of human pancreata, ATP levels were significantly increased after the TLM preservation. This study showed that additional short-term preservation by the TLM resuscitates the viability of ischemically damaged human pancreata before islet isolation, leading to improvements in islet recovery and in vitro function of isolated islets.     

Testing combinations of protease inhibitor and preservation solution to improve islet quality and yield

Pancreas preservation using an oxygenated two-layer method (TLM) has been reported to improve islet yields, as has supplementation of Liberase with Pefabloc. We hypothesized that using both TLM and Pefabloc could enhance islet yield as compared with preservation in University of Wisconsin (UW) or Histidine-Tryptophan Ketoglutarate (HTK) solution. METHODS: Ninety-eight pancreata with no significant differences of age, body mass index, or cold ischemia time preserved randomly with UW (n = 40), TLM (n = 48), or HTK (n = 10) were processed with (n = 36) or without (n = 66) Pefabloc. RESULTS: The total islet equivalent (IEQ) from TLM-preserved pancreata processed with Pefabloc (n = 12) showed lower yields versus those processed without Pefabloc (n = 36): 216,120 +/- 27,906 vs. 301,427 +/- 21,447 IEQ (P < .05). Islets from 1 of 12 (8.33%) pancreata processed with Pefabloc in TLM were transplanted, in contrast with 15/36 TLM (41.67%) pancreata processed without it. Islet yields were not significantly different among pancreata preserved in UW and processed with Pefabloc (n = 17) versus without Pefabloc (n = 23): 342,693 +/- 45,588 versus 266,609 +/- 29,006 IEQ (P = .149). The number of transplants from UW-preserved pancreata was 3/17 (17.65%) when processed with Pefabloc and 4/23 (17.39%) without. Among the HTK group, there was no significant difference in islet yields between pancreata processed with (n = 7) versus without Pefabloc (n = 3): 248,227 +/- 65,294 versus 483,555 +/- 144,070 IEQ (P = .118). CONCLUSIONS: Pefabloc showed no benefit to improve islet yields. Pancreata preserved in TLM provided better transplant quality islets when processed in the absence of Pefabloc.     

Long-term preservation of the pig pancreas by a one-layer method for successful islet isolation

Background

Pancreas preservation by two-layer method (TLM) was recently established for clinical islet transplantation. The extensive use of TLM would require enormous efforts to solve logistical and technical problems. Omitting University of Wisconsin solution (UW) as second layer would facilitate the regular application of oxygenated perfluorocarbon; (PFC). To clarify whether long-term pancreas preservation is feasible by this simplified procedure, pancreases from retired breeder pigs were subjected to 7-hour preservation utilizing PFC alone in a one-layer method (OLM, n = 8) or in combination with UW (TLM, n = 10).

Methods

Resected pancreata were intraductally flushed with cold UW. Subsequently, pancreata were promptly processed (n = 6) as previously described or stored by TLM or OLM.

Results

Compared to unstored (429200 ± 86700 IEQ) and OLM-stored pancreases (338600 ± 42100 IEQ), (P = ns vs unstored) postpurification islet yield decreased after TLM storage (238000 ± 26600 IEQ, P < .05). No significant differences were found regarding purity (>90%), adenosine triphosphate (ATP) content, and viability as determined by formazan production and trypan-blue exclusion (>95%). Glucose stimulation index of freshly isolated islets (2.5 ± 0.4) was significantly decreased after TLM storage (1.8 ± 0.2, P < .05) but not after OLM storage (2.3 ± 0.6). Islet transplantation in diabetic nude mice demonstrated sustained graft function in all experimental groups.

Conclusions

This study demonstrates that viable pig islets can be successfully isolated after prolonged ischemia utilizing PFC alone for oxygenation of cold-stored pig pancreases. The easy handling of OLM could facilitate the regular application of PFC as pancreas preservation solution.     

Improved pancreatic islet isolation outcome in autologous transplantation for chronic pancreatitis

Total or partial pancreatectomy followed by autologous islet transplantation is a therapeutic option for the treatment of refractory chronic pancreatitis (CP). Maximization of islet yields from fibrotic and inflamed organs is crucial for prevention of posttransplant diabetes. We adapted technical advancements developed for islet allotransplantation toward islet autotransplantation. Eight patients (two men, six women; ages 24-58 years) underwent total (n = 7) or partial (n = 1) pancreatectomy for the treatment of CP refractory to maximal medical management. Pancreata were preserved in UW solution (UW group) in initial three cases and the last five pancreata were preserved with pancreatic ductal injection followed by ET-Kyoto/oxygenated PFC solutions (DI+TLM group). Islets were isolated by modified Ricordi method and were purified only in one case. All islet infusions were performed under general anesthesia via direct vein injection into the portal venous system with pressure monitoring. Total islet yields (129,314 ± 51,627 vs. 572,841 ± 116,934 IEQ, p < 0.04), islet yield/pancreas weight (1,233 ± 359 vs. 6,848 ± 847 IEQ/g, p < 0.003), and islet yield/patient body weight (1,951 ± 762 vs. 7,305 ± 1,531 IEQ/kg, p < 0.05) were significantly higher in the DI+TLM group when compared to the UW group. Pellet size was also higher (5.3 ± 0.3 vs. 13.5 ± 3.4 ml) in the DI+TLM group, suggesting that this method of preservation effectively protected pancreatic tissue against autolysis. First month posttransplant basal C-peptide and the secretory unit of islet transplant objects (SUITO) index were also higher in the DI+TLM group when compared to the UW group (2.0 ± 0.3 vs. 1.4 ± 0.4 ng/ml and 42.6 ± 12.7 vs. 14.6 ± 5.6, respectively). There were no technical complications related to the infusion. Our results suggest that higher islet yields can be achieved even from chronically inflamed and fibrotic organs using DI+TLM. The techniques applied for islet isolations from normal pancreata are showing promise for fibrotic pancreata from CP patients.