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Lau H, Khosrawipour T, Li S, Alexander M, Frelkiewicz P, Labbé MK, Stieglitz S, Lakey JRT, Kielan W, Khosrawipour V. Exploring Insulin Production Following Alveolar Islet Transplantation (AIT). Int J Mol Sci 2021; 22:ijms221910185. [PMID: 34638521 PMCID: PMC8508311 DOI: 10.3390/ijms221910185] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2021] [Revised: 09/11/2021] [Accepted: 09/16/2021] [Indexed: 11/29/2022] Open
Abstract
Recent studies have demonstrated the feasibility of islet implantation into the alveoli. However, until today, there are no data on islet behavior and morphology at their transplant site. This study is the first to investigate islet distribution as well insulin production at the implant site. Using an ex vivo postmortem swine model, porcine pancreatic islets were isolated and aerosolized into the lung using an endoscopic spray-catheter. Lung tissue was explanted and bronchial airways were surgically isolated and connected to a perfusor. Correct implantation was confirmed via histology. The purpose of using this new lung perfusion model was to measure static as well as dynamic insulin excretions following glucose stimulation. Alveolar islet implantation was confirmed after aerosolization. Over 82% of islets were correctly implanted into the intra-alveolar space. The medium contact area to the alveolar surface was estimated at 60 +/− 3% of the total islet surface. The new constructed lung perfusion model was technically feasible. Following static glucose stimulation, insulin secretion was detected, and dynamic glucose stimulation revealed a biphasic insulin secretion capacity during perfusion. Our data indicate that islets secrete insulin following implantation into the alveoli and display an adapted response to dynamic changes in glucose. These preliminary results are encouraging and mark a first step toward endoscopically assisted islet implantation in the lung.
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Affiliation(s)
- Hien Lau
- Department of Surgery, University of California, Irvine (UCI), Orange, CA 92868, USA; (M.A.); (J.R.T.L.); (V.K.)
- Correspondence: (H.L.); (T.K.)
| | - Tanja Khosrawipour
- Department of Surgery (A), University-Hospital Düsseldorf, Heinrich-Heine University, Moorenstrasse 5, D-40225 Duesseldorf, Germany
- Correspondence: (H.L.); (T.K.)
| | - Shiri Li
- Department of Surgery, Weill Medical College of Cornell University, New York, NY 10065, USA;
| | - Michael Alexander
- Department of Surgery, University of California, Irvine (UCI), Orange, CA 92868, USA; (M.A.); (J.R.T.L.); (V.K.)
| | - Piotr Frelkiewicz
- Center for Experimental Diagnostics and Biomedical Innovations, Wroclaw University of Environmental and Life Sciences, 50-375 Wroclaw, Poland;
| | - Maya Karine Labbé
- School of Dentistry, Wroclaw Medical University, 50-367 Wroclaw, Poland;
| | - Sven Stieglitz
- Department Pulmonary Medicine, Petrus-Hospital Wuppertal, University of Witten-Herdecke, D-42283 Wuppertal, Germany;
| | - Jonathan Robert Todd Lakey
- Department of Surgery, University of California, Irvine (UCI), Orange, CA 92868, USA; (M.A.); (J.R.T.L.); (V.K.)
| | - Wojciech Kielan
- 2nd Department of General Surgery and Surgical Oncology, Wroclaw Medical University, 50-556 Wroclaw, Poland;
| | - Veria Khosrawipour
- Department of Surgery, University of California, Irvine (UCI), Orange, CA 92868, USA; (M.A.); (J.R.T.L.); (V.K.)
- 2nd Department of General Surgery and Surgical Oncology, Wroclaw Medical University, 50-556 Wroclaw, Poland;
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Corrales N, Park S, Lau H, Xu I, Luong C, Rodriguez S, Mönch J, Alexander M, Lakey JR. Comparison of Islet Characterization from Use of Standard Crude Collagenase to GMP-Grade Collagenase Enzyme Blends in Preweaned Porcine Islet Isolations. Cell Transplant 2021; 29:963689720977835. [PMID: 33267618 PMCID: PMC7873766 DOI: 10.1177/0963689720977835] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
For the advancement of porcine xenotransplantation for clinical use in type 1 diabetes mellitus, the concerns of a sustainable and safe digestion enzyme blend must be overcome. Incorporating good manufacturing practices (GMP) can facilitate this through utilizing GMP-grade enzymes. In conjunction, still taking into account the cost-effectiveness, a wide concern. We evaluated how GMP-grade enzyme blends impact our piglet islets and their long-term effects. Preweaned porcine islets (PPIs) were isolated from 8- to 10-day-old pigs. Digestion enzyme blends, collagenase type V (Type V), collagenase AF-1 GMP-grade with collagenase NB 6 GMP-grade (AF-1 and NB 6), and collagenase AF-1 GMP-grade with collagenase neutral protease AF GMP-grade (AF-1 and NP AF) were compared. Islet quality control assessments, islet yield, viability, and function, were performed on days 3 and 7, and cell content was performed on day 7. GMP-grade AF-1 and NB 6 (17,209 ± 2,730 islet equivalent per gram of pancreatic tissue [IE/g] on day 3, 9,001 ± 1,034 IE/g on day 7) and AF-1 and NP AF (17,214 ± 3,901 IE/g on day 3, 8,833 ± 2,398 IE/g on day 7) showed a significant increase in islet yield compared to Type V (4,618 ± 1,240 IE/g on day 3, 1,923 ± 704 IE/g on day 7). Islet size, viability, and function showed comparable results in all enzyme blends. There was no significant difference in islet cellular content between enzyme blends. This study demonstrated a comparison of GMP-grade collagenase enzyme blends and a standard crude collagenase enzyme in preweaned-aged porcine, a novel topic in this age. GMP-grade enzyme blends of AF-1 and NB 6 and AF-1 and NP AF resulted in substantially higher yields and as effective PPIs compared to Type V. In the long run, considering costs, integrity, and sustainability, GMP-grade enzyme blends are more favorable for clinical application due to high reproducibility in comparison to undefined manufacturing processes of standard enzymes.
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Affiliation(s)
- Nicole Corrales
- Department of Surgery, University of California Irvine, Orange, CA, USA
| | - Soomin Park
- Department of Surgery, University of California Irvine, Orange, CA, USA
| | - Hien Lau
- Department of Surgery, University of California Irvine, Orange, CA, USA
| | - Ivana Xu
- Department of Surgery, University of California Irvine, Orange, CA, USA
| | - Colleen Luong
- Department of Surgery, University of California Irvine, Orange, CA, USA
| | - Samuel Rodriguez
- Department of Surgery, University of California Irvine, Orange, CA, USA
| | - Johanna Mönch
- Nordmark Arzneimittel GmbH & Co. KG, Uetersen, Germany
| | - Michael Alexander
- Department of Surgery, University of California Irvine, Orange, CA, USA
| | - Jonathan Rt Lakey
- Department of Surgery, University of California Irvine, Orange, CA, USA.,Department of Biomedical Engineering, University of California Irvine, CA, USA
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Lau H, Corrales N, Rodriguez S, Luong C, Zaldivar F, Alexander M, Lakey JRT. An islet maturation media to improve the development of young porcine islets during in vitro culture. Islets 2020; 12:41-58. [PMID: 32459554 PMCID: PMC7527017 DOI: 10.1080/19382014.2020.1750933] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
BACKGROUND The use of pancreata from pre-weaned piglets has the potential to serve as an unlimited alternative source of islets for clinical xenotransplantation. As pre-weaned porcine islets (PPIs) are immature and require prolonged culture, we developed an islet maturation media (IMM) and evaluated its effect on improving the quantity and quality of PPIs over 14 days of culture. METHODS PPIs were isolated from the pancreata of pre-weaned Yorkshire piglets (8-15 days old). Each independent islet isolation was divided for culture in either control Ham's F-10 media (n = 5) or IMM (n = 5) for 14 days. On day 3, 7 and 14 of culture, islets were assessed for islet yield, isolation index, viability, insulin content, endocrine cellular composition, differentiation of beta cells, and insulin secretion during glucose stimulation. RESULTS In comparison to control islets, culturing PPIs in IMM significantly increased islet yield. PPIs cultured in IMM also maintained a stable isolation index and viability throughout 14 days of culture. The insulin content, endocrine cellular composition, and differentiation of beta cells were significantly improved in PPIs cultured in IMM, which subsequently augmented their insulin secretory capacity in response to glucose challenge compared to control islets. CONCLUSIONS Culturing PPIs in IMM increases islet yield, isolation index, viability, insulin content, endocrine cellular composition, differentiation of endocrine progenitor cells toward beta cells, and insulin secretion. Due to the improved islet quantity and quality after in vitro culture, the use of IMM in the culture of PPIs will assist to advance the outcomes of clinical islet xenotransplantation.
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Affiliation(s)
- Hien Lau
- Department of Surgery, University of California, Irvine, Orange, CA, USA
| | - Nicole Corrales
- Department of Surgery, University of California, Irvine, Orange, CA, USA
| | - Samuel Rodriguez
- Department of Surgery, University of California, Irvine, Orange, CA, USA
| | - Colleen Luong
- Department of Surgery, University of California, Irvine, Orange, CA, USA
| | - Frank Zaldivar
- Department of Pediatrics, Pediatric Exercise and Genomics Research Center, University of California, Irvine, Irvine, CA, USA
| | - Michael Alexander
- Department of Surgery, University of California, Irvine, Orange, CA, USA
| | - Jonathan R. T. Lakey
- Department of Surgery, University of California, Irvine, Orange, CA, USA
- Department of Biomedical Engineering, University of California Irvine, Irvine, CA, USA
- CONTACT Jonathan R. T. Lakey Department of Surgery and Biomedical Engineering, Clinical Islet Program, 333 City Blvd West, Suite 1600, Orange, CA92868, USA
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Dholakia S, Royston E, Sharples EJ, Sankaran V, Ploeg RJ, Friend PJ. Preserving and perfusing the allograft pancreas: Past, present, and future. Transplant Rev (Orlando) 2018; 32:127-131. [DOI: 10.1016/j.trre.2018.02.001] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2017] [Revised: 02/09/2018] [Accepted: 02/19/2018] [Indexed: 01/12/2023]
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Steffen A, Kiss T, Schmid J, Schubert U, Heinke S, Lehmann S, Bornstein S, Ludwig B, Ludwig S. Production of high-quality islets from goettingen minipigs: Choice of organ preservation solution, donor pool, and optimal cold ischemia time. Xenotransplantation 2017; 24. [PMID: 28130838 DOI: 10.1111/xen.12284] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2016] [Revised: 11/04/2016] [Accepted: 12/07/2016] [Indexed: 12/16/2022]
Abstract
BACKGROUND The transplantation of porcine islets into man might soon become reality for patients with type 1 diabetes mellitus. Therefore, porcine islets of high quality and quantity, and a scalable isolation process with strict quality control will be an unconditional prerequisite to enable the best possible transplantation graft. In this study, we provide a comparative study evaluating islet isolation outcome and in vitro survival based upon donor age, organ preservation solution (OPS), and cold ischemia time (CIT). METHODS Goettingen minipigs of younger age (1 year) and retired breeder animals (3.5 years) were studied. Pancreata were harvested according to the standards of human organ retrieval including in situ cold perfusion with either Custodiol® -HTK or Belzer® UW solution. Pancreatic tissue was characterized by quantification of apoptotic cells. Islet isolations were performed according to a modified Ricordi method, and isolation outcome was assessed by determining islet particle numbers (IP), islet equivalents (IEQ), and isolation factor (IF). Isolated islets were cultured for 24 and 48 h for the assessment of in vitro survival. RESULTS Islet viability was significantly higher in Custodiol® -HTK preserved pancreas organs compared to Belzer® UW. Furthermore, organs harvested from retired breeder preserved in Custodiol® -HTK resulted in stable islet isolation yields even after prolonged CIT and showed superior survival rates of islets in vitro compared to the Belzer® UW group. Younger porcine donor organs resulted generally in lower islet yield and survival rates. CONCLUSIONS In summary, Custodiol® -HTK solution should be preferred over Belzer® UW solution for the preservation of pancreata from porcine origin. Custodiol® -HTK allows for maintaining islet viability and promotes reproducible isolation outcome and survival even after longer CIT. The usage of retired breeder animals over young animals for islet isolation is highly advisable to yield high quality and quantity.
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Affiliation(s)
- Anja Steffen
- Department of Medicine III, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.,Paul Langerhans Institute Dresden of Helmholtz Centre Munich at University Clinic Carl Gustav Carus of TU Dresden Faculty of Medicine, Technische Universität Dresden, DZD-German Centre for Diabetes Research, Dresden, Germany
| | - Thomas Kiss
- Department of Anesthesiology and Intensive Care Medicine, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany
| | - Janine Schmid
- Department of Medicine III, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany
| | - Undine Schubert
- Department of Medicine III, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany
| | - Sophie Heinke
- Department of Medicine III, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany
| | - Susann Lehmann
- Department of Medicine III, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany
| | - Stefan Bornstein
- Department of Medicine III, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.,Paul Langerhans Institute Dresden of Helmholtz Centre Munich at University Clinic Carl Gustav Carus of TU Dresden Faculty of Medicine, Technische Universität Dresden, DZD-German Centre for Diabetes Research, Dresden, Germany.,Center for Regenerative Therapies, Technische Universität Dresden, Dresden, Germany.,Diabetes and Nutritional Sciences, Rayne Institute, King's College London, London, UK
| | - Barbara Ludwig
- Department of Medicine III, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.,Paul Langerhans Institute Dresden of Helmholtz Centre Munich at University Clinic Carl Gustav Carus of TU Dresden Faculty of Medicine, Technische Universität Dresden, DZD-German Centre for Diabetes Research, Dresden, Germany.,Center for Regenerative Therapies, Technische Universität Dresden, Dresden, Germany
| | - Stefan Ludwig
- Department of Visceral-, Thoracic- and Vascular Surgery, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany
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Yao Y, Zhang M, Liu T, Zhou J, Gao Y, Wen Z, Guan J, Zhu J, Lin Z, He D. Perfluorocarbon-Encapsulated PLGA-PEG Emulsions as Enhancement Agents for Highly Efficient Reoxygenation to Cell and Organism. ACS APPLIED MATERIALS & INTERFACES 2015; 7:18369-18378. [PMID: 26222132 DOI: 10.1021/acsami.5b04226] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/19/2023]
Abstract
Perfluorocarbon (PFC), a kind of oxygen carrier, is encapsulated in PLGA-PEG to prepare a PLGA-PEG/PFC emulsion for highly efficient reoxygenation to cell and organism. HCT 116 cells are used as a model cell, whose viability has a significant enhancement after reoxygenation with PLGA-PEG/PFC emulsion because of the sufficient and timely oxygen supply. Meanwhile, hypoxia-reoxygenation injury will happen along with cell hypoxia-reoxygenation treatment, which is reflected by increasing reactive oxygen species (ROS) in cells. However, the integration of intracellular ROS and cell viability implies that the degree of hypoxia-reoxygenation injury is sublethal to HCT116 cells when the concentration of PLGA-PEG/PFC emulsion is lower than 0.2 mg/mL. Furthermore, the change of the expression level of hypoxia-inducible factor-1α (HIF-1α) is similar to that of cell viability during reoxygenation, which suggests that HIF-1α or its downstream proteins may make a significant contribution to cell viability. In vivo oxygen supply is assessed in rats through pulmonary delivery, which shows that PLGA-PEG/PFC emulsion can supply oxygen to rats and improve rats' lung ventilation.
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Affiliation(s)
- Yanjie Yao
- School of Materials Science and Engineering, Shanghai Jiao Tong University , 800 Dongchuan Road, Shanghai 200240, P. R. China
| | - Minmin Zhang
- Department of Gastroenterology, ChangHai Hospital, Second Military Medical University , Shanghai 200433, P. R. China
| | - Tian Liu
- School of Materials Science and Engineering, Shanghai Jiao Tong University , 800 Dongchuan Road, Shanghai 200240, P. R. China
| | - Juan Zhou
- National Engineering Research Center for Nanotechnology , 28 East Jiang Chuan Road, Shanghai 200241, P. R. China
| | - Yuan Gao
- Department of Gastroenterology, ChangHai Hospital, Second Military Medical University , Shanghai 200433, P. R. China
| | - Zhengfeng Wen
- Department of Emergency and Critical Care Medicine, Shanghai Changzheng Hospital, Second Military Medical University , Shanghai 200003, P. R. China
| | - Jun Guan
- Department of Emergency and Critical Care Medicine, Shanghai Changzheng Hospital, Second Military Medical University , Shanghai 200003, P. R. China
| | - Jun Zhu
- National Engineering Research Center for Nanotechnology , 28 East Jiang Chuan Road, Shanghai 200241, P. R. China
- Key Laboratory of Organofluorine Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences , Shanghai 200032, P. R. China
| | - Zhaofen Lin
- Department of Emergency and Critical Care Medicine, Shanghai Changzheng Hospital, Second Military Medical University , Shanghai 200003, P. R. China
| | - Dannong He
- School of Materials Science and Engineering, Shanghai Jiao Tong University , 800 Dongchuan Road, Shanghai 200240, P. R. China
- National Engineering Research Center for Nanotechnology , 28 East Jiang Chuan Road, Shanghai 200241, P. R. China
- Key Laboratory of Organofluorine Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences , Shanghai 200032, P. R. China
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Establishment of a stringent large animal model of insulin-dependent diabetes for islet autotransplantation: combination of pancreatectomy and streptozotocin. Pancreas 2013; 42:329-38. [PMID: 23357925 DOI: 10.1097/mpa.0b013e318264bcdd] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/10/2022]
Abstract
OBJECTIVE A stringent porcine islet autograft diabetes model was developed to enable the assessment of autoislet safety and efficacy in either portal vein or an extrahepatic site. METHODS A 95% pancreatectomy was performed preserving the pancreaticoduodenal arcade; however, glycemic control was still maintained at 3.3 ± 0.3 days (mean ± SEM), shown by euglycemic fasting blood glucose levels of 4.9 ± 0.8 mmol/L (mean ± SEM, n = 3). To reduce surgical complications and eliminate remaining islets, pigs were dosed intravenously after a modified 90% pancreatectomy, with 150-mg/kg streptozotocin, producing a diabetic state (18.9 ± 1.8 mmol/L [mean ± SEM], n = 8; P < 0.001) within 2.0 ± 0.9 days (mean ± SEM). RESULTS Animals presented with sustained hyperglycemia, failing a glucose challenge test 12 weeks after diabetic induction, and showed no stimulated C-peptide secretion compared to nondiabetic controls (baseline: 0.479 ± 0.080 ng/mL [mean ± SEM] vs after procedure: 0.219 ± 0.055 ng/mL [mean ± SEM], P = 0.02). Diabetic animals were maintained on daily insulin. Despite an initial decline in body weight acutely after pancreatectomy and streptozotocin administration, the mean body weight increased after induction over the approximately 88-day study, indicating that the animals were in good health. CONCLUSION This stringent porcine model of diabetic induction should be used to assess autograft transplantation safety and efficacy.
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Brandhorst H, Iken M, Scott WE, Papas KK, Theisinger B, Johnson PR, Korsgren O, Brandhorst D. Quality of isolated pig islets is improved using perfluorohexyloctane for pancreas storage in a split lobe model. Cell Transplant 2012; 22:1477-83. [PMID: 23044229 DOI: 10.3727/096368912x657639] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
Pancreas transportation between donor center and islet production facility is frequently associated with prolonged ischemia impairing islet isolation and transplantation outcomes. It is foreseeable that shipment of pig pancreases from distant centralized biosecure breeding facilities to institutes that have a long-term experience in porcine islet isolation is essentially required in future clinical islet xenotransplantation. Previously, we demonstrated that perfluorohexyloctan (F6H8) is significantly more efficient to protect rat and human pancreata from ischemically induced damage compared to perfluorodecalin (PFD). To evaluate the effect of F6H8 on long-term stored pig pancreases in a prospective study, we utilized the split lobe model to minimize donor variability. Retrieved pancreases were dissected into the connecting and splenic lobe, intraductally flushed with UW solution and immersed alternately in either preoxygenated F6H8 or PFD for 8-10 h. Prior to pancreas digestion, the intrapancreatic pO2 and the ratio of ATP-to-inorganic phosphate was compared utilizing 31P-NMR spectroscopy. Isolated islets were cultured for 2-3 days at 37°C and subjected to quality assessment. Pancreatic lobes stored in preoxygenated F6H8 had a significantly higher intrapancreatic pO2 compared to pancreata in oxygen-precharged PFD (10.11 ± 3.87 vs. 1.64 ± 1.13 mmHg, p < 0.05). This correlated with a higher ATP-to-inorganic phosphate ratio (0.30 ± 0.04 vs. 0.14 ± 0.01). No effect was observed concerning yield and purity of freshly isolated islets. Nevertheless, a significantly improved glucose-stimulated insulin response, increased viability and postculture survival (57.2 ± 5.7 vs. 39.3 ± 6.4%, p < 0.01) was measured in islets isolated from F6H8-preserved pancreata. The present data suggest that F6H8 does not increase islet yield but improves quality of pig islets isolated after prolonged cold ischemia.
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Affiliation(s)
- H Brandhorst
- Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden
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Pepper AR, Hasilo CP, Melling CWJ, Mazzuca DM, Vilk G, Zou G, White DJG. The islet size to oxygen consumption ratio reliably predicts reversal of diabetes posttransplant. Cell Transplant 2012; 21:2797-804. [PMID: 22943589 DOI: 10.3727/096368912x653273] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
β-Cell replacement therapy by either whole-organ pancreas or islets of Langerhans transplantation can restore carbohydrate control to diabetic patients and reduces complications associated with the disease. One of the variables inherent in islet transplantation is the isolation of functional islets from donor pancreata. Islet isolations fail to consistently produce good-quality functional islets. A rapid pretransplant assay to determine posttransplant function of islets would be an invaluable tool. We have tested the novel hypothesis that modified oxygen consumption rates (OCR), standardized to DNA quantity (nmol/min-mg DNA), would serve as a pretransplant assessment of the metabolic potency of the islets postisolation. This study compares the ability of current in vitro assays to predict in vivo restoration of normoglycemia in a diabetic nude mouse posttransplantation of adult pig islets. There is known to be a diversity of islet sizes within each preparation. This parameter has not heretofore been effectively considered a critical factor in islet engraftment. Our results suggest a surprising finding that islet size influences the probability of restoring carbohydrate control. Based on this observation, we thus developed a novel predictor of islet graft function that combines the effects of both islet OCR and size. When OCR was divided by the islet index (size), a highly significant predictor of graft function was established (p = 0.0002, n = 75). Furthermore, when OCR/islet index values exceeded 70.0 nmol/min-mg DNA/islet index, an effective threshold of diabetes reversal was observed. This assay can be performed with as few as 1,000 islet equivalents (IEQ) and conducted in less than 60 min. Our data suggest that, using this novel method to assess islet cell function prior to transplantation, OCR/islet index thresholds provide a valuable tool in identifying which islet preparations are most likely to restore glycemic control posttransplant.
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Affiliation(s)
- Andrew R Pepper
- Department of Pathology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada
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Kuise T, Noguchi H. Recent progress in pancreatic islet transplantation. World J Transplant 2011; 1:13-8. [PMID: 24175188 PMCID: PMC3782227 DOI: 10.5500/wjt.v1.i1.13] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/14/2011] [Revised: 10/26/2011] [Accepted: 12/19/2011] [Indexed: 02/05/2023] Open
Abstract
Diabetes mellitus remains a major burden. More than 200 million people are affected worldwide, which represents 6% of the world’s population. Type 1 diabetes mellitus is an autoimmune disease, which induces the permanent destruction of the β-cells of the pancreatic islets of Langerhans. Although intensive insulin therapy has proven effective to delay and sometimes prevent the progression of complications such as nephropathy, neuropathy or retinopathy, it is difficult to achieve and maintain long term in most subjects. The successes achieved over the last few decades by the transplantation of whole pancreas and isolated islets suggest that diabetes can be cured by the replenishment of deficient β cells. However, islet transplantation efforts have various limitations, including the limited supply of donor pancreata, the paucity of experienced islet isolation teams, side effects of immunosuppressants and poor long term results. The purpose of this article is to review the recent progress in clinical islet transplantation for the treatment of diabetes and to describe the recent progress on pancreatic stem/progenitor cell research, which has opened up several possibilities for the development of new treatments for diabetes.
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Affiliation(s)
- Takashi Kuise
- Takashi Kuise, Hirofumi Noguchi, Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8558, Japan
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Matsumoto S, Noguchi H, Naziruddin B, Onaca N, Jackson A, Nobuyo H, Teru O, Naoya K, Klintmalm G, Levy M. Improvement of pancreatic islet cell isolation for transplantation. Proc (Bayl Univ Med Cent) 2011; 20:357-62. [PMID: 17948109 DOI: 10.1080/08998280.2007.11928323] [Citation(s) in RCA: 49] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
Pancreatic islet transplantation is a promising treatment for diabetes but still faces several challenges. Poor islet isolation efficiency and poor long-term insulin independence are currently two major issues, although donor shortage and the need for immunosuppressants also need to be addressed. We established the Kyoto islet isolation method (KIIM), which has enabled us to isolate and transplant islets even from non-heart-beating donors. KIIM involves 1) cooling the donor pancreas in situ, 2) preserving the ducts with modified Kyoto solution, 3) using a modified two-layer pancreas preservation method, and 4) adjusting the density of the density gradient centrifugation and using an iodixanol-based solution for purification. KIIM has enabled us to transplant 17 islet preparations out of 21 isolations (an 81% success rate). All transplanted islets functioned, and all transplanted patients had improved glycemic control without hypoglycemic unawareness. Recently, we used KIIM for islet isolation from a brain-dead donor at Baylor, which resulted in a very high islet yield (789,984 IE) with high viability (100% by fluorescein diacetate/propidium iodide staining and a stimulation index of 4.7). This preliminary evidence suggests that KIIM may also be promising for islet isolation from brain-dead donors. In addition, to assess engrafted islet mass, we developed a secretory unit of islet transplant objects (SUITO) index: fasting C-peptide (ng/dL) / [fasting blood glucose (mg/dL) - 63] x 1500. This simple index has enabled us to monitor the engrafted islet mass. This index should be useful when deciding whether to perform additional islet transplantations to maintain insulin independence. Poor islet isolation efficacy and poor long-term results could be resolved with ongoing research.
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Affiliation(s)
- Shinichi Matsumoto
- Baylor Research Institute Islet Cell Laboratory, Fort Worth, Texas, USA.
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Hosgood SA, Mohamed IH, Nicholson ML. The two layer method does not improve the preservation of porcine kidneys. Med Sci Monit 2011; 17:BR27-33. [PMID: 21169904 PMCID: PMC3524674 DOI: 10.12659/msm.881326] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022] Open
Abstract
BACKGROUND The Two layer method (TLM) has been extremely successful in the preservation of the pancreas. However, this has not been thoroughly investigated in other organs or in clinically relevant large animal models. The aim of this study was to assess the effects of TLM in a large animal model of kidney preservation. MATERIAL/METHODS Porcine kidneys were retrieved after 10 minutes of warm ischaemic injury and flushed with 300 ml UW solution at 4°C. Kidneys were then either placed in University of Wisconsin solution (UW) or TLM using pre-oxygenated perfluorodecalin and UW. Kidneys were stored for 18 hours at 4°C then reperfused with oxygenated autologous blood to assess renal function. RESULTS Renal blood flow (RBF) was significantly lower and intra-renal resistance (IRR) higher in TLM compared to UW group [Area under the curve (AUC) RBF, UW; 427±168 vs TLM; 247±55 ml/min/100g.h; P=0.041, AUC IRR, UW; 7.7±2.2 vs TLM; 10.5±1.9 ml/min/mmHg; P=0.041]. Levels of creatinine clearance (CrCl) were significantly lower in TLM group [AUC CrCl, UW; 1.8±1.0 vs TLM; 0.6±0.4 ml/min/100 g.h; P=0.034]. Levels of lipid peroxidation were significantly lower in TLM group [8-isoprostane/Cr ratio 3h; UW 3338±896 vs TLM 2072±886 pg/ml/mmol/L; P=0.04]. Levels of total nitric oxide were significantly higher in TLM group (P=0.009). CONCLUSIONS TLM did not improve the preservation condition of porcine kidneys. Furthermore, there appeared to be increased inflammation, endothelial injury and reduced renal function compared to preservation with UW. Further experimental work is needed to determine the role of PFC in kidney preservation.
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Affiliation(s)
- Sarah A Hosgood
- Department of Infection, Immunity and Inflammation, Transplant Group, University of Leicester, University Hospitals of Leicester, Leicester General Hospital, U.K
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13
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Hani H, Ibrahim TAT, Othman AM, Lila MAM, bt Allaudin ZN. Isolation, density purification, and in vitro culture maintenance of functional caprine islets of Langerhans as an alternative islet source for diabetes study. Xenotransplantation 2011; 17:469-80. [PMID: 21158948 DOI: 10.1111/j.1399-3089.2010.00616.x] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
BACKGROUND Insufficient availability of human donors makes the search for alternative source of islet cells mandatory for future developments in pancreatic transplantation. The present study investigates the potential of caprine as an alternative source of pancreatic islets. The objectives of the study were to optimize techniques for caprine islet isolation and purification for culture establishment, and to subsequently assess their viable and functional potential. METHODS Caprine pancreatic tissues were collected from a local slaughterhouse and prior transported to the laboratory by maintaining the cold chain. Islets were obtained by a collagenase-based digestion and optimized isolation technique. Islet cell purity and viability were determined by dithizone and trypan blue staining, respectively. Islet clusters of different sizes were positively identified by staining methods and demonstrated 90% viability in the culture system. Following static incubation, an in vitro insulin secretion assay was carried out and analyzed by ELISA. RESULTS The islets remained satisfactorily viable for 5 days in the culture system following regular media changes. The current study has successfully optimized the isolation, purification and culture maintenance of caprine islets. CONCLUSION The successful yield, viability and functionality of islets isolated from the optimized protocol provide promising potential as an alternative source of islets for diabetes and transplantation researches.
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Affiliation(s)
- Homayoun Hani
- Department of Medical Biotechnology, Institute of Bioscience, Universiti Putra Malaysia, Selangor, Malaysia
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14
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Abstract
Perfluorocarbons (PFCs) are inert solutions that have a high capacity for dissolving oxygen. There has been a continuing level of research into the delivery of oxygen during solid organ preservation with the use of PFCs. The one- and two-layer methods have been used as static storage techniques, proving particularly successful for pancreas preservation. They can also be formulated as an emulsion for continual perfusion or as a simple flush solution. The success of PFCs in organ preservation seems to be somewhat organ and species dependant, and further experimental evidence is needed to establish their application.
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Littlejohn GR, Gouveia JD, Edner C, Smirnoff N, Love J. Perfluorodecalin enhances in vivo confocal microscopy resolution of Arabidopsis thaliana mesophyll. THE NEW PHYTOLOGIST 2010; 186:1018-1025. [PMID: 20374500 DOI: 10.1111/j.1469-8137.2010.03244.x] [Citation(s) in RCA: 63] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/10/2023]
Abstract
*Air spaces in the leaf mesophyll generate deleterious optical effects that compromise confocal microscopy. *Leaves were mounted in the nontoxic, nonfluorescent perfluorocarbon, perfluorodecalin (PFD), and optical enhancement and physiological effect were assessed using confocal microscopy and chlorophyll fluorescence. *Mounting leaves of Arabidopsis thaliana in PFD significantly improved the optical qualities of the leaf, thereby enabling high-resolution laser scanning confocal imaging over twofold deeper into the mesophyll, compared with using water. Incubation in PFD had less physiological impact on the mounted specimen than water. *We conclude that the application of PFD as a mounting medium substantially increases confocal image resolution of living mesophyll and vascular bundle cells, with minimal physiological impact.
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Affiliation(s)
- George R Littlejohn
- School of Biosciences, The University of Exeter, Geoffrey Pope Building, Stocker Road, Exeter EX4 4QD, UK
| | - João D Gouveia
- School of Biosciences, The University of Exeter, Geoffrey Pope Building, Stocker Road, Exeter EX4 4QD, UK
| | - Christoph Edner
- School of Biosciences, The University of Exeter, Geoffrey Pope Building, Stocker Road, Exeter EX4 4QD, UK
| | - Nicholas Smirnoff
- School of Biosciences, The University of Exeter, Geoffrey Pope Building, Stocker Road, Exeter EX4 4QD, UK
| | - John Love
- School of Biosciences, The University of Exeter, Geoffrey Pope Building, Stocker Road, Exeter EX4 4QD, UK
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A New Oxygen Carrier for Improved Long-Term Storage of Human Pancreata Before Islet Isolation. Transplantation 2010; 89:155-60. [DOI: 10.1097/tp.0b013e3181c9266c] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
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Ridgway D, Manas D, Shaw J, White S. Preservation of the donor pancreas for whole pancreas and islet transplantation. Clin Transplant 2010; 24:1-19. [DOI: 10.1111/j.1399-0012.2009.01151.x] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
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Noguchi H, Levy MF, Kobayashi N, Matsumoto S. Pancreas preservation by the two-layer method: does it have a beneficial effect compared with simple preservation in University of Wisconsin solution? Cell Transplant 2009; 18:497-503. [PMID: 19775509 DOI: 10.1177/096368970901805-603] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
A large number of reports have shown that the two-layer method (TLM), which employs oxygenated perfluorochemical (PFC) and University of Wisconsin (UW) solution, is superior to simple cold storage in UW in islet transplantation. However, two recent large-scale studies showed no beneficial effect of TLM compared with UW storage in human islet transplantation. We reevaluated the effect of TLM by following three groups: group 1: UW simple storage; group 2: TLM performed by multiorgan procurement teams (not specialists of islet isolation); and group 3: TLM performed by specialists of islet isolation (Noguchi and Matsumoto). There were no significant differences between groups 1 and 2, whereas islet yields were significantly higher in group 3 compared with either group 1 or 2. Our data suggest that exact, complete performance of TLM could improve the outcome of islet isolation and transplantation. In this review, we describe the mechanisms of the TLM, the procedure of preoxygenated TLM, and the several possibilities for the reasons of the discrepancy.
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Affiliation(s)
- Hirofumi Noguchi
- Baylor Institute for Immunology Research/Baylor All Saints Medical Center, Baylor Research Institute, Dallas, TX, USA.
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19
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Brandhorst H, Theisinger B, Yamaya H, Henriksnäs J, Carlsson PO, Korsgren O, Brandhorst D. Perfluorohexyloctane improves long-term storage of rat pancreata for subsequent islet isolation. Transpl Int 2009; 22:1017-22. [DOI: 10.1111/j.1432-2277.2009.00905.x] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023]
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20
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Kinasiewicz A, Śmietanka A, Gajkowska B, Weryński A. Impact of Oxygenation of Bioartificial Liver Using Perfluorocarbon Emulsion Perftoran on Metabolism of Human Hepatoma C3A Cells. ACTA ACUST UNITED AC 2009; 36:525-34. [DOI: 10.1080/10731190802554380] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/21/2022]
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21
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Iken M, Brandhorst H, Korsgren O, Brandhorst D. Pig pancreas oxygenation at 20 degrees C increases islet ATP generation but deteriorates islet function. Cell Transplant 2009; 18:745-51. [PMID: 19520045 DOI: 10.3727/096368909x470838] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
Successful pancreas preservation during storage in oxygenated perfluorodecalin (PFD) is mainly related to oxidative ATP generation during storage. Increasing the storage temperature would accelerate this process essential for resuscitation of ischemically damaged pancreatic tissue. The present study aimed at comparing islet isolation outcome from adult pig pancreata preserved in oxygenated PFD by means of a one-layer method during storage on ice or at 20 degrees C. Resected pancreata were intraductally flushed with cold UW solution and promptly processed (n = 6) or stored for 3 h in continuously oxygenated PFD at 4 degrees C (n = 5) or 20 degrees C (n = 7). Prior to digestion-filtration pancreata were intraductally injected with UW supplemented with Serva collagenase NB8 and neutral protease. Islet quality assessment determined viability, glucose stimulation index, mitochondrial activity, intracellular ATP content, and transplant function in diabetic nude mice. Pancreata oxygenated for 3 h at 20 degrees C yielded islet numbers similar to organs oxygenated at 4 degrees C. Compared to a storage temperature of 20 degrees C, preservation at 4 degrees C reduced islet ATP content (p < 0.05) as well as islet viability (p < 0.05). Nevertheless, PFD storage at 20 degrees C decreased insulin response to glucose compared to unstored pancreata (p < 0.05). In contrast to unstored pancreata or cold-stored organs, transplantation of islets isolated after oxygenation at 20 degrees C was characterized by an early loss of transplant function in 50% of recipients (p < 0.05). The present study demonstrates that PFD storage at 20 degrees C enhances islet ATP synthesis within a short period of oxygenation but deteriorates islet function. We conclude that the present data reflect an equilibration between reduced depression of metabolic activity resulting in damage of islets and temperature-stimulated acceleration of ATP synthesis. Future studies are required to adjust the optimum storage temperature for pancreas oxygenation in different species.
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Affiliation(s)
- M Iken
- Department of Gastroenterology, Hepatology & Endocrinology, Medical School, Hanover, Germany
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22
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Ikemoto T, Noguchi H, Shimoda M, Naziruddin B, Jackson A, Tamura Y, Fujita Y, Onaca N, Levy MF, Matsumoto S. Islet cell transplantation for the treatment of type 1 diabetes in the USA. JOURNAL OF HEPATO-BILIARY-PANCREATIC SURGERY 2009; 16:118-23. [PMID: 19110650 DOI: 10.1007/s00534-008-0019-9] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/15/2008] [Accepted: 07/15/2008] [Indexed: 11/25/2022]
Abstract
Islet cell transplantation (ICTx) is one of the most effective treatments for type 1 diabetes and is less invasive compared to whole organ transplantation. The US has been the leader in the research and clinical applications of ICTx for the last 40 years. ICTx requires complex procedures, including pancreas procurement and preservation; pancreas digestion; islet purification; and transplantation. Even with the dramatic progresses in each of the procedures listed above, there are still challenges to make ICTx the standard therapy. These challenges are: (1) obtaining enough islets from a single donor and (2) preventing graft loss due to allogenic rejection and recurrence of autoimmune islet destruction. A new preservation strategy for pancreata and pancreatic ducts using ET-Kyoto solution as well as a new islet purification method using iodixanol has substantially improved islet yields. Continuous research to improve the efficacy of islet isolation will solve the issue of obtaining enough islets from a single donor. Immunological tolerance is an ideal solution for the issue of rejection and autoimmune recurrence and a regulatory T cell strategy seems promising. Moreover, the SUITO index is a simple and powerful tool to assess engrafted islet mass and is, therefore, useful for evaluating the efficacy of new immunosuppressant strategies. Once ICTx becomes a standard treatment, the donor shortage will become the next challenge. Marginal or living donor islet transplantations could help alleviate this issue; however, bio-artificial islet transplantation with animal islets could be the ultimate solution.
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Affiliation(s)
- Tetsuya Ikemoto
- Baylor Research Institute, Baylor Institute for Immunology Research, Dallas, TX 76104, USA
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23
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Abstract
PURPOSE OF REVIEW Organ preservation aims at reducing ischemia-reperfusion injury and maintains or even improves its function, and, therefore, increases transplant safety and efficiency. With the chronic lack of organs for transplantation, marginal donors are more and more frequently used in Western countries. New challenges, therefore, have to be met in organ preservation. RECENT FINDINGS We summarize the effects of cold preservation on various organ grafts, with particular emphasis on the pancreas. We review the different preservation solutions currently available in the clinic, and we present the current knowledge and clinical experience in pancreas and islet transplantation. SUMMARY Overall, in whole pancreas and islet transplantation, current cold preservation solutions (University of Wisconsin solution, Celsior, histidine-tryptophan-ketoglutarate) seem to be equivalent, with only few studies showing better results with University of Wisconsin solution. Regarding preservation with the two-layer method, conflicting results have been reported, and proper prospective controlled studies have yet to be performed to gather evidence on its impact on islet yield and function. Some recent developments and future strategies in general organ preservation not yet applied to pancreas preservation are reviewed at the end of the article.
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24
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Abstract
PURPOSE OF REVIEW To summarize advances and limitations in pancreas procurement and preservation for pancreas and islet transplantation, and review advances in islet protection and preservation. RECENT FINDINGS Pancreases procured after cardiac death, with in-situ regional organ cooling, have been successfully used for islet transplantation. Colloid-free Celsior and histidine-tryptophan-ketoglutarate preservation solutions are comparable to University of Wisconsin solution when used for cold storage before pancreas transplantation. Colloid-free preservation solutions are inferior to University of Wisconsin solution for pancreas preservation prior to islet isolation and transplantation. Clinical reports on pancreas transplants suggest that the two-layer method may not offer significant benefits over cold storage with the University of Wisconsin solution: improved oxygenation may depend on the graft size; benefits in experimental models may not translate to human organs. Improvements in islet yield and quality occurred from pancreases treated with inhibitors of stress-induced apoptosis during procurement, storage, isolation or culture desirable before islet isolation and transplantation and may improve islet yield and quality. Methods for real-time, noninvasive assessment of pancreas quality during preservation have been implemented and objective islet-potency assays have been developed and validated. These innovations should contribute to objective evaluation and establishment of improved pancreas-preservation and islet-isolation strategies. SUMMARY Cold storage may be adequate for preservation before pancreas transplants, but insufficient when pancreases are processed for islets or when expanded donors are used. Supplementation of cold-storage solutions with cytoprotective agents and perfusion may improve pancreas and islet transplant outcomes.
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25
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Iwanaga Y, Sutherland DE, Harmon JV, Papas KK. Pancreas preservation for pancreas and islet transplantation. Curr Opin Organ Transplant 2008; 13:445-51. [PMID: 18685343 PMCID: PMC2858000 DOI: 10.1097/mot.0b013e328303df04] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
PURPOSE OF REVIEW To summarize advances and limitations in pancreas procurement and preservation for pancreas and islet transplantation, and review advances in islet protection and preservation. RECENT FINDINGS Pancreases procured after cardiac death, with in-situ regional organ cooling, have been successfully used for islet transplantation. Colloid-free Celsior and histidine-tryptophan-ketoglutarate preservation solutions are comparable to University of Wisconsin solution when used for cold storage before pancreas transplantation. Colloid-free preservation solutions are inferior to University of Wisconsin solution for pancreas preservation prior to islet isolation and transplantation. Clinical reports on pancreas and islet transplants suggest that the two-layer method may not offer significant benefits over cold storage with the University of Wisconsin solution: improved oxygenation may depend on the graft size; benefits in experimental models may not translate to human organs. Improvements in islet yield and quality occurred from pancreases treated with inhibitors of stress-induced apoptosis during procurement, storage, isolation or culture. Pancreas perfusion may be desirable before islet isolation and transplantation and may improve islet yields and quality. Methods for real-time, noninvasive assessment of pancreas quality during preservation have been implemented and objective islet potency assays have been developed and validated. These innovations should contribute to objective evaluation and establishment of improved pancreas preservation and islet isolation strategies. SUMMARY Cold storage may be adequate for preservation before pancreas transplants, but insufficient when pancreases are processed for islets or when expanded donors are used. Supplementation of cold storage solutions with cytoprotective agents and perfusion may improve pancreas and islet transplant outcomes.
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Affiliation(s)
- Yasuhiro Iwanaga
- Transplantation Unit, Kyoto University Hospital, Kyoto, Japan
- Department of Surgery, Division of Transplantation, University of Minnesota, Minneapolis, USA
| | - David E.R. Sutherland
- Department of Surgery, Division of Transplantation, University of Minnesota, Minneapolis, USA
| | - James V. Harmon
- Department of Surgery, Division of Transplantation, University of Minnesota, Minneapolis, USA
| | - Klearchos K. Papas
- Department of Surgery, Division of Transplantation, University of Minnesota, Minneapolis, USA
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Brandhorst H, Muehling B, Yamaya H, Henriksnaes J, Carlsson PO, Korsgren O, Brandhorst D. New class of oxygen carriers improves islet isolation from long-term stored rat pancreata. Transplant Proc 2008; 40:393-4. [PMID: 18374079 DOI: 10.1016/j.transproceed.2008.01.012] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
OBJECTIVE Pancreas shipment is frequently associated with prolonged ischemia deteriorating islet graft function. The strategy to prevent ischemic damage utilizing perfluorodecalin (PFD) for human pancreas oxygenation does not seem to improve isolation outcome. The present study investigated the efficiency of perfluorohexyloctane (F6H8), a hyperoxygen carrier characterized by low specific density (1.33 g/cm3) and lipophilic qualities, to facilitate islet isolation from long-term stored rat pancreata. MATERIALS AND METHODS Prior to islet isolation, pancreata were intraductally flushed in situ with Kyoto solution (KS) and stored for 24 hours in KS, oxygenated PFD, or F6H8. RESULTS Islet isolation performed after 24-hour storage in KS failed completely. The intrapancreatic pO2 in PFD- and F6H8-incubated pancreata was almost the same. In correspondence, the ATP content and viability of isolated islets were similar as well. In contrast, islet yield and in vitro function were significantly reduced after storage in PFD compared with F6H8. CONCLUSION This study suggested that islet isolation performed after long-term pancreas preservation can be significantly improved utilizing semifluorinated alkanes as oxygen carriers.
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Affiliation(s)
- H Brandhorst
- Department of Clinical Immunology, Uppsala University Hospital, Uppsala, Sweden
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27
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Abstract
OBJECTIVES Although the stimulation index (SI) is widely used as a parameter that reflects the insulin releasing function of isolated islets, comparison between the outcomes of different institutions is problematic. This basic study was conducted for 2 objectives as we aimed to determine the optimal high glucose level for rat islets and clarify the available evidence in this field. METHODS We isolated islets of 3 rat strains (Lewis, Sprague-Dawley, and Wistar--n = 5 per strain) and measured the viability of the islets. We measured the amount of insulin released after high and low glucose stimulation. The SI was calculated as a ratio of the insulin value after high glucose stimulation divided by insulin value after low glucose stimulation. We examined the correlation between SI and viability at the high glucose levels. RESULTS Isolated islets released insulin corresponding to the changes of high glucose level. The highest correlation coefficient between SI and viability was seen at a glucose level of 16.5 mmol/L (R2 = 0.546, correlation coefficient = 0.154) followed by 19.3 mmol/L (R2 = 0.541, correlation coefficient = 0.169). CONCLUSIONS We could optimize the high glucose level at 16.5 to 19.3 mmol/L (300-350 mg/dL) for isolated rat islets, with evidence acquired from correlation studies between SI and viability.
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Balibrea del Castillo JM, Vara Ameigeiras E, Arias-Díaz J, García Martín MC, García-Pérez JC, Balibrea Cantero JL. Estado actual del trasplante de islotes pancreáticos. Cir Esp 2007; 81:177-91. [PMID: 17403353 DOI: 10.1016/s0009-739x(07)71297-6] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Abstract
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.
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Kim JH, Kim HI, Lee KW, Yu JE, Kim SH, Park HS, Park CG, Ihm SH, Ha J, Kim SJ, Lee HK, Ahn C, Park KS. Influence of strain and age differences on the yields of porcine islet isolation: extremely high islet yields from SPF CMS miniature pigs. Xenotransplantation 2007; 14:60-6. [PMID: 17214705 DOI: 10.1111/j.1399-3089.2006.00364.x] [Citation(s) in RCA: 53] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
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.
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Affiliation(s)
- Jae Hyeon Kim
- Department of Internal Medicine, Seoul National University Boramae Hospital, Seoul, Korea
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Brandhorst D, Iken M, Bretzel RG, Brandhorst H. Pancreas storage in oxygenated perfluorodecalin does not restore post-transplant function of isolated pig islets pre-damaged by warm ischemia. Xenotransplantation 2007; 13:465-70. [PMID: 16925671 DOI: 10.1111/j.1399-3089.2006.00340.x] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
BACKGROUND Cold storage in oxygenated perfluorodecalin (PFD) restores transplant function of ischemically damaged dog pancreata and reduces the impact of cold ischemia on recovery of isolated human islets. Whether PFD storage can improve islet isolation from pancreata exposed to significant warm ischemia (WI) is unclear yet. The present study aimed to clarify this question in adult pigs. METHODS After exsanguination, the intestine was removed immediately or left in the cavity for 30 min of WI. Resected pancreata were intraductally flushed with cold University of Wisconsin solution. Subsequently, pancreata were processed immediately by digestion-filtration (group I: 0 min WI, n=6; II: 30 min WI, n=6) or first stored for 3 h in oxygenated PFD (III: 0 min WI+PFD, n=5; IV: 30 min WI+PFD, n=6). RESULTS Pancreata subjected to 30 min of WI yielded significantly less islets compared with the corresponding non-ischemic organs (I vs. II, P<0.01; III vs. IV, P<0.05). Oxygenation did not ameliorate the loss in islet yield (II vs. IV, NS). Ischemic islets were characterized by depleted ATP stores (388+/-73 (I) vs. 133+/-22 ng/1000 IEQ (II), P<0.01) and diminished insulin response to glucose calculated as stimulation index (SI; 2.47+/-0.36 (I) vs. 0.25+/-0.17 (II), P<0.05). PFD storage of ischemic organs partially restored ATP content (217+/-23 ng/1000 IEQ, II vs. IV, P<0.05) and glucose SI (1.60+/-0.09, II vs. IV, P<0.05) to a significant extent that reached the level of corresponding PFD-stored, non-ischemic pancreata (III vs. IV, NS). Sustained normoglycemia was exclusively observed in diabetic nude mice transplanted with islets isolated from non-ischemic organs. The significantly reduced graft function of ischemic islets (I vs. II, III vs. IV, P<0.001) was not increased by pancreatic oxygenation (II vs. IV, NS). CONCLUSIONS The present study suggests that pancreas short-term storage in oxygenated PFD improves in vitro but not the in vivo function of ischemically damaged pig islets.
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Affiliation(s)
- Daniel Brandhorst
- Department of Oncology, Radiology & Clinical Immunology, University Hospital, Uppsala, Sweden.
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31
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Tanaka H, Kobayashi E. Education and research using experimental pigs in a medical school. J Artif Organs 2006; 9:136-43. [PMID: 16998697 DOI: 10.1007/s10047-006-0343-2] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2006] [Accepted: 06/07/2006] [Indexed: 01/20/2023]
Abstract
Medium-sized animals such as miniature pigs are considered to be important for education and training in medical schools to master the skills required in surgical treatment. Much still remains to be done to establish total management for animal experiments using pigs. Improvement of the effective utilization of pigs is also required from the economical and ethical points of view. We have been providing a support system at a facility for experimental animals in a medical school for 3 years, and herein we introduce our personal experiments as an instructional lecture. Before starting surgical training using live pigs, sufficient education concerning animal ethics and dry laboratory training was completed. Four kinds of miniature pigs have been used as experimental animals; porcine rearing pens have been improved and a postoperative care system has been implemented. Moreover, staff at the center offer a preoperative service of anesthesia for surgical education, training, and research. Chronic experiments have increased to represent 35% and 48% of experiments using pigs in 2003 and 2004, respectively. Experimental pigs have undergone secondary use after being killed to reduce the number of animals used in experiments. Sharing and reuse have allowed effective use of miniature pig tissues and cells for research, and have reduced the number of animals used. We recommend that researchers consider use of our total systems because they can improve the quality of medical education and research and facilitate effective use of tissues and cells by sharing and reuse among different departments.
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Affiliation(s)
- Hozumi Tanaka
- Center for Experimental Medicine, Jichi Medical University, 3311-1 Yakushiji, Shimotuke, Tochigi 329-0498, Japan
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Rood PPM, Buhler LH, Bottino R, Trucco M, Cooper DKC. Pig-to-nonhuman primate islet xenotransplantation: a review of current problems. Cell Transplant 2006; 15:89-104. [PMID: 16719044 DOI: 10.3727/000000006783982052] [Citation(s) in RCA: 48] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
Islet allotransplantation has been shown to have potential as a treatment for type 1 diabetic patients. Xenotransplantation, using the pig as a donor, offers the possibility of an unlimited number of islets. This comprehensive review focuses on experience obtained in pig-to-nonhuman primate models, particularly with regard to the different types of islets (fetal, neonatal, adult) and isolation procedures used, and the methods to determine islet viability. The advantages and disadvantages of the methods to induce diabetes (pancreatectomy, streptozotocin) are discussed. Experience in pig-to-nonhuman primate islet transplantation studies is reviewed, including discussion of the possible mechanisms of rejection and the immunosuppressive regimens used. The research carried out to date has led to workable animal models to study islet xenotransplantation, but several questions regarding methodology remain unanswered, and details of these practicalities require to be adequately addressed. The encouraging porcine islet survival reported recently provides an indicator for future immunosuppressive regimens.
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Affiliation(s)
- P P M Rood
- Starzl Transplantation Institute, University of Pittsburgh Medical Center, Pittsburgh, PA, USA
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