Two-layer Cold Storage Method Research Articles

Pancreatic Islets from Non-Heart-Beating Donor Pig: Two-layer Preservation Method in an in Vitro Porcine Model

Pancreata from non-heart beating donors could represent an unlimited source of islets if their cell viability can be efficiently preserved during the time necessary to process the organs by the use of a better solution of preservation compared to the classic University of Wisconsin solution. The aim of this study was to determine whether it is possible to obtain functioning "alive islets" from non-heart-beating donors by comparing, on a porcine model, the classic "UW ice-store" method with a two-layer cold storage method (TLM) using oxygenated Perfluorocarbons (PFC) and UW. Whole pancreata were harvested from 20 NHBDs female pigs with similar characteristics and preserved for 4 h in UW solution (n = 10) or TLM (UW/PFC) solution (n=10). The isolated islets were then evaluated for number, viability, purity, and insulin secretion, also estimated after 8 weeks of cryopreservation. The total number of islets obtained from isolation, and their function assayed by the insulin stimulation index, before and after cryopreservation, showed a higher value in the TLM group. No significative differences in terms of purity and viability before and after cryopreservation were found when comparing the two groups. TLM solution for NHBDs porcine pancreata with cold ischemia time lower than 4 h offers significant advantages over UW solution storage, thereby increasing the isolation yield and isolation success rate of the pancreatic porcine islets.

Two-layer cold storage method for pancreas and islet cell transplantation

The two-layer cold storage method (TLM) was first reported in 1988, consisting of a perfluorochemical (PFC) and initially Euro-Collins' solution, which was later replaced by University of Wisconsin solution (UW). PFC is a biologically inert liquid and acts as an oxygen-supplying agent. A pancreas preserved using the TLM is oxygenated through the PFC and substrates are supplied by the UW solution. This allows the pancreas preserved using the TLM to generate adenosine triphosphate during storage, prolonging the preservation time. In a canine model, the TLM was shown to repair and resuscitate warm ischemically damaged pancreata during preservation, improve pancreas graft survival after transplantation, and also improve the islet yield after isolation. Clinical trials using the TLM in pancreas preservation before whole-pancreas transplantation and islet isolation have shown promising outcomes. We describe the role of the TLM in pancreas and islet transplantation.

The Start of an Islet Transplantation Program in Japan

In Japan, pancreas donation had become possible from cadaveric donor sources, both heart-beating or non–heart-beating (NHB). Pancreas allografts have been distributed in the organ allocation system of the Japan Organ Transplant Network. Meanwhile, islet transplantation has been categorized as a tissue transplantation; it is free from legal restraints. Thus, pancreata for islet isolation must be obtained from NHB donors. Herein we report the starting program and preliminary results of islet transplantation in Japan. Selection and listing criteria for transplantation include regional priority, ABO blood type, previous islet transplant status with insulin independence, and a longer waiting time. Five institutes in Japan (Fukushima, Chiba, Kyoto, Kobe, and Fukuoka) are prepared to start programs. A two-layer cold storage method using perfluorocarbons and UW solution is recommended for pancreas preservation. Islet isolation and purification procedures are performed according to institute-specific protocol. Immunosuppression is based on sirolimus/tacrolimus combined with basiliximab induction. Two or three consecutive infusions of >5000 IE/kg are planned for each recipient until achieving insulin independence. Twenty-seven isolations and 14 transplants were performed in eight non–insulin-dependent diabetes mellitus (IDDM) recipients. Almost all (26 of 27) were NHB donors. All recipients are free from hypoglycemic episode after transplantation. One of these recipients is insulin independent; the others are currently on minimal doses of exogenous insulin. The feasibility of islet transplantation using NHB donors was confirmed using a two-layer cold storage method and a steroid-free immunosuppressive protocol, with a high rate of graft function.

Superiority of the Two-Layer Method Before Islet Isolation Confirmed by In Vivo Viability Assessment

Although the outcome of islet transplantation has improved, there remains a major obstacle in isolating viable islets from prolonged preserved pancreas. We previously reported that the two-layer cold storage method (TLM) improved the yield and in vitro function. In this study, we performed in vivo accurate functional analyses of islets from TLM-preserved pancreas and investigated pancreatic duct cell viability, which may critically affect islet isolation. Rat islets isolated from fresh pancreas (group 1), after preservation in the University of Wisconsin (UW) solution (group 2) or by the TLM (group 3), were examined by assessing islet yields, stimulation indices, cure rates after transplantation to diabetic nude mice, and trypan blue uptake of pancreatic duct cells. TLM significantly improved the islet yield compared with UW cold storage. The cure rates after transplantation were 100%, 0%, and 80% for groups 1, 2, and 3, respectively. This indicates that islet viability was well maintained even after 24 hr of TLM preservation. The percentages of nonviable duct cells were 4.1%+/-1.9%, 48.3%+/-8.0%, and 26.1%+/-21.4% in groups 1, 2, and 3, respectively, showing that the TLM was superior to UW as seen by this duct cell viability assessment. The TLM used for pancreas preservation before islet isolation results in excellent islet function in addition to improved islet yield comparable to freshly isolated islets. The underlying mechanism may be duct cell viability maintained during TLM preservation. Therefore the TLM is an excellent preservation technique for isolating sufficient numbers of highly viable islets.

Ultrastructural analyses of pancreatic grafts preserved by the two-layer cold-storage method and by simple cold storage in University of Wisconsin solution.

The two-layer cold storage method (TLM) using University of Wisconsin (UW) solution supplies sufficient oxygen to pancreatic grafts during preservation and extends pancreas preservation time to up to 96 h in the canine model. Simple cold storage in UW (UWM) on the other hand, preserves canine pancreas grafts for up to 72 h by preventing cell swelling, mainly because of its high osmotic pressure. The aim of this study is to analyze morphologically dog pancreatic grafts preserved by these two methods with their different mechanisms. Immediately after preservation of canine pancreata by TLM for 72 h and 96 h (group 1 and group 3, respectively), and by UWM for 72 h and 96 h (group 2 and group 4, respectively), tissue ATP levels were determined using high-performance liquid chromatography (HPLC), and detailed morphological analyses of intragraft components were performed using light- and electron microscopy. The mean areas of one mitochondrion and rough endoplasmic reticulum (RER) vacuolization were calculated by computer-graphic analyses using NIH image 1.62 f soft. The tissue ATP levels were significantly higher in groups 1 and 3 than groups 2 and 4 ( P < 0.05). Light microscopy demonstrated no marked difference among the 4 groups. By electron microscopy however, mitochondrial swelling and RER vacuolization were observed in acinar cells to various extents in the 4 groups. They were significantly more evident in group 2 than group 1 ( P < 0.05), and in group 4 than group 3 ( P < 0.05). In conclusion, TLM demonstrated excellent protection of intracellular organelles, mitochondria, and RER, up to 72-96 h. Well-maintained graft ATP levels in TLM groups may result in maintaining the integrity of intracellular organelle membranes as well as cellular membranes.

Objective and rapid assessment of pancreas graft viability using 31P-nuclear magnetic resonance spectroscopy combined with two- layer cold storage method.

With the current shortage of donors, there is a critical need to optimally use "less-than-ideal" donors for pancreas transplantation. Objective and rapid means for assessing graft viability and suitability for transplantation are mandatory. This study examined the possibility of graft viability assessment and posttransplant outcome prediction using (31)P-nuclear magnetic resonance (NMR) spectroscopy combined with the two-layer cold-storage method (TLM). Canine pancreas grafts were preserved with TLM for 24 hours after 0, 60, or 120 minutes of warm ischemia (groups 1, 2, or 3, respectively). After preservation, we determined intragraft phosphate metabolites noninvasively using (31)P-NMR spectroscopy. Time required for this assessment was 5 minutes. Because our previous studies demonstrated that all grafts in groups 1 and 2 were successfully transplanted (the viable group), whereas all in group 3 failed to survive (the nonviable group), the possibility of posttransplant outcome prediction was examined on the basis of the comparison between these two groups. The ratios of inorganic phosphate/gamma-adenosine triphosphate (Pi/gamma-ATP) and Pi/beta-ATP reflected the extent of graft damage, and the differences were statistically significant among groups 1, 2, and 3. On the basis of analyses of receiver operator characteristic curves, the optimum cutoff levels between the viable and nonviable groups were 1.6 and 2.2 for Pi/gammaATP and Pi/betaATP, respectively. The accuracy rates of these ratios were both 83%. (31)P-NMR spectroscopy combined with TLM preservation could provide an objective, rapid, and possibly noninvasive means to assess pancreas graft viability and to determine suitability of damaged pancreata for organ transplantation.

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.

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 °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.

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.

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

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. 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). 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. 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.

Outcomes in clinical pancreas transplantation with the two-layer cold storage method versus simple storage in University of Wisconsin solution

Outcomes in clinical pancreas transplantation with the two-layer cold storage method versus simple storage in University of Wisconsin solution

Ultrastructural analyses of pancreatic grafts preserved by the two-layer cold-storage method and by simple cold storage in University of Wisconsin solution

Ultrastructural analyses of pancreatic grafts preserved by the two-layer cold-storage method and by simple cold storage in University of Wisconsin solution

Successful 24-h Preservation of Canine Small Bowel Using the Cavitary Two-layer (University of Wisconsin Solution/Perfluorochemical) Cold Storage Method

We previously developed the two-layer cold storage method (TLM), which allows sufficient oxygen delivery to the canine pancreas during preservation, and successfully achieved 96-h preservation. In this study, we applied a modified TLM (cavitary TLM) to small bowel preservation in a canine heterotopic transplant model. Using simple storage in University of Wisconsin solution (UWM, group 1, n = 12) or cavitary TLM (group 2, n = 8), 40 cm segments of the jejunum were preserved for 24 h at 4 degrees C. The nonpreservation group served as the control (group 3, n = 8). The grafts were implanted heterotopically as a Thiry-Vella loop. Eleven of 12 dogs in group 1 died within 3 days post-transplant as a result of graft intraluminal hemorrhage, while all dogs in groups 2 and 3 survived until day 7. Histological analyses showed almost normal structures of the graft mucosa in groups 2 and 3 at day 7. Results from maltose and acetaminophen absorption tests in group 2 were comparable to those in group 3. Only one survivor in group 1 showed distinct graft mucosal damage, confirmed by histological and functional analyses. In our transplant model, the canine small bowel was successfully preserved by cavitary TLM for at least 24 h, while this preservation time was beyond the limit with UWM.

Human Pancreas Preservation Prior to Islet Isolation

Islet transplantation has become a feasible treatment option for patients with type 1 diabetes mellitus after improvements regarding less diabetogenic immunosuppression and preparation of sufficient quantities of highly viable islets for transplantation. While the current techniques of multiorgan harvesting and transportation of organs to distant hospitals often result in 12 h or more of cold storage time before transplantation, University of Wisconsin (UW) solution has proven to be effective in pancreas preservation, leading to successful clinical whole pancreas transplantation, and human pancreatic grafts can be preserved by UW solution for periods exceeding 24 h. In the case of clinical islet transplantation, however, prolonged cold ischemia in UW solution before islet isolation significantly reduced recovery of viable islets, and it is still difficult to recover sufficient numbers of islets from a single cadaveric donor pancreas to achieve insulin independence after transplantation. Therefore, preservation of the pancreas has been an important factor in the subsequent success of clinical islet transplantation. We review pancreas preservation prior to islet isolation and discuss a two-layer (UW solution/perfluorochemical) cold storage method of the human pancreas as a potential method for pancreas rescue.

Optimal temperature in pancreas preservation by the two-layer cold storage method before islet isolation

COLD organ storage is based on the effect of hypothermia, which is basically used to reduce the metabolism at low temperatures. However, the low temperature itself may be deleterious to the cell membrane during organ preservation. The two-layer cold storage method (TL), which employs UW solution (UW) and perfluorochemical (PFC), has been demonstrated to be superior to simple UW storage and to be feasible for pancreas preservation before islet isolation using a canine model. It has been clarified that a pancreas graft is directly oxygenated with PFC during preservation by TL and maintains high tissue ATP level, which keeps cellular integrity. Because ATP synthesis in TL is an enzymatic reaction, it seems likely that increasing temperature makes ATP synthesis faster and decreases the damage of the cell membrane due to the low temperature during storage by TL. The aim of this study is to determine the optimal temperature for TL in pancreas preservation before islet isolation.

Successful pancreas preservation before islet isolation by the simplified two-layer cold storage method

Successful pancreas preservation before islet isolation by the simplified two-layer cold storage method

The effect of the two-layer cold storage method on islet isolation from ischemically damaged pancreas.

Previous studies have shown that the two-layer cold storage method (TL) resuscitates the function of warm ischemically (WI) damaged pancreas when transplanted as a segmental graft in a dog model. In this study, we evaluated the effect of TL on yield and viability of islets isolated from WI damaged pancreas. In group 1 (n = 10), dog pancreases were processed into islets immediately after total pancreatectomy. In group 2 (n = 5), excised pancreases were left in the abdominal cavity for 60 minutes as WI then processed. In group 3 (n = 5), WI damaged pancreases were preserved in TL at 4 degrees C for 24 hours before islet isolation. Islets were isolated by the digestion methods described by Ricordi, et al. And purified on Euro-Ficoll discontinuous gradients. We compared three groups in terms of islet yields before and after purification, expressed as islet equivalents (IEQ, diameter standardizing to 150 microns) per pancreas weight (IEQ/g pancreas). Viability of islets was assessed with in vitro static incubation test. Islet yields after purification were 2500 +/- 1100 (mean +/- SD) in group 1, 1200 +/- 800 in group 2, (p = 0.04 vs. group 1, p = 0.03 vs. group 3), and 2200 +/- 300 in group 3 (p = 0.38 vs. group 1). Static incubation test revealed that islets in group 1 and 3 showed sufficient insulin release in response to glucose stimuli. These data clearly demonstrated that TL can regain islet yield from WI damaged pancreas up to similar level to that from fresh pancreas. Viability of those islets was well maintained. Thus temporary TL applied before islet isolation may allow to use islets from non-heartbeating cadavers in the clinical setting.

THE POSSIBILITY OF ISLET ISOLATION FROM PANCREAS HARVESTED FROM NON-HEART BEATING CADAVER

342 Introduction: Pancreas from non-heartbeating cadaver can be available in pancreas islet transplantation if its damage from warm ischamia (WI) could be reduced by a suitable method. The two-layer cold storage method (TL), which utilizes perfluorochemical and UW solution, has previously demonstrated to have unique characteristics of supplying oxygen to the graft, effectively maintaining the viability of whole pancreas grafts. In this study we evaluated the effect of TL on islet yield and viability of WI damaged pancreas. Method: Dog pancreases were divided into following five groups (group 1; n=10, groups 2∼5; n=5). In group 1, pancreases were processed immediately after total pancreatectomy. In groups 2 (4), pancreases were left in the abdominal cavity for 60 (90) minutes as WI, then processed into islets. In groups 3 (5), pancreases were left in the abdominal cavity for 60 (90) minutes as WI followed by TL for 24 hours at 4°C, then processed into islets. Islet isolation was performed by the automated collagenase digestion method of Ricordi, purified on Euro-Ficoll discontinuous gradients. We compared five groups in terms of islet yield, expressed as islet equivalents (IEQ, diameter standardizing to 150 μm) per pancreas weight (IEQ/g pancreas). Results: Islet yield was 2700 ± 1200 (mean±SD) in group 1, 1500 ± 700 in group 2, (p=0.03 vs. group 1, p=0.02 vs. group 3), and 2600 ± 600 in group 3 (p=0.80 vs. group 1). In group 4 and 5, almost no islets were obtained after purification. In static incubation islets in group 1 and 3 showed sufficient insulin release in response to glucose stimuli and this means that viability of islets in group 1 and 3 was maintained. Conclusions: These data clearly demonstrates that TL following WI can regain islet yield and viability almost at the same level as those of fresh pancreas. So it could be possible to isolate islets from pancreas harvested from non-heartbeating cadaver by TL.

Successful 14-day preservation of rat skin using a two-layer cold-storage method

Skin-graft preservation for the purpose of delayed autografting to burn patients is a basic tool of plastic and burn surgery. This study was undertaken to evaluate the efficacy of the two-layer (physiological saline/perfluorochemical (PFC)) cold-storage method for skin preservation. In Wistar rats, a full-thickness skin graft, 3 cm in diameter, was harvested from the back, and separated from the dermis to obtain the epidermal sheet. This epidermal sheet was preserved at 4°C using physiological saline (group 1, n = 15), physiological saline bubbled with a 95 per cent oxygen, 5 per cent carbon dioxide mixture (group 2, n = 15) or a two-layer method with bubbling with 95 per cent oxygen and 5 per cent carbon dioxide mixture (group 3, n = 15). The epidermal skin sheets were then autotransplanted to the backs of the original donor rats 7, 10 and 14 days after the start of preservation. The success rate of the skin autotransplantation was determined by measuring the ratio of viable area to total graft area 7 days after grafting. In group 1, the success rates after 7, 10 and 14 days' perservation were 56.4, 47.4 and 0.09 per cent, respectively. In group 2, the corresponding success rates were 62.0, 48.4 and 0.8 per cent respectively, showing no improvement with oxygenated saline. In clear contrast, the success rates were significantly improved by the two-layer method, the values being 92.1, 87.9 and 77.6 per cent after 7, 10 and 14 days of perservation, respectively ( P < 0.01 vs. groups 1 and 2 for all preservation durations). Based on these results, we concluded that the two-layer method is useful for not only improving the quality of the skin graft but also extending the preservation time of the skin graft up to 14 days.

Small bowel preservation using a cavitary two-layer (University of Wisconsin solution/perfluorochemical) cold storage method.

Preservation of the small bowel by a cavitary two-layer (University of Wisconsin solution [UW]/perfluorochemical) cold storage method was evaluated in the heterotopic rat segmental small bowel transplant model. Simple cold storage UW was effective only for 24-hr preservation. However, the cavitary two-layer method using UW made it possible to prolong preservation time up to 48 hr. Furthermore, without oxygen bubbling, the cavitary two-layer method was not effective for 48-hr preservation. Histologic studies of grafts preserved by the cavitary two-layer method for 48 hr at 7 days after transplantation showed normal architecture of the intestinal mucosa. This study demonstrates that the oxygenation of the small bowel during preservation by the cavitary two-layer method using UW makes it possible to extend preservation time up to 48 hr in the heterotopic rat segmental small bowel transplant model.