Clinical application of the two-layer (University of Wisconsin solution/perfluorochemical plus O2) method of pancreas preservation before transplantation

BACKGROUND: The two-layer method [University of Wisconson solution (UW)/perfluorochemical plus O2] for pancreas preservation has been demonstrated to be superior to simple UW storage alone in the canine model. For the first time, we applied the two-layer method to clinical whole-pancreas transplantation. METHODS: Pancreases were placed in the two-layer method in 10 cases and UW alone in 44 cases before transplant. The mean cold ischemic time was 16.5 hr in the two-layer group versus 18.1 hr in the UW group (P=NS). We compared the condition of graft at the time of reperfusion, and then 3 months posttransplant graft function and complications. RESULTS: At the time of reperfusion, no grafts in the two-layer group were edematous, compared with 10(23.3%) of 43 in the UW group (P=0.18). Seven (70%) of 10 grafts in the two-layer group obtained the best overall quality score, compared with 24(57.1%) of 42 in the UW group (P=0.72). Nine (90%) of 10 recipients in the two-layer group became insulin-independent during hospitalization, compared with 31(70.5%) of 44 in the UW group (P=0.26). Time to insulin independence was no different between the two groups. No pancreas grafts preserved by the two-layer method suffered acute rejection. Conclusions. The two-layer preservation method is feasible in human clinical transplantation. It was at least equivalent and may be superior to UW alone in both morphologic and functional assessment of the transplanted pancreas.     

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.     

Human islet isolation outcomes from pancreata preserved with Histidine-Tryptophan Ketoglutarate versus University of Wisconsin solution

This study was designed to compare Histadine-Tryptophan-Ketogluterate (HTK) with University of Wisconsin (UW) solution. Pancreata from extended criteria donors were flushed and transported with HTK (n=41) or UW (n=45). Isolation outcomes were determined by islet yields, viability and in vitro and in vivo function. Final yields were similar between two groups (HTK: 383,085 vs. UW: 328,514 EIN, P=0.14). In the HTK group, 63.4% (26/41) of isolations resulted in a yield of over 300,000, and in the UW group this was achieved in 46.7% (21/45; P=0.12). Viability results were similar (HTK: 82.9 vs. UW: 82.7%, P=0.93). Stimulation index in the HTK and UW groups were comparable (5.28 vs. 4.91, P=0.62). Ten out of 41 islet preparations in HTK and 4 of 45 in UW group were suitable for clinical transplantation (P=0.05). Our study shows HTK is equivalent to UW solution in the preservation of pancreata for islet isolation.     

Experimental pancreatic preservation prior to islet isolation and transplantation

Preservation of the Lewis rat pancreas prior to islet isolation was accomplished by initial intraductal distension with the University of Wisconsin (UW) hydroxyethyl starch-lactobionate solution to which collagenase had been added, followed by simple cold storage at 4 degrees C for 0, 3, 12, 24, and 48 hr (n = 16-21 at each interval). The pancreases were then processed by digestion and mechanical dispersion to produce free islets of Langerhans. The mean islet yields (+/- standard errors of the means) were controls = 819 +/- 58 (n = 21), 3 hr = 867 +/- 51 (n = 20), 12 hr = 770 +/- 71 (n = 16), 24 hr = 805 +/- 62 (n = 18), and 48 hr = 722 +/- 55 (n = 16). None of these means differed significantly. The islets from pairs of donor pancreases (mean dose of islets = 1586 +/- 72) were transplanted intraportally into single isogeneic recipients with streptozotocin-induced diabetes (plasma glucose greater than 400). The preservation interval directly influenced the outcome of these islet isografts in the following manner: (i) Rates of functional success (nonfasting glucose less than 200 mg/dl) were 100% after storage times of 0 hr (n = 10), 3 hr (n = 8), and 12 hr (n = 8); 86% with a storage time of 24 hr (n = 7); and 0% after 48 hr (n = 8). (ii) Return of euglycemia was increasingly delayed with increasing preservation intervals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Seventy-two-hour preservation of the canine pancreas by the two-layer (Euro-Collins' solution/perfluorochemical) cold storage method

A 72-hr preservation of canine pancreas by a 2-layer (Euro-Collins'/Perfluorochemical) cold storage method was tested in the canine model of segmental pancreas autotransplantation. The functional recovery of the grafts by this method (group 1) was determined by daily fasting blood glucose concentration and intravenous glucose tolerance test at 2 weeks after autotransplantation and compared with simple cold storage with Euro-Collins' solution (group 2) and control (no preservation) (group 3). Maintenance of normoglycemia for at least 5 days after transplantation was considered a successful preservation. The functional success rates after 72-hr preservation were 100%, 0%, and 100% for groups 1, 2, and 3, respectively. The mean K values of group 1 after 72-hr preservation was 1.78 +/- 0.42 compared with 2.05 +/- 0.32 for group 3 at 2 weeks after transplantation. Biopsies of grafts of group 2 after 72-hr preservation showed remarkable autolytic changes in exocrine and endocrine tissues. In contrast, biopsies of grafts of group 1 after 72-hr preservation showed almost normal architecture in both tissues. In addition, biopsies of 72-hr preserved grafts of group 1 at 4 weeks after after autotransplantation showed almost normal pancreas architecture with minimal fibrotic changes in the exocrine tissue. This study demonstrated the possibility of 72-hr preservation of the pancreas for transplantation.     

Mechanism of oxygenation of pancreas during preservation by a two-layer (Euro-Collins' solution/perfluorochemical) cold-storage method

To clarify the mechanism of oxygenation of the pancreas during preservation by two-layer (Euro-Collins' solution [EC]/perfluorochemical [PFC]) cold-storage method, the pancreas viability in the canine model of the pancreatic autotransplantation and tissue concentration of adenosine triphosphate were examined after 24-hr preservation by original and modified two-layer methods with respect to the position of the pancreas and oxygen bubbling into the PFC. Namely, the pancreas was in EC and on the surface of PFC with (group 1, original method) or without (group 2) oxygen bubbling into PFC. The pancreas was floated in EC with oxygen bubbling into PFC (group 3); compared with simple cold storage of the pancreas in EC (group 4); and nonpreserved pancreas (control, group 5). The preserved pancreas grafts by each method functioned immediately after transplantation and maintained normoglycemia for at least 5 days except that 1 of 5 dogs in group 4 died of a cause unrelated to the pancreas graft. The functional success rates of groups 1, 2, 3, 4, and 5 were 100%, 100%, 100%, 80%, and 100%, respectively. It was clear that mitochondrial function was well-preserved during 24-hr preservation regardless of the preservation method. In the condition that the mitochondrial function is well-preserved the tissue concentration of ATP was mostly dependent on the tissue oxygenation. The tissue concentration of ATP of group 1, 7.92 +/- 1.06 mumol/g dry weight, was significantly higher than that of nonpreserved pancreas (group 5), 4.44 +/- 0.49 mumol/g dry weight (P less than 0.01). It was apparent that the two-layer method was excellent to supply oxygen to the pancreas and maintain high ATP concentration of the pancreas during preservation. In contrast, ATP concentration of the pancreas of group 2 was 1.83 +/- 0.30 mumol/g dry weight, and there was no significant difference between group 2 and group 4, 1.19 +/- 0.33 mumol/g dry weight, thus meaning that PFC was biologically inert without oxygenation. In addition, when the pancreas was not contacted with oxygenated PFC and floated in EC (group 3) ATP concentration of the pancreas, 2.24 +/- 0.90 mumol/g dry weight, was significantly lower than group 1 (P less than 0.01), and no significant different was found as compared to groups 2 and 4. It was essential that the pancreas was contacted with oxygenated PFC to maintain high ATP tissue concentration during preservation by the two-layer method.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

The mechanism of action of the two-layer (Euro-Collins' solution/perfluorochemical) cold storage method in canine pancreas preservation

To clarify the mechanism of action of a two-layer [Euro-Collins' solution (EC)/perfluorochemical (PFC) ] cold storage method in the preservation of the pancreas, pancreatic viability and tissue concentrations of adenosine triphosphate (ATP) were examined in the canine model of pancreatic autotransplantation after preservation for 24 and 48 h by simple cold storage in EC (group 1), the two-layer, EC/PFC, method (group 2) and the two-layer, EC + 2, 4 dinitrophenol (DNP)/PFC, method (group 3). DNP is an uncoupler of oxidative phosphorylation. Maintenance of normoglycemia for at least 5 days after transplantation was considered a successful preservation. After preservation for 24 h, the functional success rates of groups 1, 2 and 3 were 100% (4/4), 100% (5/5) and 80% (4/5) respectively. One of five dogs in group 3 died of a cause unrelated to the pancreas. ATP tissue concentrations in group 2 were significantly higher than in group 1 (7.47 ± 0.47 gmol/g dry weight vs 1.41 ± 0.53 gmol/g dry weight, P < 0.01) and ATP tissue concentrations in group 3 were significantly lower than in group 2 (1.25 ± 0.37 gmol/g dry weight vs 7.47 ± 0.47 gmol/g dry weight, P < 0.01). It was apparent that ATP was not an essential factor for successful 24-hour preservation of the canine pancreas in EC because all the pancreatic grafts except one of five grafts in group 3 remained viable after preservation for 24 h, regardless of ATP tissue concentrations. On the other hand, after preservation for 48 h, the functional success rates for groups 1, 2 and 3 were 0% (0/4), 100% (4/4) and 0% (0/3) respectively. ATP tissue concentrations in group 2 were significantly higher than in group 1 (7.91 ± 1.21 gmol/g dry weight vs 1.21 ± 0.314tmol/g dry weight, P < 0.01) and ATP tissue concentrations in group 3 were significantly lower than in group 2 (0.61 ± 0.07 gmol/g dry weight vs 7.91 ± 1.21 µmol/g dry weight, P < 0.01). It was clear that preservation of the pancreas for 48 h was unsuccessful by simple cold storage in EC (group 1) and the two-layer method (group 2) made preservation for 48 h possible by increasing ATP tissue concentrations. However, DNP (group 3) inhibited the synthesis of ATP and the effectiveness of the two-layer method for 48-hour preservation of the pancreas. It was clear that maintenance of high ATP tissue concentrations during preservation was essential for the successful preservation of the canine pancreas in EC by the two-layer method for more than 48 h. We concluded that an adequate supply of oxygen to the pancreas during preservation by the two-layer method led to sufficient production of ATP to maintain cellular integrity and permitted the improvement of pancreatic preservation.     

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.     

Effect of the two-layer (University of Wisconsin solution-perfluorochemical plus O2) method of pancreas preservation on human islet isolation,as assessed by the Edmonton Isolation Protocol

BACKGROUND: Current techniques for isolating islets require that pancreata stored with University of Wisconsin solution (UW) are processed within 12 hours of cold storage. In this study, we hypothesized that the two-layer method (TLM) could extend the acceptable preservation period of pancreata before islet isolation and increase islet yields. METHODS: In the first experimental set, eight pancreata were maintained for an average of 8.3+/-1.2 hours in UW and transferred into the TLM for an additional 14.3+/-1.1 hours for a total cold ischemic period of 22.6+/-1.6 hours (prolonged TLM). Four pancreata were maintained as a control group in UW alone for a total of 21.3+/-2.0 hours. In the second experimental set, six pancreata were maintained for an average of 6.4+/-1.8 hours in UW followed by 4.8+/-0.8 hours with the TLM for a total preservation time of 11.3+/-2.5 hours (short TLM). The control organs for the short TLM group were stored for an average of 9.5+/-1.3 hours in UW alone. Islets were isolated and evaluated according to the Edmonton protocol. RESULTS: Between each group of the two experimental sets, there was no significant difference in donor-related factors (i.e. gender, age, body mass index [BMI], etc.). The TLM as compared with UW preservation resulted in a significant increase in islet yields postpurification for both short (3,353+/-394 islet equivalents [IE] vs. 2,027+/-415 IE; mean+/-SEM) and prolonged (2,404+/-503 IE vs. 514+/-180 IE) periods of storage. Furthermore, islet yields after prolonged storage with the TLM were not significantly different from organs maintained for only a short period with UW (P=0.17). The quality of islets as assessed by size, postculture viability, survival rates, insulin content, and insulin secretion were similar for each of the four groups. CONCLUSION: In comparison with UW organ preservation, exposure of pancreata to the TLM result in greater islet yields and extended preservation times.     

Comparison of histidine-tryptophan-ketoglutarate solution and University of Wisconsin solution for organ preservation in clinical pancreas transplantation

BACKGROUND: University of Wisconsin (UW) solution is currently the standard preservation solution used for abdominal organ transplantation. This study assesses the efficacy of histidine-tryptophan-ketoglutarate (HTK) compared with UW in pancreas transplantation. METHODS: Between October 2002 and August 2003, 20 pancreas transplants were performed. Patients were divided into two groups: UW (n=10) and HTK (n=10). Donor and recipient demographics were similar in both groups. The mean cold ischemia time for both groups was 11 +/-3 hr. RESULTS: There was an anticipated difference between total preservative volumes used (HTK: 4.5 +/- 1.2 L vs. UW: 3.4 +/-0.8 L; P =0.03). Patient and graft survivals to date were 100% in both groups. Serum fasting blood glucose, peak amylase, and serial amylase levels remained comparable at all intervals posttransplantation. CONCLUSIONS: Within this range of cold ischemia time, UW and HTK demonstrate similar efficacy in pancreas preservation.     

Two-layer method (UW solution/perfluorochemical plus O2) for lung preservation in rat lung transplantation

A new preservation method using perfluorochemicals (PFC) with oxygen administered continuously was developed for lung preservation and compared with traditional cold preservation methods for rat lung transplantation. Male Sprague-Dawley rats underwent orthotopic left lung transplantations of grafts preserved in lactiated Ringers solution (LR), University of Wisconsin solution (UW), Celsior solution, or a two-layer (PFC plus O2) solution for 6 hours. One hour after reperfusion, the right pulmonary artery and bronchus were clamped and 5 minutes later we recorded peak airway pressure and PaO2 level. The isograft was excised for measurement of myeloperoxidase activity, wet-to-dry ratio, and histologic examination to evaluate isograft function. The mean peak airway pressure was 29.80+/-6.72 mm H2O in the LR group, 28.80+/-5.76 mm H2O in the UW group, 33.60+/-5.17 mm H2O in the Celsior group, and 32.40+/-2.60 in the two-layer group. The mean PaO2 level was 99.78+/-76.09 mm Hg in the LR group, 87.84+/-33.58 mm Hg in the UW group, 104.50+/-72.93 mm Hg in the Celsior group, and 62.08+/-31.34 mm Hg in PFC and UW solution plus O2 group (two layers). The mean net myeloperoxidase activity OD level was 0.110+/-0.104 in the LR group, 0.392+/-0.328 in the UW group, 0.351+/-0.620 in the Celsior group, and 0.532+/-0.616 in the two-layer group. The mean wet-to-dry ratio was 7.47+/-1.60 in the LR group, 6.56+/-0.62 in the UW group, 7.54+/-2.19 in the Celsior group, and 5.32+/-2.20 in the two-layer group. The differences between groups in these parameters were not significant. Upon histologic examination, more inflammatory cell aggregates were seen in the two-layer group, less in the LR and the Celsior groups. The function of the lung graft after 6 hours of storage was not better using this two-layer method for preservation than traditional preservation methods in rat lung transplantation. Histologic examination revealed more inflammatory cell aggregates in the lung graft preserved using a two-layer method.     

Characterization of islet-infiltrating immunocytes after pancreas preservation by two-layer (UW/perfluorochemical) cold storage method

Clinical islet transplantation offers the prospect of good blood glycemic control without major surgical risks. Nevertheless, long-term function of the transplanted islets is seldom appreciated because rejection is followed by the graft failure. Although it has been implicated that islets have high immunogenicity, characterization of the islet-infiltrating immunocytes, such as leukocytes and macrophages, has not been extensively studied. Rat islets were isolated by the collagenase digestion method and separated by handpicking under the microscope. The islets were further dispersed into individual cells for flow cytometric analysis. Monoclonal antibodies directed toward T cells, B cells, and macrophages as well as ICAM-1, and MHC class I and II were used to enumerate cells. Pancreatic islets contained 6.3 +/- 2.9% immunocytes; T cells (39.6 +/- 4.2%), B cells (44.7 +/- 5.8%), and macrophages (1.7 +/- 0.6%). MHC class I was expressed on 85.6 +/- 2.8%, MHC class II on 36.8 +/- 2.9%, and ICAM-1 on 39.9 +/- 7.0%. The results of islets from preserved pancreas also showed the same tendency. As these islet-infiltrating immunocytes within the grafts may contribute to the rejection, one potential strategy to prevent early graft loss might start to eliminate or inactivate the islet-infiltrating immunocytes.     

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.