Islet isolation and transplantation outcomes of pancreas preserved with University of Wisconsin solution versus two-layer method using preoxygenated perfluorocarbon

BACKGROUND: Previous small clinical trials indicate that the two-layer method (TLM) for pancreas preservation improves islet isolation outcome. However, the effect of TLM has not been evaluated in large-scale study. In addition, a direct benefit of TLM on islet transplantation outcome has not been addressed in the setting of any randomized controlled trials. METHODS: Between April 2003 and October 2005, human pancreata from brain-dead donors were preserved by TLM using preoxygenated perfluorocarbon (n = 75) or in University of Wisconsin (UW) solution (n = 91) prior to islet isolation. Islet isolation and transplantation outcomes were compared between the two groups. RESULTS: We did not find any significant differences in adenosine triphosphate content in pancreatic tissue after preservation, pre and postpurification islet yields, in vitro insulin secretory function, or utilization ratio of transplantation between the two groups. Transplanted mass and functional viability of islet isolated from TLM-preserved pancreas were similar to those from UW-preserved pancreas. Patients receiving the TLM-islet or the UW-islet showed a marked decrease in insulin requirement after transplantation. However, no significant difference was observed in a decrease in insulin requirement between patients receiving the TLM-islet and the UW-islet. CONCLUSIONS: No beneficial effect of TLM on islet isolation and transplantation outcomes was observed. Our findings bring into question the true merit of routine use of TLM prior to islet isolation.     

Improvement of pancreatic islet isolation outcomes using glutamine perfusion during isolation procedure

During procurement, isolation, and transplantation, islets are exposed to high levels of oxidative stress triggering a variety of signaling pathways that can ultimately lead to cell death. Glutamine is an important cellular fuel and an essential precursor for the antioxidant glutathione. The aim of this study was to examine the role of intraductal glutamine administration in facilitating recovery of isolated rat islets from pancreases subjected to a clinically relevant period of warm ischemia. Islets were isolated in Sprague-Dawley (SD) rats (n = 18 per group). Pancreata in groups 1 and 2 were procured immediately while groups 3 and 4 were subjected to 30-min warm ischemia. Groups 2 and 4 were treated intraductally with 5 mM glutamine prior to pancreatectomy. Exposure to 30-min warm ischemia significantly reduced islet yield [groups 1 & 2 (nonischemia): 503 +/- 29 islets/rat vs. groups 3 & 4 (ischemia): 247 +/- 26 islets/rat; p < 0.05]. Intraductal glutamine treatment significantly improved islet yield when pancreata were subjected to 30-min warm ischemia [144 +/- 16 islets/rat without glutamine (group 3) vs. 343 +/- 36 islets/rat with glutamine (group 4), p < 0.05]. Glutamine also significantly improved islet viability (values were 50 +/- 4% in group 4 vs. 27 +/- 3% in group 3, p < 0.05). Similarly, glutathione (reduced) levels were significantly elevated in both glutamine-treated groups; however, this increase was greatest in tissues exposed to ischemia (2.76 +/- 0.04 nmol/mg protein in group 4 vs. 1.66 +/- 0.04 nmol/mg protein in group 3, p < 0.05). Intraductal glutamine administration considerably improves the islet yield, viability, and augments endogenous glutathione levels in pancreata procured after a clinically relevant period of ischemia. Intraductal administration of glutamine at the time of digestive enzyme delivery into the harvested pancreas may represent a simple yet effective tool to improve islet yields in clinical isolations.     

Development of a novel digestion chamber for human and porcine islet isolation

BACKGROUND: The current technique of human pancreas digestion for islet isolation relies on selective distribution of collagenase delivered via the pancreatic duct to produce digestion and removal of peri-acinar fibrous tissue. However, the collagenase has relatively little effect on the interlobular fibrous tissue, which must therefore be broken down by mechanical means within the digestion chamber so as to release the contained acini and islets. The current way of achieving this in the Ricordi chamber is to place five or six stainless steel balls within the chamber and shake vigorously. The shaking presumably breaks down the interlobular fibrous tissue by a combination of shear force induced by the movement of tissue through the shaking process, assisted by numerous blows from the steel balls. Intuitively, one would expect some islets would be destroyed rather than released by such a battering. METHODS: In an attempt to improve the efficiency of islet isolation we have designed a new digestion/filtration chamber that consists of a glass cylinder, sealed with Teflon plates holding in mesh filters at each end, secured in place by a central threaded tie-rod and external knurled nuts. A ring-shaped piston within the cylinder can be pushed up and down the travel by two rods passing out through sealed ports in the Teflon disk at one end and connected to an external handle. The handle is used to gently push the piston up and down the travel of the cylinder, which pushes the fluid and tissue through the central lumen of the ring-piston. A series of hooks attached to the central tie-rod catch the fibrous strands of the passing tissue; the shearing forces produced cause disruption by a process thought to be similar to teasing the tissue apart with fine forceps. RESULTS: A series of initial experiments with human pancreas showed the prototype to be too large, causing temperature control problems, and a redesigned smaller chamber was produced, maintaining the crucial design features. Experience processing five human pancreata has now demonstrated that in three of five pancreata the new chamber produced a good yield (>200,000 I.E.) of remarkably well separated and intact islets, the entire dispersion process being under 1 hour. However, in two isolations the collagenase digestion was poor, with few free islets. A copy of the new chamber (reserved for porcine work only) has been produced, as well as a copy of the Ricordi chamber. We have confirmed that the new chamber can isolate porcine islets in large numbers (>5000 islets/g pancreas [n = 2], but note that pig islets are small). CONCLUSION: These preliminary studies are sufficiently encouraging to justify further direct comparison with the Ricordi chamber for the purpose of animal and human islet isolation.     

Adenine nucleotide levels in a closed enzymatic digestion system for porcine islet isolation

Obtaining viable islets is a crucial step for successful islet transplantation. Adenosine triphosphate (ATP) is a marker of cell viability. However, little is known about any changes in the energy status of the tissues that are being digested during the digestion phase. We herein examined whether the ATP content in serially digested pancreatic tissue samples could be specific objective parameters that signal the optimal point to stop the digestion process. We obtained partial pancreata (body to tail) from 4- to 5-year-old pigs from a slaughterhouse. The tissue samples were preserved in M-Kyoto solution for less than 3 h. They were digested using an automated enzymatic and mechanical dissociation system at 37°C for 90 min following intraductal injection of Liberase HI. Samples were collected from the digestive circuit every 5 or 10 min to determine the ATP level, total adenine nucleotide (TAN) level, islet count (count/g), and yield of islet equivalent (IEQ) in the serial digestive fluids. The ATP and TAN levels, IEQ and islet count were increased and then decreased during digestion process. The profile of these parameters differed from case to case. However, when ATP changing ratio (respective value/precedent value) was compared with IEQ changing ratio, a greater than threefold increase in the ATP changing ratio followed by an increase in the islet count changing ratio within 5 min was consistently observed, indicating the optimal time to stop the digestion. The ATP levels of the handpicked islets in the digested samples were lower in the overdigested phase in comparison to those in the earlier digested phase. These results indicate that the ATP level in digested fluid could be an effective indicator to estimate the viability of cells as well as determine the optimal time to terminate the digestion process in order to obtain viable islets.     

Significant progress in porcine islet mass isolation utilizing liberase HI for enzymatic low-temperature pancreas digestion.

BACKGROUND: Frequent success in human islet isolation is prevented by the large variability of scarce organ donors; this favors the future utilization of pigs as donors for clinical islet xenotransplantation. Porcine-specific difficulties of islet isolation are attributed to the intrinsic fragility of islets during pancreas digestion. METHODS: To preserve islet integrity during efficient pancreas dissociation, porcine pancreata (n=48) were distended after cold storage with cold University of Wisconsin solution containing Liberase HI and digested at 24-28 degrees C using digestion-filtration. Pancreata distended with University of Wisconsin solution containing well-proven crude collagenase and digested at 32-34 degrees C served as controls (n=46). Monolayer Ficolldiatrizoate gradient purification was performed in a Cobe 2991. RESULTS: Purified yield of islet equivalents per pancreas (mean+/-SEM) was almost doubled by Liberase HI compared with crude collagenase (526,480+/-46,560 vs. 270,270+/-19,420; P < 0.0001) and also significantly increased comparing islet equivalents per gram of pancreas (4,210+/-320 vs. 2,640+/-245; P=0.0004). Islet integrity was better preserved during Liberase HI digestion compared with crude collagenase digestion as indicated by isolation index (2.1+/-0.1 vs. 1.4+/-0.1; P<0.0001). Purity, viability, and in vitro function of islets did not differ between experimental groups. Preserved in vivo function of islets isolated by Liberase HI was demonstrated after subcapsular transplantation into 16 diabetic nude rats. CONCLUSIONS: If the problems related to xenograft rejection and xenosis could be solved, low-temperature digestion of porcine pancreata using Liberase HI could serve as an essential prerequisite for successful 1:1 xenotransplantation of pig islets into type 1 diabetic human recipients.     

Cell therapy for diabetes using piscine islet tissue

The islet tissue, called Brockmann bodies, in certain teleost fish is anatomically distinct from the pancreatic exocrine tissue and can be easily identified macroscopically. Expensive islet isolation procedures, such as required when procuring islet tissue from mammalian pancreases, are unnecessary. Tilapia islets transplanted into diabetic athymic nude mice will produce long-term normoglycemia and mammalian-like glucose tolerance profiles. Encapsulated tilapia islets function well after transplantation into euthymic recipients. Additionally, tilapia are potentially ideal xenogeneic donors because of markedly lower donor production costs, minimal islet procurement costs, and possibly decreased xenozoonotic potential relative to mammalian donors. Tilapia islets appear to be appropriately glucose responsive with high insulin output, can be cryopreserved, and are much more resistant to hypoxia than mammalian islets. Because tilapia and human insulin differ by 17 amino acids, we have cloned, sequenced, and modified the tilapia insulin gene by site-directed mutagenesis resulting in a tilapia insulin gene that codes for "humanized" insulin while still maintaining all of the tilapia regulatory (noncoding) sequences. We have now produced small numbers of founder transgenic tilapia with incorporation of our humanized tilapia insulin gene construct, and we have shown transmission and expression of the transgene in the beta cells and serum of their Fl offspring. Our ultimate goal is to achieve homologous recombination and to breed for homozygosity for the hybrid insulin gene. Subsequent generations of transgenic tilapia will undergo a program of genomic optimization selecting for growth, survival, and fecundity as well as stability of the transgene. Islets from the resulting transgenic fish, after extensive characterization, could be harvested, encapsulated, and then transplanted into diabetic patients.     

Ameliorating injury during preservation and isolation of human islets using the two-layer method with perfluorocarbon and UW solution

This study assessed the effects of a two-layer method (TLM), using perfluorocarbon and UW solution, on the quality of human pancreata following storage and islet yield/function after isolation. In part A, TLM was applied immediately after procurement and the energetic profile was compared to a group treated with UW solution only (control) throughout 24-h storage. In part B, cadaveric human pancreata were procured and subjected to a TLM after cold storage in UW solution (TLM group) or UW solution (control group). Energetics, lipid peroxidation, and islet recovery/function were assessed after preservation at 4 degrees C. In part A, after 9-h storage, the energetic profile (ATP, ATP/ADP, energy charge) for the TLM group was superior to controls. In part B, TLM treatment resulted in consistently greater ATP, ATP/ADP, and energy charge values than with storage in UW solution alone (p < 0.05). UW treatment resulted in 40% greater peroxidative damage than in the TLM group (p < 0.05). Islet recovery and functional viability were 30-40% higher following TLM treatment (p < 0.05). These data support the hypothesis that islet viability and yields can be significantly improved using a brief period of TLM treatment following conventional UW storage; reduced energetic and oxidative stress are implicated as potential mechanisms.     

Validation of the scoring system for standardization of the pancreatic donor for islet isolation as used in a new islet isolation center

BACKGROUND: The aim of this study was to evaluate the effectiveness of the Edmonton Donor Scoring System for use in our much less active islet center. Because the ability to recognize an appropriate donor may help to achieve consistent and predictable success of pancreatic islet isolation, it should lead to increased effectiveness and lower cost. MATERIAL AND METHODS: Charts of 36 consecutive pancreas donors were reviewed to assess the donor points (DP). DP ranged from 0 to 100 based on donor age, body mass index, cause of death, social and medical history, hospital stay, vasopressor dosages, laboratory tests, cold ischemia time and procurement team, as well as pancreas size, consistency, fat content, damage, and quality of procurement and packing. RESULTS: Successful isolation was achieved in 39% of donors (14 of 36), a value similar to that achieved in Edmonton (40%). We used the optimal cutoff value (DP = 79) proposed by the Edmonton group. Sensitivity, specificity, positive predictive value, negative predictive value, and accuracy were 66%, 75%, 57%, 82% and 72%, respectively. Successful islet isolation from poor or marginal donors (DP < 49.5 and 50 to 59.5) was 0% and 28.6% respectively; it was 63% and 100% in optimal donors (DP = 80 to 89.5 and 90 to 100). We concluded that islet isolation success correlated with the previously proposed donor scoring system. CONCLUSIONS: The Donor Scoring System can be successfully implemented regardless of the level of activity of an experienced isolation center. This system permits identification of a suitable donor prior to organ processing. It may guide a center's donor selection strategy based on its goals and its budget.     

Evaluating the effect of serine proteases on collagenase activity during human islet isolation

Inconsistencies in human islet yields after collagenase digestion have been attributed to the activation of endogenous enzymes of the donor pancreas. It has been suggested that pancreatic serine proteases contribute to the proteolysis of collagenase. This study defined the effects of endogenous enzymes within the pancreas on pancreas dissociation during collagenase digestion. Levels of collagenase activity from samples taken throughout several steps in islet isolation procedures, both with and without the addition of the serine protease inhibitor Pefabloc, were determined by a spectrophotometric assay using N-[3-(2-furyl)acryloyl]-Leu-Gly-Pro-Ala as the substrate. Results clearly demonstrated that the level of collagenase activity remains stable throughout the isolation procedure despite differences in the donor factors from several cadaveric donor pancreases. This was further demonstrated by observing no difference in activity levels after incubating commercial collagenase preparations with serine proteases and analyzing by means of collagenase activity and SDS-PAGE. These data show that the presence of serine proteases does not affect the level of collagenase activity; however, they likely damage the islet cells upon prolonged digestion of the pancreatic tissue. Further efforts at examining exogenous and endogenous enzyme levels may result in the development of an enzyme cocktail that is both stable and effective for digesting the human pancreas while preserving islet function and viability.     

Application of the two-layer method on pancreas digestion results in improved islet yield and maintained viability of isolated islets

BACKGROUND: Oxygenation of the pancreas during preservation by the two-layer method (TLM) has shown beneficial effects in islet transplantation. Here, we apply this concept (oxygenation) to the isolation process. METHODS: Rat pancreases were digested using four different methods. Pancreases were digested with preoxygenated perfluorocarbon (PFC) in group 2 and without it in group 1. Additionally, adenosine was included in the collagenase solution in subgroups B but not in subgroups A. Islet yields and viability were compared between groups. RESULTS: Tissue oxygen tension in group 1 was essentially zero during digestion, but rapidly reached around 300 mm Hg and was maintained in group 2. The tissue adenosine triphosphate (ATP) level in rat pancreas just after laparotomy (control) was 4.2+/-0.7 micromol/g dry weight; after digestion, it was 0.12+/-0.03 micromol/g, 0.70+/-0.10 micromol/g, 0.30+/-0.18 micromol/g, and 2.90+/-0.80 micromol/g in groups 1A, 1B, 2A, and 2B, respectively. No significant differences were observed between group 2B and control (P=0.19). Islet yields (IEQ/pancreas) were 1600+/-400, 1400+/-400, 1300+/-400, and 2400+/-100 in groups 1A, 1B, 2A, and 2B, respectively. The islet yield of group 2B was significantly higher than other groups (P<0.05). The cure rate after transplanting 200 islets into athymic nude mice did not differ (80% in all groups). The stimulation indices in the four groups were also the same. CONCLUSIONS: Tissue ATP levels after digestion were well maintained using TLM with adenosine digestion method. Consequently, greater numbers of islets could be retrieved. The new method was at least equivalent to islet function isolated by conventional method. Clinical study is therefore warranted.     

Pancreatic islet isolation after gastric bypass in a rat model: technique and initial results for a promising research tool

BackgroundRoux-en-Y gastric bypass (RYGB) affords a high remission rate of type 2 diabetes mellitus among morbidly obese diabetic patients. We report the use of the isolated islet technique to assess pancreatic function and glucoregulatory mechanisms after RYGB surgery.MethodsA total of 15 adult, male, Sprague Dawley diet-induced obese rats were randomly divided into 3 experimental groups: sham, RYGB, and pair-fed, with 5 rats in each group. The body weight was measured at baseline and every week for 4 weeks. Pancreatic islet function was assessed in vitro according to the amount of insulin secreted from isolated islets incubated in 2 mM and 20 mM glucose for 1 hour at 37°C. Fasting plasma glucose, insulin, glucagon-like peptide-1, PYY3-36, and glucose-dependent insulinotropic peptide were measured at baseline and 28 days after surgery.ResultsThe baseline body weight was 917 ± 61, 831 ± 42, and 927 ± 43 g for the sham, RYGB, and pair-fed groups, respectively. The RYGB group lost 32% body weight compared with 16% for the sham and 24% for the pair-fed groups. Glucose-stimulated insulin secretion from the isolated islets in the RYGB group was greater than in the comparison groups (P = .04) at 4 weeks after surgery. Fasting plasma glucagon-like peptide-1 and PYY3-36 were significantly increased at 4 weeks in the RYGB group.ConclusionIslet isolation and stimulation in the present animal model was feasible, affords a direct measurement of pancreatic islet function, and might provide a useful tool to study the effects of RYGB on pancreatic function and the relationship between islet cell function and incretin production after bariatric surgery.     

Refinement of the automated method for human islet isolation and presentation of a closed system for in vitro islet culture

BACKGROUND: The procedure of human islet isolation needs further optimization and standardization. Here, we describe techniques to enhance enzymatic digestion and minimize mechanical forces during the digestion process. The isolation protocol has also been modified to meet current GMP (cGMP) standards. Moreover, the impact of donor- and process-related factors was correlated to the use of islets for clinical transplantation. METHODS: One hundred twelve standardized consecutive islet isolations were evaluated. Metyltioninklorid and indermil (topical tissue adhesive) were applied to detect leakage of collagenase injected and to repair the damaged pancreatic glands. The effects of dye and glue were evaluated in terms of islet yield, islet function using the perifusion assay, and success rate of the isolation. To analyze key factors for successful isolations, both univariate and multivariate regression analysis were performed. RESULTS: Both Metyltioninklorid and Indermil were effective to prevent leakage of enzyme solutions from the pancreatic glands. Both islet yield and success rate were higher when these tools were applied (4,516.1+/-543.0 vs. 3,447.7+/-323.5, P=0.02; 50.0% vs. 21.3%, P=0.02, respectively). No adverse effects on islet function or collagenase activity were observed. Multivariate regression analysis identified the maximal recorded amylase >100 U/L (P=0.026), BMI (P=0.03), and the use of catecholamine (P=0.04) as crucial donor-related factors. In addition, cold ischemia time (P=0.005), the dissection procedure using whole glands with duodenum (P=0.02), and the local procurement team (P=0.03) were identified as crucial isolation-related variables. CONCLUSIONS: A standardized technique of islet isolation is presented applying novel means to improve enzymatic digestion and to meet cGMP standards.     

Amylase levels in preservation solutions as a marker of exocrine tissue injury and as a prognostic factor for pancreatic islet isolation

Occurrence of primary graft nonfunction of pancreatic islets demands research for new methods of organ preservation during cold ischemia conditions. Digestive enzymes released during preservation injure the islets for subsequent rewarming and islet isolation processes. The aim of our study was to assess the amylase level in preservation solution as a marker of exocrine tissue injury, allowing the prognosis of islet yield and viability. The experiments undertaken on rats used three commercially available preservation solutions: ViaSpan (UW); Custodiol (HTK); and Euro-Collins (EC). After 180 minutes of cold ischemia, the highest islet recovery was observed among pancreata stored in UW solution (508 +/- 139 vs HTK 344 +/- 103; P <.05 vs EC 322 +/- 113; P <.05). These islets also revealed the highest insulin stimulation index in glucose static tests (1.19 +/- 0.30 vs HTK, 0.87 +/- 0.43; P <.01, vs EC.25 +/-.06; P <.001). The highest amylase level in the preservation solution was associated with a decreased yield of islets during the isolation process and lowest insulin stimulation index (increasing 139 +/- 18% for EC, 108 +/- 12% for HTK; P <.05 vs 87 +/- 10% for UW; P <.05). Our data strongly suggest, that the dynamic of amylase release during pancreas preservation at 4 degrees C correlates with a reduced number and viability of isolated islets. These results suggest that measurement of amylase levels after pancreas preservation may have potential clinical application as a marker to evaluate pancreatic tissue injury.     

小鼠胰岛的分离及胰岛移植

目的研究小鼠胰岛分离和移植的方法。方法在Gotoh等所介绍的小鼠胰岛分离方法的基础上作了一些改进,由原来经胆总管注入胶原酶消化液改为由胆囊注入,并在消化液和Ficoll分离液中加入了胰酶抑制剂和BSA。结果使分离纯化后的胰岛产量由原来方法的41.7±13.2个提高到了266.5±32.1个(P<0.01),活性在95%以上,除了少量导管外几乎不含腺泡组织。结论改进后的方法可以在肉眼条件下注入消化液,而不需要解剖显微镜,既方便了操作又提高了成功率;避免了整个消化和分离过程中胰酶对胰岛的消化作用,提高了得率,且具有很好的重复性。     

Influence of strain and age differences on the yields of porcine islet isolation: extremely high islet yields from SPF CMS miniature pigs

BACKGROUND: Porcine pancreas is a potential source of material for islet xenotransplantation. However, the difficulty in isolating islets, because of their fragility and the variability of isolation outcome in donor age and breed, represents a major obstacle to porcine islet xenotransplantation. In this study, we compared the islet isolation yield of specific pathogen-free (SPF) Chicago Medical School (CMS) miniature pigs with that of another miniature pig breed and market pigs from a local slaughterhouse. METHODS: Nine adult CMS miniature (ACM) pigs (>12 months), six young CMS miniature (YCM) pigs (6-7 months), four adult Prestige World Genetics (PWG) miniature (APM) pigs (>12 months), and 13 adult market (AM) pigs from a local slaughterhouse were used for islet isolation. RESULTS: The islet yield per gram of pancreas from ACM pigs (9589 +/- 2823 IEQ/g) was significantly higher than that from APM pigs (1752 +/- 874 IEQ/g, P < 0.05), AM pigs (1931 +/- 947 IEQ/g, P < 0.05), or YCM pigs (3460 +/- 1985 IEQ/g, P < 0.05). Isolated islets from ACM pigs were significantly larger than those from AM pigs or YCM pigs. The in vitro and in vivo function of isolated islets showed no difference among experimental groups. The pancreases of ACM pigs contained higher mean islet volume density percentages and larger size of islets than those of AM or APM pigs. CONCLUSIONS: We isolated extremely high yields of well-functioning islets from ACM pigs bred under SPF conditions. SPF CMS miniature pigs should be one of the best porcine islet donors for clinical porcine islet xenotransplantation.     

Effect of core pancreas temperature during cadaveric procurement on human islet isolation and functional viability

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.     

Achievement of insulin independence in three consecutive type-1 diabetic patients via pancreatic islet transplantation using islets isolated at a remote islet isolation center

BACKGROUND: As a result of advances in both immunosuppressive protocols and pancreatic islet isolation techniques, insulin independence has recently been achieved in several patients with type 1 diabetes mellitus via pancreatic islet transplantation (PIT). Although the dissemination of immunosuppressive protocols is quite easy, transferring the knowledge and expertise required to isolate a large number of quality human islets for transplantation is a far greater challenge. Therefore, in an attempt to centralize the critical islet processing needed for islet transplantation and to avoid the development of another islet processing center, we have established a collaborative islet transplant program between two geographically distant transplant centers. PATIENTS AND METHODS: Three consecutive patients with type 1 diabetes mellitus with a history of severe hypoglycemia and metabolic instability underwent PIT at the Methodist Hospital (TMH), Houston, Texas, using pancreatic islets. All pancreatic islets were isolated from pancreata procured in Houston and subsequently transported for isolation to the Human Islet Cell Processing Facility of the Diabetes Research Institute (DRI) at the University of Miami, Miami, Florida. Pancreatic islets were isolated at DRI after enzymatic ductal perfusion (Liberase-HI) by the automated method (Ricordi Chamber) using endotoxin-free and xenoprotein-free media. After purification, the islets were immediately transported back to TMH and transplanted via percutaneous transhepatic portal embolization. Immunosuppression consisted of sirolimus, tacrolimus, and daclizumab. RESULTS: After donor cross-clamp in Houston, donor pancreata arrived at DRI and the isolation process began within 6.5 hr in all cases (median, 5.4 hr; range, 4.8-6.5 hr). At the completion of the isolation process, the islets were immediately transported back to TMH and transplanted. All three patients attained sustained insulin independence after transplantation of 395,567, 394,381, and 563,206 pancreatic islet equivalents (IEQ), respectively. Despite insulin independence, the first two patients received less than 10,000 IEQ/kg; therefore, to increase their functional pancreatic islet reserve, they underwent a second islet transplant with 326,720 and 768,132 IEQ, respectively. Posttransplantation follow-up for these three patients is 4, 3, and 0.5 months, respectively. The mean glycosylated hemoglobin values have been dramatically reduced in the first two patients. In addition, the mean amplitude of glycemic excursions have also been reduced in all three recipients (patient 1: before transplantation 197 mg/dL vs. after transplantation 61 mg/dL; patient 2: before transplantation 202 mg/dL vs. after transplantation 52 mg/dL; patient 3: before transplantation 245 mg/dL vs. after transplantation 58 mg/dL) after PIT. All pancreatic islet allografts demonstrated the ability to respond to an in vitro glucose stimulus at the DRI before shipment and at TMH after shipment and final processing with a median stimulation index of 2.1 and 2.2, respectively. None of the transplant recipients have had a hyper- or hypoglycemic episode since PIT and no complications have occurred. CONCLUSIONS: These early data demonstrate that (1) pancreatic islets remain viable after shipment to remote transplant sites; (2) pancreatic islet isolation techniques and experience can be concentrated at a small number of regional facilities that could supply islets to remote transplant centers; and (3) insulin independence via PIT can be achieved using a remote pancreatic islet isolation center.