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

doi:10.1016/j.transproceed.2004.12.058

Transplantation Proceedings

Volume 37, Issue 1, January–February 2005, Pages 229-230

https://doi.org/10.1016/j.transproceed.2004.12.058 Get rights and content

Abstract

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.

Section snippets

Materials and methods

Resected pancreases were intraductally flushed with cold UW. Subsequently, organs were distended with 4.4 PZ-U/mL per gram of UW-dissolved collagenase NB-8 (Serva) supplemented with neutral protease adjusted according to preliminary experiments to 1.1 DMC-U/mL per gram for freshly procured pancreases (n = 6) or 0.8 DMC-U/mL per gram for pancreases stored by TLM (n = 10) or OLM (n = 8).

For TLM storage, pancreases were fixed at the interface between preoxygenated PFC and UW.⁵ Pancreases preserved

Results

As shown in Table 1, prolonged cold ischemia significantly decreased postpurification islet yield in TLM-preserved, but not OLM-stored, pancreases compared to unstored organs. Quality control revealed consistently high viability as determined by trypan-blue exclusion (>95%) or formazan production (Table 1). The intracellular ATP content, a significant predictor for islet graft function, was maintained during TLM and OLM storage on the level of freshly processed islets (Table 1). Although the

Discussion

The present study demonstrates that PFC alone can be used for successful pig pancreas preservation, suggesting that the presence of UW as second layer is not essential during oxygenation of a PFC-immersed pancreas. The easy handling of OLM could facilitate the application of PFC as regularly used preservation solution for pancreatic tissue.

References (6)

T. Tsujimura et al.
Human islet transplantation from pancreases with prolonged cold ischemia using additional preservation by the two-layer (UW solution/perfluorochemical) cold-storage method
Transplantation
(2002)
S. Matsumoto et al.
Efficacy of the oxygen-charged static two-layer method for short-term pancreas preservation and islet isolation from nonhuman primate and human pancreata
Cell Transplant
(2002)
Y. Kuroda et al.
Excellence of perfluorochemical with simple oxygen bubbling as a preservation medium for simple cold storage of canine pancreas
Transplantation
(1990)

There are more references available in the full text version of this article.

Cited by (14)

Pancreas and islet preservation
2019, Transplantation, Bioengineering, and Regeneration of the Endocrine Pancreas: Volume 1
Pancreatic islets contain endocrine beta and alpha cells that produce insulin and glucagon, respectively, two hormones key to regulating blood sugar. Recent technological advances in human islet transplantation (ITx) have triggered renewed interest in whole organ and islet preservation methods aimed at protecting the structural and functional integrity of islets during the process of islet manufacturing, from the point of organ procurement to the point of islet infusion into the recipient. Islet isolation is a complex, multistep and laborious procedure influenced by numerous factors that require highly trained and skillful personnel. Due to a high islet attrition rate during isolation and limited islet viability after purification, most current ITx protocols require islets from more than one pancreas. The need for multiple donors represents a major economical and clinical barrier to expand the number of diabetic patients that could benefit from ITx. Ischemia and hypoxia have been identified as prominent factors contributing to islet losses during isolation and after ITx. Delivery of appropriate amounts of oxygen and other preservation measures must be implemented at the organ, tissue and cellular level to improve islet viability and preserve beta and alpha cell function. The current chapter reviews the mechanistic impact of ischemia and hypoxia on islet function and viability, as well as current methods of islet preservation prior, during and after islet isolation, purification, culture, and distribution.
Mimicking oxygen delivery and waste removal functions of blood
2017, Advanced Drug Delivery Reviews
In addition to immunological and wound healing cell and platelet delivery, ion stasis and nutrient supply, blood delivers oxygen to cells and tissues and removes metabolic wastes. For decades researchers have been trying to develop approaches that mimic these two immediately vital functions of blood. Oxygen is crucial for the long-term survival of tissues and cells in vertebrates. Hypoxia (oxygen deficiency) and even at times anoxia (absence of oxygen) can occur during organ preservation, organ and cell transplantation, wound healing, in tumors and engineering of tissues. Different approaches have been developed to deliver oxygen to tissues and cells, including hyperbaric oxygen therapy (HBOT), normobaric hyperoxia therapy (NBOT), using biochemical reactions and electrolysis, employing liquids with high oxygen solubility, administering hemoglobin, myoglobin and red blood cells (RBCs), introducing oxygen-generating agents, using oxygen-carrying microparticles, persufflation, and peritoneal oxygenation. Metabolic waste accumulation is another issue in biological systems when blood flow is insufficient. Metabolic wastes change the microenvironment of cells and tissues, influence the metabolic activities of cells, and ultimately cause cell death. This review examines advances in blood mimicking systems in the field of biomedical engineering in terms of oxygen delivery and metabolic waste removal.
Preoxygenation of Different Preservation Solutions for Porcine Pancreas Preservation
2010, Transplantation Proceedings
Organ preservation quality impacts porcine islet cell isolation and transplantation success. Among several preservation methods, the two-layer method is promising, but technically demanding and fails to deliver sufficient oxygen. The use of hyperbaric oxygenation may be an easier, more effective method to supply high partial pressure of oxygen (pO₂) for organ storage. Therefore, the aim of this study was to test the capability of preoxygenation of various preservation solutions with HBO to maintain high pO₂ levels.
University of Wisconsin (UW), Custodiol, Perfadex, or Celsior solutions were preoxygenated in a pressure chamber. NaCl served as the control. pO₂ levels were measured at defined times. The oxygen storage capability was evaluated by leaving the storage bottles open for 2 minutes.
It was feasible to preoxygenate preservation solutions. The best solution to maintain high pO₂ tensions was Perfadex, followed by Celsior, and UW.
The greater the amount of oxygen in the preservation solution, the more oxygen can be delivered to the preserved pancreas. Further studies on the influence of preoxygenated preservation solutions on the porcine pancreas are warranted to improve organ quality, porcine islet cell isolation, and transplantation success.
Ductal Injection of University of Wisconsin Solution Prior to Pancreas Preservation Prevents Oxidative Cell Damage
2009, Transplantation Proceedings
Citation Excerpt :
A recent study11 showed that ductal injection of preservation solution increased ATP levels in pancreatic tissue and therefore augmented cell viability and functionality. Several studies18–20 have shown a positive effect of intraductally administered UW solution on pancreatic organ quality after organ procurement. Therefore, we investigated the effect of different volumes of UW solution infused via the pancreatic duct on the occurrence of apoptosis, high–cellular energy phosphate status, as well as generation of oxidative stress–induced DNA, protein, and lipid damage as quantified by measurements of MDA and CP in the PP.
Several studies have been carried out investigating different preservation methods and preservation solutions for the pancreata of various species. Attention has to be drawn to the extreme vulnerability of porcine pancreata (PP) to oxidative stress due to the lack of endogenous antioxidants. This study sought to evaluate the influence of cannulation and infusion of different volumes of University of Wisconsin (UW) solution immediately after organ retrieval on PP organ quality.
PP from 24 slaughterhouse pigs were harvested with immediate cannulation of the pancreatic duct for infusion of 10 mL, 20 mL, 50 mL, or 100 mL UW solution. The organs were stored in cold UW solution. Control organs were only stored in UW. After 6 hours of cold ischemia, tissue and supernate samples were analyzed for markers of oxidative cell damage, adenosine triphosphate (ATP) levels, and occurrence of apoptosis.
The fewest apoptotic cells were detected in the PP infused with 50 mL UW via the pancreatic duct (PP 50) as compared with all other groups. Oxidative cell damage was lowest and ATP levels were highest in the PP 50 group.
Because PP 50 showed significantly better results when compared with all other groups, we suggest that infusion of 50 mL UW via the pancreatic duct immediately after organ retrieval may be useful to minimize oxidative cell damage and cell death in PP.
Current status of islet transplantation
2007, Cirugia Espanola
El trasplante de islotes pancreáticos mejora día a día sus resultados clínicos gracias a numerosas mejoras en el proceso desarrolladas en los últimos años. Tanto los nuevos protocolos de inmunosupresión como la selección de los donantes y receptores, así como un especial cuidado en la obtención, preservación y procesamiento de los páncreas, han hecho posible conseguir controles glucémicos prolongados. Uno de los objetivos principales de esta revisión es presentar la evolución de los resultados en humanos a medida que estos cambios han ido introduciéndose. Asimismo, se revisa el empleo de nuevas fuentes de islotes, como son el donante vivo y el donante a corazón parado, junto con los nuevos hallazgos en el conocimiento de los mecanismos de rechazo y las nuevas opciones terapéuticas desarrolladas para prevenirlo.
Due to the numerous advances in islet transplantation in the last few years, clinical outcomes following this procedure are continually improving. Novel immunosuppression protocols, improved donor and recipient selection, and careful attention to the process of organ extraction, preservation and islet isolation have contributed to long-term success. The present article reviews the results of clinical islet transplantation and their relationship with the different advances introduced. The use of new islet sources such as living and non heart beating donors, as well as recent advances in our knowledge of the mechanisms of rejection and its prevention, are also reviewed.
Pancreas oxygenation is limited during preservation with the two-layer method
2005, Transplantation Proceedings
The two-layer method (TLM) for pancreas preservation reportedly improves islet yield and transplantation outcome relative to previous methods. Increased ATP concentrations support the hypothesis that these improvements are related to better oxygenation from the perfluorocarbon solution. However, there are limited direct measurements of oxygen partial pressure, (pO₂) in pancreata preserved with the TLM. Theory predicts that only a small fraction of a human pancreas can be oxygenated externally. In this report we examine pancreas oxygenation with the TLM using theory and direct pO₂ measurements.
pO₂ profiles in cylindrical pancreata were calculated at various temperatures with a diffusion-reaction model. The pO₂ was measured using fiber optic sensors in the core of porcine pancreatic tissue preserved with the TLM in media saturated with 100% oxygen.
The model predicts that at 8°C, even in the absence of an external pO₂ gradient, oxygen penetration depth is about 1 mm and insensitive to pancreas diameter, while the oxygenated volume fraction is about 15% for a 2.5-cm-diameter pancreas. Experimental measurements verified that pO₂ is virtually zero in the core of a 1-cm-thick pancreatic piece preserved with the TLM. Penetration of solution around the sensor may be responsible for the observed lag and for the previously reported nonzero pO₂ measurements. Reoxygenation of heat-treated tissue took several hours.
The TLM can oxygenate only a small volume fraction of a human pancreas. Pancreas oxygenation through the native vasculature should be explored to further improve yield of viable islets.

View all citing articles on Scopus

: Supported in part by the Juvenile Diabetes Research Foundation International and the NIDDK (DK 56962-2).

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

Abstract

Background

Methods

Results

Conclusions

Section snippets

Materials and methods

Results

Discussion

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

Transplantation

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

Cell Transplant

Excellence of perfluorochemical with simple oxygen bubbling as a preservation medium for simple cold storage of canine pancreas

Transplantation