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1
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Liu C, Chen X, Liu Y, Sun L, Yu Z, Ren Y, Zeng C, Li Y. Engineering Extracellular Matrix-Bound Nanovesicles Secreted by Three-Dimensional Human Mesenchymal Stem Cells. Adv Healthc Mater 2023; 12:e2301112. [PMID: 37225144 PMCID: PMC10723826 DOI: 10.1002/adhm.202301112] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2023] [Revised: 05/19/2023] [Indexed: 05/26/2023]
Abstract
Extracellular matrix (ECM) in the human tissue contains vesicles, which are defined as matrix-bound nanovesicles (MBVs). MBVs serve as one of the functional components in ECM, recapitulating part of the regulatory roles and in vivo microenvironment. In this study, extracellular vesicles from culture supernatants (SuEVs) and MBVs are isolated from the conditioned medium or ECM, respectively, of 3D human mesenchymal stem cells. Nanoparticle tracking analysis shows that MBVs are smaller than SuEVs (100-150 nm). Transmission electron microscopy captures the typical cup shape morphology for both SuEVs and MBVs. Western blot reveals that MBVs have low detection of some SuEV markers such as syntenin-1. miRNA analysis of MBVs shows that 3D microenvironment enhances the expression of miRNAs such as miR-19a and miR-21. In vitro functional analysis shows that MBVs can facilitate human pluripotent stem cell-derived forebrain organoid recovery after starvation and promote high passage fibroblast proliferation. In macrophage polarization, 2D MBVs tend to suppress the pro-inflammatory cytokine IL-12β, while 3D MBVs tend to enhance the anti-inflammatory cytokine IL-10. This study has the significance in advancing the understanding of the bio-interface of nanovesicles with human tissue and the design of cell-free therapy for treating neurological disorders such as ischemic stroke.
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Affiliation(s)
- Chang Liu
- Department of Chemical and Biomedical Engineering, FAMU-FSU College of Engineering, Florida State University
| | - Xingchi Chen
- Department of Chemical and Biomedical Engineering, FAMU-FSU College of Engineering, Florida State University
- High Performance Materials Institute, FAMU-FSU College of Engineering, Florida State University
| | - Yuan Liu
- Department of Chemical and Biomedical Engineering, FAMU-FSU College of Engineering, Florida State University
| | - Li Sun
- Department of Chemical and Biomedical Engineering, FAMU-FSU College of Engineering, Florida State University
- Department of Biomedical Sciences, College of Medicine, Florida State University
| | - Zhibin Yu
- High Performance Materials Institute, FAMU-FSU College of Engineering, Florida State University
- Department of Industrial and Manufacturing Engineering, FAMU-FSU College of Engineering, Florida State University
| | - Yi Ren
- Department of Biomedical Sciences, College of Medicine, Florida State University
| | - Changchun Zeng
- High Performance Materials Institute, FAMU-FSU College of Engineering, Florida State University
- Department of Industrial and Manufacturing Engineering, FAMU-FSU College of Engineering, Florida State University
| | - Yan Li
- Department of Chemical and Biomedical Engineering, FAMU-FSU College of Engineering, Florida State University
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2
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Kalsi RS, Kreger AM, Saleh M, Yoshida S, Sharma K, Fusco J, Saloman JL, Zhang T, Thomas M, Sehrawat A, Wang Y, Reif J, Mills J, Raad S, Zengin B, Gomez A, Singhi A, Tadros S, Slivka A, Esni F, Prasadan K, Gittes G. Chemical pancreatectomy in non-human primates ablates the acini and ducts and enhances beta-cell function. Sci Rep 2023; 13:9113. [PMID: 37277426 PMCID: PMC10241801 DOI: 10.1038/s41598-023-35820-2] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2023] [Accepted: 05/24/2023] [Indexed: 06/07/2023] Open
Abstract
Chronic pancreatitis is a debilitating disease affecting millions worldwide. These patients suffer from bouts of severe pain that are minimally relieved by pain medications and may necessitate major surgeries with high morbidity and mortality. Previously, we demonstrated that "chemical pancreatectomy," a pancreatic intraductal infusion of dilute acetic acid solution, ablated the exocrine pancreas while preserving the endocrine pancreas. Notably, chemical pancreatectomy resolved chronic inflammation, alleviated allodynia in the cerulein pancreatitis model, and improved glucose homeostasis. Herein, we extensively tested the feasibility of a chemical pancreatectomy in NHPs and validated our previously published pilot study. We did serial computed tomography (CT) scans of the abdomen and pelvis, analyzed dorsal root ganglia, measured serum enzymes, and performed histological and ultrastructural assessments and pancreatic endocrine function assays. Based on serial CT scans, chemical pancreatectomy led to the loss of pancreatic volume. Immunohistochemistry and transmission electron microscopy demonstrated exocrine pancreatic ablation with endocrine islet preservation. Importantly, chemical pancreatectomy did not increase pro-nociceptive markers in harvested dorsal root ganglia. Also, chemical pancreatectomy improved insulin secretion to supranormal levels in vivo and in vitro. Thus, this study may provide a foundation for translating this procedure to patients with chronic pancreatitis or other conditions requiring a pancreatectomy.
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Affiliation(s)
- Ranjeet S Kalsi
- Division of Pediatric Surgery, UPMC Children's Hospital of Pittsburgh, 4401 Penn Avenue, Pittsburgh, PA, 15224, USA
| | - Alexander M Kreger
- Division of Pediatric Surgery, UPMC Children's Hospital of Pittsburgh, 4401 Penn Avenue, Pittsburgh, PA, 15224, USA
| | - Mohamed Saleh
- Division of Pediatric Surgery, UPMC Children's Hospital of Pittsburgh, 4401 Penn Avenue, Pittsburgh, PA, 15224, USA
- Division of Pediatric Endocrinology, UPMC Children's Hospital of Pittsburgh, Pittsburgh, PA, USA
| | - Shiho Yoshida
- Division of Pediatric Surgery, UPMC Children's Hospital of Pittsburgh, 4401 Penn Avenue, Pittsburgh, PA, 15224, USA
| | - Kartikeya Sharma
- Division of Pediatric Surgery, UPMC Children's Hospital of Pittsburgh, 4401 Penn Avenue, Pittsburgh, PA, 15224, USA
| | - Joseph Fusco
- Division of Pediatric Surgery, UPMC Children's Hospital of Pittsburgh, 4401 Penn Avenue, Pittsburgh, PA, 15224, USA
| | - Jami L Saloman
- Department of Medicine, Division of Gastroenterology, Hepatology, and Nutrition, Department of Neurobiology, Pittsburgh Center for Pain Research, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
| | - Ting Zhang
- Division of Pediatric Surgery, UPMC Children's Hospital of Pittsburgh, 4401 Penn Avenue, Pittsburgh, PA, 15224, USA
| | - Madison Thomas
- Division of Pediatric Surgery, UPMC Children's Hospital of Pittsburgh, 4401 Penn Avenue, Pittsburgh, PA, 15224, USA
| | - Anuradha Sehrawat
- Division of Pediatric Surgery, UPMC Children's Hospital of Pittsburgh, 4401 Penn Avenue, Pittsburgh, PA, 15224, USA
| | - Yan Wang
- Division of Pediatric Surgery, UPMC Children's Hospital of Pittsburgh, 4401 Penn Avenue, Pittsburgh, PA, 15224, USA
| | - Jason Reif
- Division of Pediatric Surgery, UPMC Children's Hospital of Pittsburgh, 4401 Penn Avenue, Pittsburgh, PA, 15224, USA
| | - Juliana Mills
- Division of Pediatric Surgery, UPMC Children's Hospital of Pittsburgh, 4401 Penn Avenue, Pittsburgh, PA, 15224, USA
| | - Sarah Raad
- Division of Pediatric Surgery, UPMC Children's Hospital of Pittsburgh, 4401 Penn Avenue, Pittsburgh, PA, 15224, USA
| | - Bugra Zengin
- Division of Pediatric Surgery, UPMC Children's Hospital of Pittsburgh, 4401 Penn Avenue, Pittsburgh, PA, 15224, USA
| | - Ana Gomez
- Division of Pediatric Pathology, UPMC Children's Hospital of Pittsburgh, Pittsburgh, PA, USA
| | - Aatur Singhi
- Divisions of Anatomic Pathology and Molecular Genomic Pathology, University of Pittsburgh Medical Center, Pittsburgh, PA, USA
| | - Sameh Tadros
- Department of Pediatric Radiology, UPMC Children's Hospital of Pittsburgh, Pittsburgh, PA, USA
| | - Adam Slivka
- Division of Gastroenterology, Hepatology and Nutrition, University of Pittsburgh Medical Center, Pittsburgh, PA, USA
| | - Farzad Esni
- Division of Pediatric Surgery, UPMC Children's Hospital of Pittsburgh, 4401 Penn Avenue, Pittsburgh, PA, 15224, USA
| | - Krishna Prasadan
- Division of Pediatric Surgery, UPMC Children's Hospital of Pittsburgh, 4401 Penn Avenue, Pittsburgh, PA, 15224, USA
| | - George Gittes
- Division of Pediatric Surgery, UPMC Children's Hospital of Pittsburgh, 4401 Penn Avenue, Pittsburgh, PA, 15224, USA.
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3
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Corder RD, Vachieri RB, Martin ME, Taylor DK, Fleming JM, Khan SA. Linear and nonlinear rheology of liberase-treated breast cancer tumors. Biomater Sci 2023; 11:2186-2199. [PMID: 36744734 PMCID: PMC10023448 DOI: 10.1039/d3bm00038a] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
Abstract
Extracellular matrix (ECM) rigidity has been shown to increase the invasive properties of breast cancer cells, promoting transformation and metastasis through mechanotransduction. Reducing ECM stiffness via enzymatic digestion could be a promising approach to slowing breast cancer development by de-differentiation of breast cancer cells to less aggressive phenotypes and enhancing the effectiveness of existing chemotherapeutics via improved drug penetrance throughout the tumor. In this study, we examine the effects of injectable liberase (a blend of collagenase and thermolysin enzymes) treatments on the linear and nonlinear rheology of allograft 4T1 mouse mammary tumors. We perform two sets of in vivo mouse studies, in which either one or multiple treatment injections occur before the tumors are harvested for rheological analysis. The treatment groups in each study consist of a buffer control, free liberase enzyme in buffer, a thermoresponsive copolymer called LiquoGel (LQG) in buffer, and a combined, localized injection of LQG and liberase. All tumor samples exhibit gel-like linear rheological behavior with the elastic modulus significantly larger than the viscous modulus and both independent of frequency. Tumors that receive a single injection of localized liberase have significantly lower tumor volumes and lower tissue moduli at both the center and edge compared to buffer- and free liberase-injected control tumors, while tissue viscoelasticity remains relatively unaffected. Tumors injected multiple times with LQG and liberase also have lower tissue volumes but possess higher tissue moduli and lower viscoelasticities compared to the other treatment groups. We propose that a mechanotransductive mechanism could cause the formation of smaller but stiffer tumors after repeated, localized liberase injections. Large amplitude oscillatory shear (LAOS) experiments are also performed on tissues from the multiple injection study and the results are analyzed using MITlaos. LAOS analysis reveals that all 4T1 tumors from the multiple injection study exhibit nonlinear rheological behavior at high strains and strain rates. Examination of the Lissajous-Bowditch curves, Chebyshev coefficient ratios, elastic moduli, and dynamic viscosities demonstrate that the onset and type of nonlinear behavior is independent of treatment type and elastic modulus, suggesting that multiple liberase injections do not affect the nonlinear viscoelasticity of 4T1 tumors.
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Affiliation(s)
- Ria D Corder
- Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC, 27695, USA.
| | - Robert B Vachieri
- Department of Chemistry and Biochemistry, North Carolina Central University, Durham, NC, 27707, USA
| | - Megan E Martin
- Department of Biological and Biomedical Sciences, North Carolina Central University, Durham, NC, 27707, USA.
| | - Darlene K Taylor
- Department of Chemistry and Biochemistry, North Carolina Central University, Durham, NC, 27707, USA
| | - Jodie M Fleming
- Department of Biological and Biomedical Sciences, North Carolina Central University, Durham, NC, 27707, USA.
| | - Saad A Khan
- Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC, 27695, USA.
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4
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Kalsi RS, Kreger AM, Saleh M, Yoshida S, Sharma K, Fusco J, Saloman JL, Zhang T, Thomas M, Sehrawat A, Wang Y, Reif J, Mills J, Raad S, Zengin B, Gomez A, Singhi A, Tadros S, Slivka A, Esni F, Prasadan K, Gittes G. Chemical pancreatectomy in non-human primates ablates the acini and ducts and enhances beta-cell function. RESEARCH SQUARE 2023:rs.3.rs-2618133. [PMID: 36945494 PMCID: PMC10029118 DOI: 10.21203/rs.3.rs-2618133/v1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 03/11/2023]
Abstract
Chronic pancreatitis is a debilitating disease affecting millions worldwide. These patients suffer from bouts of severe pain that are minimally relieved by pain medications and may necessitate major surgeries with high morbidity and mortality. Previously, we demonstrated that "chemical pancreatectomy," a pancreatic intraductal infusion of dilute acetic acid solution, ablated the exocrine pancreas while preserving the endocrine pancreas. Notably, chemical pancreatectomy resolved chronic inflammation, alleviated allodynia in the cerulein pancreatitis model, and improved glucose homeostasis. Herein, we extensively tested the feasibility of a chemical pancreatectomy in NHPs and validated our previously published pilot study. We did serial computed tomography (CT) scans of the abdomen and pelvis, analyzed dorsal root ganglia, measured serum enzymes, and performed histological and ultrastructural assessments and pancreatic endocrine function assays. Based on serial CT scans, chemical pancreatectomy led to the loss of pancreatic volume. Immunohistochemistry and transmission electron microscopy demonstrated exocrine pancreatic ablation with endocrine islet preservation. Importantly, chemical pancreatectomy did not increase pro-nociceptive markers in harvested dorsal root ganglia. Also, chemical pancreatectomy improved insulin secretion to supranormal levels in vivo and in vitro. Thus, this study may provide a foundation for translating this procedure to patients with chronic pancreatitis or other conditions requiring a pancreatectomy.
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Affiliation(s)
| | | | | | | | | | | | | | | | | | | | - Yan Wang
- UPMC Children's Hospital of Pittsburgh
| | | | | | | | | | - Ana Gomez
- UPMC Children's Hospital of Pittsburgh
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5
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Lee SM, Kim D, Kwak KM, Khin PP, Lim OK, Kim KW, Kim BJ, Jun HS. Comparison of the Effects of Liraglutide on Islet Graft Survival Between Local and Systemic Delivery. Cell Transplant 2021; 29:963689720971245. [PMID: 33172296 PMCID: PMC7784585 DOI: 10.1177/0963689720971245] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022] Open
Abstract
Islet transplantation has emerged as a promising treatment for type 1 diabetes mellitus. Liraglutide, a glucagon-like peptide-1 (GLP-1) receptor agonist, protects beta cells after islet transplantation by improving glycemic control through several mechanisms. In this study, we compared the effects of local pretreatment and systemic treatment with liraglutide on islet transplantation in a diabetic mouse model. Streptozotocin (STZ)-induced diabetic C57BL/6 mice were transplanted with syngeneic islets under the kidney capsule. Isolated islets were either locally treated with liraglutide before transplantation or mice were treated systemically by intraperitoneal injection after islet transplantation. Local pretreatment of islets with liraglutide was more effective in increasing body weight, decreasing hemoglobin A1c levels, and lowering blood glucose levels in STZ-diabetic mice transplanted with islets. Local pretreatment was also more effective in increasing insulin secretion and islet survival in STZ-diabetic mice. Histological analysis of the transplantation site revealed fewer apoptotic cells following local pretreatment compared with systemic injection of liraglutide. These findings indicate that liraglutide administered once locally before transplantation might have superior effects on islet preservation than systemic administration.
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Affiliation(s)
- Song Mi Lee
- College of Pharmacy and Gachon Institute Pharmaceutical Science, Gachon University, Yeonsu-gu, Incheon, Korea.,Lee Gil Ya Cancer and Diabetes Institute, Gachon University, Yeonsu-gu, Incheon, Korea.,Both the authors contributed equally to this article
| | - Donghee Kim
- Lee Gil Ya Cancer and Diabetes Institute, Gachon University, Yeonsu-gu, Incheon, Korea.,Both the authors contributed equally to this article
| | - Kyung Min Kwak
- Lee Gil Ya Cancer and Diabetes Institute, Gachon University, Yeonsu-gu, Incheon, Korea
| | - Phyu Phyu Khin
- College of Pharmacy and Gachon Institute Pharmaceutical Science, Gachon University, Yeonsu-gu, Incheon, Korea.,Lee Gil Ya Cancer and Diabetes Institute, Gachon University, Yeonsu-gu, Incheon, Korea
| | - Oh Kyung Lim
- Department of Rehabilitation Medicine, Gachon University Gil Medical Center, Namdong-gu, Incheon, Korea
| | - Kwang-Won Kim
- Department of Internal Medicine, Gachon University Gil Medical Center, Namdong-gu, Incheon, Korea
| | - Byung-Joon Kim
- Department of Internal Medicine, Gachon University Gil Medical Center, Namdong-gu, Incheon, Korea
| | - Hee-Sook Jun
- College of Pharmacy and Gachon Institute Pharmaceutical Science, Gachon University, Yeonsu-gu, Incheon, Korea.,Lee Gil Ya Cancer and Diabetes Institute, Gachon University, Yeonsu-gu, Incheon, Korea.,Gil Medical Research Institute, Gil Hospital, Namdong-gu, Incheon, Korea
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6
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Kwak K, Park JK, Shim J, Ko N, Kim HJ, Lee Y, Kim JH, Alexander M, Lakey JRT, Kim H, Choi K. Comparison of islet isolation result and clinical applicability according to GMP-grade collagenase enzyme blend in adult porcine islet isolation and culture. Xenotransplantation 2021; 28:e12703. [PMID: 34176167 PMCID: PMC8459292 DOI: 10.1111/xen.12703] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2021] [Revised: 06/06/2021] [Accepted: 06/09/2021] [Indexed: 12/11/2022]
Abstract
Background Porcine islet xenotransplantation is a promising treatment for type 1 diabetes as an alternative to human pancreatic islet transplantation and long‐term insulin therapy. Several research groups have explored porcine islets as an alternative to the inconsistent and chronic shortage of pancreases from human organ donors. Studies have confirmed successful transplant of porcine islets into non‐human primate models of diabetes; however, in most cases, they require more than one adult porcine donor to achieve sufficient viable islet mass for sustained function. The importance of GMP‐grade reagents includes the following: specific enzymes utilized in the pancreatic isolation process were identified as a key factor in successful human clinical islet transplantation trials using cadaveric islets. As xenotransplantation clinical research progresses, isolation reagents and digestion enzymes play a key role in the consistency of the product and ultimately the outcome of the islet xenotransplant. In this study, we evaluated several commercially available enzyme blends that have been used for islet isolation. We evaluated their impact on islet isolation yield and subsequent islet function as part of our plan to bring xenotransplantation into clinical xenotransplantation trials. Methods Adult porcine islets were isolated from 16 to 17‐month‐old Yucatan miniature pigs following standard rapid procurement. Pigs weighed on average 48.71 ± 2.85 kg, and the produced pancreases were 39.51 ± 1.80 grams (mean ± SEM). After ductal cannulation, we evaluated both GMP‐grade enzymes (Collagenase AF‐1 GMP grade and Liberase MTF C/T GMP grade) and compared with standard non‐GMP enzyme blend (Collagenase P). Islet quality control assessments including islet yield, islet size (IEQ), membrane integrity (acridine orange/propidium iodide), and functional viability (GSIS) were evaluated in triplicate on day 1 post‐islet isolation culture. Results Islet yield was highest in the group of adult pigs where Collagenase AF‐1 GMP grade was utilized. The mean islet yield was 16 586 ± 1391 IEQ/g vs 8302 ± 986 IEQ/g from pancreases isolated using unpurified crude Collagenase P. The mean islet size was higher in Collagenase AF‐1 GMP grade with neutral protease than in Collagenase P and Liberase MTF C/T GMP grade. We observed no significant difference between the experimental groups, but in vitro islet function after overnight tissue culture was significantly higher in Collagenase AF‐1 GMP grade with neutral protease and Liberase MTF C/T GMP grade than the crude control enzyme group. As expected, the GMP‐grade enzyme has significantly lower endotoxin levels than the crude control enzyme group when measured. Conclusions This study validates the importance of using specifically blended GMP grade for adult pig islet isolation for xenotransplantation trials and the ability to isolate a sufficient number of viable islets from one adult pig to provide a sufficient number for islets for a clinical islet transplantation. GMP‐grade enzymes are highly efficient in increasing islet yield, size, viability, and function at a lower and acceptable endotoxin level. Ongoing research transplants these islets into animal models of diabetes to validate in vivo function. Also, these defined and reproducible techniques using GMP‐grade enzymes allow for continuance of our plan to advance to xenotransplantation of isolated pig islets for the treatment of type 1 diabetes.
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Affiliation(s)
| | | | | | | | | | | | | | - Michael Alexander
- Department of Biomedical Engineering, University of California Irvine, Irvine, CA, USA
| | - Jonathan R T Lakey
- Department of Biomedical Engineering, University of California Irvine, Irvine, CA, USA.,Department of Surgery, University of California Irvine, Orange, CA, USA
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Alcazar O, Alvarez A, Ricordi C, Linetsky E, Buchwald P. The Effect of Recovery Warm-up Time Following Cold Storage on the Dynamic Glucose-stimulated Insulin Secretion of Isolated Human Islets. Cell Transplant 2021; 29:963689720908278. [PMID: 32223315 PMCID: PMC7444215 DOI: 10.1177/0963689720908278] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022] Open
Abstract
Standardized islet characterization assays that can provide results in a timely manner are essential for successful islet cell transplantation. A critical component of islet cell quality is β-cell function, and perifusion-based assessments of dynamic glucose-stimulated insulin secretion (GSIS) are the most informative method to assess this, as they provide the most complex in vitro evaluation of GSIS. However, protocols used vary considerably among centers and investigators as they often use different low- and high-glucose concentrations, exposure-times, flow-rates, oxygen concentrations, islet numbers, analytical methods, measurement units, and instruments, which result in different readouts and make comparisons across platforms difficult. Additionally, the conditions of islet storage and shipment prior to assessment may also affect islet function. Establishing improved standardized protocols for perifusion GSIS assays should be an integral part of the ongoing effort to increase the rigor of human islet studies. Here, we performed detailed evaluation of GSIS of human islets using a fully automated multichannel perifusion instrument following various warm-up recovery times after cold storage that corresponds to current shipping conditions (8°C). We found that recovery times shorter than 18 h (overnight) resulted in impaired insulin secretion. While the effects were relatively moderate on second-phase insulin secretion, first-phase peaks were restored only following 18-h incubation. Hence, the biphasic profile of dynamic GSIS was considerably affected when islets were not allowed to recover for a sufficient time after being maintained in cold. Accordingly, while cold storage might improve islet cell survival during shipment and prolong the length of culture, functional assessments should be performed only after allowing for at least overnight recovery at physiological temperatures.
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Affiliation(s)
- Oscar Alcazar
- Diabetes Research Institute, Miller School of Medicine, University of Miami, FL, USA
| | - Alejandro Alvarez
- Diabetes Research Institute, Miller School of Medicine, University of Miami, FL, USA
| | - Camillo Ricordi
- Diabetes Research Institute, Miller School of Medicine, University of Miami, FL, USA.,Department of Surgery, Division of Cellular Transplantation, cGMP Advanced Cell and Biologic Manufacturing Facility, Miller School of Medicine, University of Miami, FL, USA
| | - Elina Linetsky
- Diabetes Research Institute, Miller School of Medicine, University of Miami, FL, USA.,Department of Surgery, Division of Cellular Transplantation, cGMP Advanced Cell and Biologic Manufacturing Facility, Miller School of Medicine, University of Miami, FL, USA
| | - Peter Buchwald
- Diabetes Research Institute, Miller School of Medicine, University of Miami, FL, USA.,Department of Molecular and Cellular Pharmacology, Miller School of Medicine, University of Miami, FL, USA
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8
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Quijano LM, Naranjo JD, El-Mossier SO, Turner NJ, Pineda Molina C, Bartolacci J, Zhang L, White L, Li H, Badylak SF. Matrix-Bound Nanovesicles: The Effects of Isolation Method upon Yield, Purity, and Function. Tissue Eng Part C Methods 2020; 26:528-540. [PMID: 33012221 PMCID: PMC7869881 DOI: 10.1089/ten.tec.2020.0243] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2020] [Accepted: 09/25/2020] [Indexed: 12/11/2022] Open
Abstract
Identification of matrix-bound nanovesicles (MBV) as ubiquitous components of the extracellular matrix (ECM) raises questions regarding their biologic functions and their potential theranostic application. Unlike liquid-phase extracellular vesicles (e.g., exosomes), MBV are tightly bound to the ECM, which makes their isolation and harvesting more challenging. The indiscriminate use of different methods to harvest MBV can alter or disrupt their structural and/or functional integrity. The objective of the present study was to compare the effect of various MBV harvesting methods upon yield, purity, and biologic activity. Combinations of four methods to solubilize the ECM (collagenase [COL], liberase [LIB], or proteinase K [PK] and nonenzymatic elution with potassium chloride) and four isolation methods (ultracentrifugation, ultrafiltration [UF], density barrier, and size exclusion chromatography [SEC]) were used to isolate MBV from urinary bladder-derived ECM. All combinations of solubilization and isolation methods allowed for the harvesting of MBV, however, distinct differences were noted. The highest yield, purity, cellular uptake, and biologic activity were seen with MBV isolated by a combination of liberase or collagenase followed by SEC. The combination of proteinase K and UF was shown to have detrimental effects on bioactivity. The results show the importance of selecting appropriate MBV harvesting methods for the characterization and evaluation of MBV and for analysis of their potential theranostic application. Impact statement Identification of matrix-bound nanovesicles (MBV) as ubiquitous components of the extracellular matrix (ECM) has raised questions regarding their biologic functions and their potential theranostic application. This study demonstrates that the harvesting methods used can result in samples with physical and biochemical properties that are unique to the isolation and solubilization methods used. Consequently, developing harvesting methods that minimize sample contamination with ECM remnants and/or solubilization agents will be essential in determining the theranostic potential of MBV in future studies.
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Affiliation(s)
- Lina M. Quijano
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
- Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
| | - Juan D. Naranjo
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
- Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
| | - Salma O. El-Mossier
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
| | - Neill J. Turner
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
- Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
| | - Catalina Pineda Molina
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
- Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
| | - Joseph Bartolacci
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
| | - Li Zhang
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
- Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
| | - Lisa White
- School of Pharmacy, University of Nottingham, Nottingham, United Kingdom
| | - Hui Li
- Department of Obstetrics, Gynecology, and Reproductive Sciences, Magee-Womens Research Institute, Pittsburgh, Pennsylvania, USA
| | - Stephen F. Badylak
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
- Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
- Department of Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
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9
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Heterogeneity of Human Pancreatic Islet Isolation Around Europe: Results of a Survey Study. Transplantation 2020; 104:190-196. [PMID: 31365472 DOI: 10.1097/tp.0000000000002777] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
BACKGROUND Europe is currently the most active region in the field of pancreatic islet transplantation, and many of the leading groups are actually achieving similar good outcomes. Further collaborative advances in the field require the standardization of islet cell product isolation processes, and this work aimed to identify differences in the human pancreatic islet isolation processes within European countries. METHODS A web-based questionnaire about critical steps, including donor selection, pancreas processing, pancreas perfusion and digestion, islet counting and culture, islet quality evaluation, microbiological evaluation, and release criteria of the product, was completed by isolation facilities participating at the Ninth International European Pancreas and Islet Transplant Association (EPITA) Workshop on Islet-Beta Cell Replacement in Milan. RESULTS Eleven islet isolation facilities completed the questionnaire. The facilities reported 445 and 53 islet isolations per year over the last 3 years from deceased organ donors and pancreatectomized patients, respectively. This activity resulted in 120 and 40 infusions per year in allograft and autograft recipients, respectively. Differences among facilities emerged in donor selection (age, cold ischemia time, intensive care unit length, amylase concentration), pancreas procurement, isolation procedures (brand and concentration of collagenase, additive, maximum acceptable digestion time), quality evaluation, and release criteria for transplantation (glucose-stimulated insulin secretion tests, islet numbers, and purity). Moreover, even when a high concordance about the relevance of one parameter was evident, thresholds for the acceptance were different among facilities. CONCLUSIONS The result highlighted the presence of a heterogeneity in the islet cell product process and product release criteria.
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10
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Tsuang FY, Chen MH, Lin FH, Yang MC, Liao CJ, Chang WH, Sun JS. Partial enzyme digestion facilitates regeneration of crushed nerve in rat. Transl Neurosci 2020; 11:251-263. [PMID: 33335765 PMCID: PMC7711954 DOI: 10.1515/tnsci-2020-0112] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2020] [Revised: 05/23/2020] [Accepted: 06/12/2020] [Indexed: 11/15/2022] Open
Abstract
Peripheral nerve injury is a life-changing disability with significant socioeconomic consequences. In this rat model, we propose that partial enzyme digestion can facilitate the functional recovery of a crushed nerve. The sciatic nerves were harvested and in vitro cultured with the addition of Liberase to determine the appropriate enzyme amount in the hyaluronic acid (HA) membrane. Then, the sciatic nerve of adult male Sprague-Dawley rats was exposed, crushed, and then treated with partial enzyme digestion (either 0.001 or 0.002 unit/mm2 Liberase-HA membrane). The sciatic function index (SFI) for functional recovery of the sciatic nerve was evaluated. After 2 h of in vitro digestion, fascicles and axons were separated from each other, with the cells mobilized. Greater destruction of histology structures occurred in the high enzyme (Liberase-HA membrane at 0.002 unit/mm2) group at 24 h than in the low enzyme (0.001 unit/mm2) group at 48 h. In the SFI evaluation, the improvement in 0.001 unit/mm2 Liberase group was significantly better than control and 0.002 unit/mm2 Liberase group. Our study demonstrated that appropriate enzyme digestion had a significantly faster and earlier recovery.
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Affiliation(s)
- Fon-Yih Tsuang
- Institute of Biomedical Engineering, College of Engineering and College of Medicine, National Taiwan University, Taipei, Taiwan.,Department of Surgery, National Taiwan University Hospital, Taipei City, Taiwan
| | - Ming-Hong Chen
- Department of Surgery, Division of Neurosurgery, WanFang Hospital, Taipei, Taiwan
| | - Feng-Huei Lin
- Institute of Biomedical Engineering, College of Engineering and College of Medicine, National Taiwan University, Taipei, Taiwan.,Division of Medical Engineering, National Health Research Institute, Miaoli County, Taiwan
| | - Ming-Chia Yang
- Orthopedic Device Technology Division, Industrial Technology Research Institute, Hsinchu County, Taiwan
| | - Chun-Jen Liao
- Orthopedic Device Technology Division, Industrial Technology Research Institute, Hsinchu County, Taiwan
| | - Wen-Hsiang Chang
- Orthopedic Device Technology Division, Industrial Technology Research Institute, Hsinchu County, Taiwan
| | - Jui-Sheng Sun
- Department of Orthopedic Surgery, College of Medicine, National Taiwan University, Taipei, Taiwan.,Department of Orthopedic Surgery, National Taiwan University Hospital, Taipei, Taiwan
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11
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Loganathan G, Balamurugan AN, Venugopal S. Human pancreatic tissue dissociation enzymes for islet isolation: Advances and clinical perspectives. Diabetes Metab Syndr 2020; 14:159-166. [PMID: 32088647 DOI: 10.1016/j.dsx.2020.01.010] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/18/2019] [Revised: 01/26/2020] [Accepted: 01/27/2020] [Indexed: 02/06/2023]
Abstract
BACKGROUND AND AIMS Successful clinical human allo or auto-islet transplantation requires the recovery of a sufficient number of functional islets from either brain-dead or chronic pancreatitis pancreases respectively. METHODS In the last two decades (2000-2019), significant progress has been made in improving the human islet isolation procedures and in standardizing the use of different tissue dissociation enzyme (TDE; a mixture of collagenase and protease enzymes) blends to recover higher islet yields. RESULTS AND CONCLUSIONS This review presents information focusing on properties and role of TDE blends during the islet isolation process, particularly emphasizing on the current developments, associated challenges and future perspectives within the field.
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Affiliation(s)
- Gopalakrishnan Loganathan
- Clinical Islet Cell Laboratory, Cardiovascular Innovation Institute, Department of Surgery, University of Louisville, Louisville, KY, USA; School of Biosciences and Technology, Vellore Institute of Technology, Vellore, India
| | - Appakalai N Balamurugan
- Clinical Islet Cell Laboratory, Cardiovascular Innovation Institute, Department of Surgery, University of Louisville, Louisville, KY, USA
| | - Subhashree Venugopal
- School of Biosciences and Technology, Vellore Institute of Technology, Vellore, India.
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12
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Carnosine, pancreatic protection, and oxidative stress in type 1 diabetes. Diabetes 2020. [DOI: 10.1016/b978-0-12-815776-3.00020-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 10/24/2022]
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13
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Miceli V, Pampalone M, Frazziano G, Grasso G, Rizzarelli E, Ricordi C, Casu A, Iannolo G, Conaldi PG. Carnosine protects pancreatic beta cells and islets against oxidative stress damage. Mol Cell Endocrinol 2018; 474:105-118. [PMID: 29496567 DOI: 10.1016/j.mce.2018.02.016] [Citation(s) in RCA: 33] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/04/2017] [Revised: 02/22/2018] [Accepted: 02/23/2018] [Indexed: 02/07/2023]
Abstract
Islet transplantation is a valid therapeutic option for type 1 diabetes treatment. However, in this procedure one of the major problems is the oxidative stress produced during pancreatic islet isolation. The aim of our study was to evaluate potential protective effects of L-carnosine and its isomer D-carnosine against oxidative stress. We evaluated the carnosine effect on cell growth, cell death, insulin production, and the main markers of oxidative stress in rat and murine stressed beta cell lines as well as in human pancreatic islets. Both isomers clearly inhibited hydrogen peroxide induced cytotoxicity, with a decrease in intracellular reactive oxygen and nitrogen species, prevented hydrogen peroxide induced apoptosis/necrosis, nitrite production, and reduced glucose-induced insulin secretion. In addition, NF-κB expression/translocation and nitrated protein induced in stressed cells was significantly reduced. Furthermore, both isomers improved survival and function, and decreased reactive oxygen and nitrogen species, and nitrite and nitrotyrosine production in human islets cultured for 1, 3, and 7 days. These results seem to indicate that both L and D-carnosine have a significant cytoprotective effect by reducing oxidative stress in beta cell lines and human islets, suggesting their potential use to improve islet survival during the islet transplantation procedure.
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Affiliation(s)
- Vitale Miceli
- Department of Laboratory Medicine and Advanced Biotechnologies, IRCCS-ISMETT (Istituto Mediterraneo per i Trapianti e Terapie ad alta specializzazione), Palermo, Italy.
| | | | | | - Giuseppe Grasso
- Department of Chemical Sciences, University of Catania, Catania, Italy
| | - Enrico Rizzarelli
- Institute of Biostructures and Bioimages, National Council of Research (CNR), Catania, Italy
| | - Camillo Ricordi
- Diabetes Research Institute and Cell Transplant Program, University of Miami Miller School of Medicine, Miami, FL, USA
| | - Anna Casu
- Diabetes and Islet Transplantation Unit, Department for the Study and Treatment of Abdominal Diseases and Abdominal Transplantation, IRCCS-ISMETT (Istituto Mediterraneo per i Trapianti e Terapie ad alta specializzazione), Palermo, Italy
| | - Gioacchin Iannolo
- Department of Laboratory Medicine and Advanced Biotechnologies, IRCCS-ISMETT (Istituto Mediterraneo per i Trapianti e Terapie ad alta specializzazione), Palermo, Italy
| | - Pier Giulio Conaldi
- Department of Laboratory Medicine and Advanced Biotechnologies, IRCCS-ISMETT (Istituto Mediterraneo per i Trapianti e Terapie ad alta specializzazione), Palermo, Italy
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14
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Huang HH, Harrington S, Stehno-Bittel L. The Flaws and Future of Islet Volume Measurements. Cell Transplant 2018; 27:1017-1026. [PMID: 29954219 PMCID: PMC6158542 DOI: 10.1177/0963689718779898] [Citation(s) in RCA: 31] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2018] [Revised: 03/09/2018] [Accepted: 04/01/2018] [Indexed: 11/17/2022] Open
Abstract
When working with isolated islet preparations, measuring the volume of tissue is not a trivial matter. Islets come in a large range of sizes and are often contaminated with exocrine tissue. Many factors complicate the procedure, and yet knowledge of the islet volume is essential for predicting the success of an islet transplant or comparing experimental groups in the laboratory. In 1990, Ricordi presented the islet equivalency (IEQ), defined as one IEQ equaling a single spherical islet of 150 μm in diameter. The method for estimating IEQ was developed by visualizing islets in a microscope, estimating their diameter in 50 μm categories and calculating a total volume for the preparation. Shortly after its introduction, the IEQ was adopted as the standard method for islet volume measurements. It has helped to advance research in the field by providing a useful tool improving the reproducibility of islet research and eventually the success of clinical islet transplants. However, the accuracy of the IEQ method has been questioned for years and many alternatives have been proposed, but none have been able to replace the widespread use of the IEQ. This article reviews the history of the IEQ, and discusses the benefits and failings of the measurement. A thorough evaluation of alternatives for estimating islet volume is provided along with the steps needed to uniformly move to an improved method of islet volume estimation. The lessons learned from islet researchers may serve as a guide for other fields of regenerative medicine as cell clusters become a more attractive therapeutic option.
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Affiliation(s)
- Han-Hung Huang
- Angelo State University, Texas Tech University System, San Angelo, TX, USA
| | | | - Lisa Stehno-Bittel
- Likarda, LLC, Kansas City, MO, USA
- University of Kansas Medical Center, Kansas City, KS, USA
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15
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Thompson EM, Sollinger JL, Opara EC, Adin CA. Selective Osmotic Shock for Islet Isolation in the Cadaveric Canine Pancreas. Cell Transplant 2018; 27:542-550. [PMID: 29869518 PMCID: PMC6038033 DOI: 10.1177/0963689717752947] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022] Open
Abstract
Currently, islet isolation is performed using harsh collagenases that cause nonspecific injury to both islets and exocrine tissue, negatively affecting the outcome of cell transplantation. We evaluated a novel islet isolation protocol utilizing high concentrations of glucose to cause selective osmotic shock (SOS). Islets have a membrane glucose transporter that allows adaptation to changes in glucose concentrations while exocrine tissue can be selectively destroyed by these osmolar shifts. Canine pancreata were obtained within 15 min after euthanasia from animals ( n = 6) euthanized for reasons unrelated to this study. Each pancreas was divided into 4 segments that were randomized to receive 300 mOsm glucose for 20 min (group 1), 600 mOsm for 20 min (group 2), 300 mOsm for 40 min (group 3), or 600 mOsm for 40 min (group 4). Islet yield, purity, and viability were compared between groups. Mean ± standard error of the mean islet yield for groups 1 to 4 was 428 ± 159, 560 ± 257, 878 ± 443, and 990 ± 394 islet equivalents per gram, respectively. Purity ranged from 37% to 45% without the use of density gradient centrifugation and was not significantly different between groups. Islet cell viability was excellent overall (89%) and did not differ between treatment protocol. Islet function was best in groups treated with 300 mOsm of glucose (stimulation index [SI] = 3.3), suggesting that the lower concentration of glucose may be preferred for use in canine islet isolation. SOS provides a widely available means for researchers to isolate canine islets for use in islet transplantation or in studies of canine islet physiology.
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Affiliation(s)
- Elizabeth M Thompson
- 1 Department of Clinical Sciences, North Carolina State University, Raleigh, NC, USA
| | - Jennifer L Sollinger
- 1 Department of Clinical Sciences, North Carolina State University, Raleigh, NC, USA
| | - Emmanuel C Opara
- 2 Institute for Regenerative Medicine Center on Diabetes, Obesity, and Metabolism Biomedical Engineering, Wake Forest University, Winston Salem, NC, USA
| | - Christopher A Adin
- 1 Department of Clinical Sciences, North Carolina State University, Raleigh, NC, USA
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16
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Khiatah B, Tucker A, Chen KT, Perez R, Bilbao S, Valiente L, Medrano L, Rawson J, Forouhar E, Omori K, Kandeel F, Qi M, Al-Abdullah IH. Evaluation of collagenase gold plus BP protease in isolating islets from human pancreata. Islets 2018; 10:51-59. [PMID: 29381419 PMCID: PMC5895173 DOI: 10.1080/19382014.2017.1417716] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/26/2023] Open
Abstract
Selection of enzymes for optimal pancreas digestion is essential for successful human islet isolations. The aim of this study was to evaluate the efficacy and outcome of using Collagenase Gold plus BP protease (VitaCyte) (n = 8) by comparing it to two commercially available enzymes, Liberase MTF C/T (Roche) (n = 48) and Collagenase NB1/NP (Serva) (n = 15). The isolation outcomes were assessed by islet counting, viability, glucose-stimulated oxygen consumption rate (OCR), and successful graft-rate following transplantation in diabetic NOD scid mice. The pancreas donor characteristics were not significantly different between the tested enzyme groups regarding their BMI, pancreas weight, cold ischemia time (CIT) and HbA1c. The results show that digested tissue volume was not statistically significant between the VitaCyte enzyme (34.25 ± 5.4 mL) and the Roche enzyme (55.25 ± 3.42 mL, p = 0.073), however, this was significant with Serva enzyme (64.07 ± 7.95 mL, p = 0.020). Interestingly, the islet yields were not statistically different between all enzyme groups. Moreover, when islets were transplanted into NOD scid mice, the reversal rate of diabetes for the VitaCyte enzyme group was similar to all enzyme groups. In conclusion, the effectiveness of Collagenase Gold plus BP protease is comparable to the MTF C/T and the Collagenase NB1/NP enzymes; the low cost could facilitate the use of more pancreata for islet isolations.
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Affiliation(s)
- Bashar Khiatah
- Department of Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research Institute, Beckman Research Institute of the City of Hope, Duarte, CA, USA
| | - Amber Tucker
- Department of Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research Institute, Beckman Research Institute of the City of Hope, Duarte, CA, USA
| | - Kuan-Tsen Chen
- Department of Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research Institute, Beckman Research Institute of the City of Hope, Duarte, CA, USA
| | - Rachel Perez
- Department of Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research Institute, Beckman Research Institute of the City of Hope, Duarte, CA, USA
| | - Shiela Bilbao
- Department of Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research Institute, Beckman Research Institute of the City of Hope, Duarte, CA, USA
| | - Luis Valiente
- Department of Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research Institute, Beckman Research Institute of the City of Hope, Duarte, CA, USA
| | - Leonard Medrano
- Department of Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research Institute, Beckman Research Institute of the City of Hope, Duarte, CA, USA
| | - Jeffrey Rawson
- Department of Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research Institute, Beckman Research Institute of the City of Hope, Duarte, CA, USA
| | - Elena Forouhar
- Department of Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research Institute, Beckman Research Institute of the City of Hope, Duarte, CA, USA
| | - Keiko Omori
- Department of Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research Institute, Beckman Research Institute of the City of Hope, Duarte, CA, USA
| | - Fouad Kandeel
- Department of Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research Institute, Beckman Research Institute of the City of Hope, Duarte, CA, USA
| | - Meirigeng Qi
- Department of Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research Institute, Beckman Research Institute of the City of Hope, Duarte, CA, USA
| | - Ismail H. Al-Abdullah
- Department of Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research Institute, Beckman Research Institute of the City of Hope, Duarte, CA, USA
- CONTACT Ismail H. Al-Abdullah Department of Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research Institute, Beckman Research Institute of the City of Hope, 1500 E. Duarte Rd, Duarte, CA 91010
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17
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Abstract
PURPOSE OF REVIEW Transplantation of allogenic pancreatic islets is a minimally invasive treatment option to control severe hypoglycemia and dependence on exogenous insulin among type 1 diabetes (T1D) patients. This overview summarizes the current issues and progress in islet transplantation outcomes and research. RECENT FINDINGS Several clinical trials from North America and other countries have documented the safety and efficacy of clinical islet transplantation for T1D patients with impaired hypoglycemia awareness. A recently completed phase 3 clinical trial allows centres in the United States to apply for a Food and Drug Administration Biologics License for the procedure. Introduction of anti-inflammatory drugs along with T-cell depleting induction therapy has significantly improved long-term function of transplanted islets. Research into islet biomarkers, immunosuppression, extrahepatic transplant sites and potential alternative beta cell sources is driving further progress. SUMMARY Allogeneic islet transplantation has vastly improved over the past two decades. Success in restoration of glycemic control and hypoglycemic awareness after islet transplantation has been further highlighted by clinical trials. However, lack of effective strategies to maintain long-term islet function and insufficient sources of donor tissue still impose limitations to the widespread use of islet transplantation. In the United States, wide adoption of this technology still awaits regulatory approval and, importantly, a financial mechanism to support the use of this technology.
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18
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Barra JM, Tse HM. Redox-Dependent Inflammation in Islet Transplantation Rejection. Front Endocrinol (Lausanne) 2018; 9:175. [PMID: 29740396 PMCID: PMC5924790 DOI: 10.3389/fendo.2018.00175] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/24/2018] [Accepted: 04/03/2018] [Indexed: 12/19/2022] Open
Abstract
Type 1 diabetes is an autoimmune disease that results in the progressive destruction of insulin-producing pancreatic β-cells inside the islets of Langerhans. The loss of this vital population leaves patients with a lifelong dependency on exogenous insulin and puts them at risk for life-threatening complications. One method being investigated to help restore insulin independence in these patients is islet cell transplantation. However, challenges associated with transplant rejection and islet viability have prevented long-term β-cell function. Redox signaling and the production of reactive oxygen species (ROS) by recipient immune cells and transplanted islets themselves are key players in graft rejection. Therefore, dissipation of ROS generation is a viable intervention that can protect transplanted islets from immune-mediated destruction. Here, we will discuss the newly appreciated role of redox signaling and ROS synthesis during graft rejection as well as new strategies being tested for their efficacy in redox modulation during islet cell transplantation.
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19
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Green ML, Breite AG, Beechler CA, Dwulet FE, McCarthy RC. Effectiveness of different molecular forms of C. histolyticum class I collagenase to recover islets. Islets 2017; 9:177-181. [PMID: 28933628 PMCID: PMC5710699 DOI: 10.1080/19382014.2017.1365996] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
One factor that may contribute to variability between different lots of purified collagenase to recover islets is the molecular form of C. histolyticum class I (C1) collagenase used in the isolation procedure. Two different enzyme mixtures containing C1, class II (C2) collagenase and BP Protease were compared for their effectiveness to recover islets from split adult porcine pancreas. The same enzyme activities per g trimmed tissue were used for all isolations with the only difference being the mass of C1 required to achieve 25,000 collagen degradation activity U/g tissue. The results show no differences in performance of the two enzyme mixtures. The only significant difference is 19 fold more truncated C1 was required to achieve the same result as intact C1.
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Affiliation(s)
| | | | | | | | - Robert C. McCarthy
- VitaCyte LLC, Indianapolis, IN, USA
- CONTACT Robert C. McCarthy VitaCyte LLC, 1341 Sadlier Circle West Drive, Indianapolis, IN 46239, USA
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20
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Bondoc AJ, Abu-El-Haija M, Nathan JD. Pediatric pancreas transplantation, including total pancreatectomy with islet autotransplantation. Semin Pediatr Surg 2017; 26:250-256. [PMID: 28964481 DOI: 10.1053/j.sempedsurg.2017.07.004] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Unlike other solid-organ transplants, whole pancreas transplantation in children is relatively rare, and it occurs more frequently in the context of multivisceral or composite organ transplantation. Because children only infrequently suffer severe sequelae of type 1 diabetes mellitus, pancreas transplantation is rarely indicated in the pediatric population. More commonly, pediatric pancreas transplant occurs in the setting of incapacitating acute recurrent or chronic pancreatitis, specifically islet autotransplantation after total pancreatectomy. In this clinical scenario, total pancreatectomy removes the nidus of chronic pain and debilitation, while autologous islet transplantation aims to preserve endocrine function. The published experiences with pediatric total pancreatectomy with islet autotransplantation (TPIAT) in children has demonstrated excellent outcomes including liberation from chronic opioid use, as well as improved mental and physical quality of life with good glycemic control. Given the complexity of the operation, risk of postoperative complication, and long-term physiologic changes, appropriate patient selection and comprehensive multidisciplinary care teams are critical to ensuring optimal outcomes.
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Affiliation(s)
- Alexander J Bondoc
- Division of Pediatric General and Thoracic Surgery, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Mail Location 2023, Cincinnati, Ohio 45229.
| | - Maisam Abu-El-Haija
- Division of Gastroenterology, Hepatology and Nutrition, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio
| | - Jaimie D Nathan
- Division of Pediatric General and Thoracic Surgery, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Mail Location 2023, Cincinnati, Ohio 45229
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21
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Lakey JR, Cavanagh TJ, Zieger MA, Wright M. Evaluation of a Purified Enzyme Blend for the Recovery and Function of Canine Pancreatic Islets. Cell Transplant 2017; 7:365-72. [PMID: 9710305 DOI: 10.1177/096368979800700404] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022] Open
Abstract
Recently developed technologies enabling the production of a reproducible, purified enzyme blend for optimal human pancreatic islet isolation has renewed interest in clinical islet transplantation. The canine model has been an ideal preclinical model for the development of islet transplantation protocols. As seen in other species, the application of crude collagenase for isolating canine islets resulted in highly variable islet yields, extensive islet fragmentation, and variable islet functionality. We compared the function of commercially available crude collagenases with that of Liberase™-CI purified enzyme blend for canine islet isolation. We also compared two manufacturing runs of Liberase-CI enzyme (lots 1 and 2) to demonstrate reproducibility of islet recovery and function. We report on the improved recovery and function of islets isolated using Liberase-CI enzyme. No difference in dog age, mean body weight, or pancreas weight were observed between the experimental groups. We observed a significantly higher postpurification recovery of islet equivalent number (IE) from pancreases processed using two lots of Liberase-CI enzyme (189 ± 20 × 103 IE, n = 4) and lot 2 (234 ± 39 × 103 IE, n = 7) than that obtained from pancreases processed with Sigma Type V (116.8 ± 27 × 103 IE, n = 5), Serva collagenase (49 ± 11.6 × 103 IE, n = 5, p < 0.05) or Boehringer–Mannheim (BM) Type P collagenase (85.4 ± 25 × 103 IE, n = 5, p < 0.05, ANOVA). No significant differences were observed in islet yield recovery from pancreases processed using the two production lots of Liberase-CI enzyme. Islet survival after 48 h in culture at 37°C was significantly higher from islets isolated using Liberase-CI enzyme (88 ± 3.7% survival) when compared to Sigma Type V (81.8 ± 3.3%), Serva (71.7 ± 2.8%), and BM Type P (77 ± 7.2%) (p < 0.05). Islet functional testing in vitro demonstrated islets isolated using crude collagenase had an increased insulin basal release and a reduced insulin stimulated response when compared with islets isolated using the two lots of Liberase-CI enzyme. The calculated stimulation index was 7.8 ± 1.7, 3.1 ± 0.6, and 4.8 ± 1.1 for Sigma Type V, Serva, and BM Type P isolated islets, respectively, compared to 15.7 ± 1.6 and 16.2 ± 1.9 for islets isolated with Liberase-CI enzyme production lots 1 and 2, respectively (p < 0.05). This evaluation demonstrates that a purified enzyme blend can significantly improve islet recovery and function. It also demonstrates the manufacturing reproducibility of Liberase-CI enzyme lots resulting in the isolation of canine islets with the same degree of efficacy. A blend of purified enzymes, specifically formulated for canine islet isolation, can consistently yield large numbers of islets that survive longer in culture and demonstrate an improved insulin response in vitro.
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Affiliation(s)
- J R Lakey
- Comprehensive Tissue Center, Department of Surgery, Laboratory Medicine and Pathology, University of Alberta, Edmonton, Canada
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22
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Affiliation(s)
- James R. Wright
- Departments of Pathology, Biomedical Engineering, and Surgery, Izaak Walton Killam Health Centre, Dalhousie University Faculty of Medicine, 5850 University Ave., Halifax, Nova Scotia, Canada B3J 3G9
| | - Bill Pohajdak
- Department of Biology, 5076 Life Sciences Bldg., Dalhousie University, Halifax, Nova Scotia, Canada B3H 4J1
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23
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Georges P, Muirhead RP, Williams L, Holman S, Tabiin MT, Dean SK, Tuch BE. Comparison of Size, Viability, and Function of Fetal Pig Islet-Like Cell Clusters after Digestion Using Collagenase or Liberase. Cell Transplant 2017. [DOI: 10.3727/000000002783985477] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
Liberase is a highly purified blend of collagenases that has been specifically developed to eliminate the numerous problems associated with the conventional use of crude collagenase when isolating islet-like cell clusters (ICCs) from pancreases of different species. The influence of Liberase on yield, size, viability, and function of ICCs has been documented when this enzyme was used to digest adult but not fetal pancreases. In this study, we compared the effects of collagenase and Liberase on fetal pig ICCs. A total of eight fetal pig pancreas digestions were analyzed. Fetuses were obtained from Large White Landrace pigs of gestational age 80 ± 2.1 days. The pancreases were digested with either 3 mg/ml collagenase P or 1.2 mg/ml Liberase HI. The time taken to digest the pancreas was shorter for collagenase when compared with Liberase (22 ± 2 vs. 31 ± 2 min). The size of ICCs was similar for both collagenase (83 ± 0.5 μm) and Liberase (79 ± 0.4 μm) as was the number of ICCs produced per pancreas (7653 ± 1297 vs. 8101 ± 1177). Viability, as assessed using fluorescent markers, was slightly greater for Liberase (79 ± 1% vs. 76 ± 1%, p < 0.05). Responsiveness to β-cell stimulus (20 mM KCl) was similar for both methods of isolation, as was the insulin content of the ICCs, both in vitro and at 1 month after transplantation of 1500 ICCs beneath the renal capsule of immunoincompetent mice. Despite the high content of endotoxins in collagenase, the above results show that this enzyme was equally as efficient as Liberase in isolating functional ICCs from fetal pig pancreas.
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Affiliation(s)
- Pauline Georges
- Diabetes Transplant Unit, Prince of Wales Hospital and The University of New South Wales, Sydney, New South Wales, 2031, Australia
| | - Roslyn P. Muirhead
- Diabetes Transplant Unit, Prince of Wales Hospital and The University of New South Wales, Sydney, New South Wales, 2031, Australia
| | - Lindy Williams
- Diabetes Transplant Unit, Prince of Wales Hospital and The University of New South Wales, Sydney, New South Wales, 2031, Australia
| | - Sara Holman
- Diabetes Transplant Unit, Prince of Wales Hospital and The University of New South Wales, Sydney, New South Wales, 2031, Australia
| | - Muhammad Tani Tabiin
- Diabetes Transplant Unit, Prince of Wales Hospital and The University of New South Wales, Sydney, New South Wales, 2031, Australia
| | - Sophia K. Dean
- Diabetes Transplant Unit, Prince of Wales Hospital and The University of New South Wales, Sydney, New South Wales, 2031, Australia
| | - Bernard E. Tuch
- Diabetes Transplant Unit, Prince of Wales Hospital and The University of New South Wales, Sydney, New South Wales, 2031, Australia
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Matsumoto S, Rigley TH, Qualley SA, Kuroda Y, Reems JA, Stevens RB. 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 2017. [DOI: 10.3727/000000002783985332] [Citation(s) in RCA: 76] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
Previous reports indicate that the two-layer method (TLM) of human pancreas preservation is superior to University of Wisconsin solution (UW) when pancreata are preserved for extended periods (i.e., >24 h) prior to islet isolation. In this study, the efficacy of using the TLM for preserving pancreata for short periods (i.e., <13 h) was evaluated using both nonhuman primate and human pancreata preserved with a TLM kit precharged with oxygen. An oxygen precharged TLM (static TLM) was established and compared with the original TLM with continuous oxygen supply. For the static TLM, the perfluorochemical was fully oxygenated and the oxygen supply removed prior to pancreas preservation. In the primate model, pancreata were preserved by the static TLM, the original TLM, and UW for 5 h prior to islet isolation. In the human model, pancreata were preserved with the static TLM or the original TLM or UW for 4–13 h. Both primate and human pancreata were processed by intraductal collagenase injection and digestion followed by continuous density gradient purification to isolate islets. Islets were assessed for islet yield, purity, viability, and in vitro functionality. In the primate model, islet yield, viability, and in vitro functionality were significantly improved by both the static TLM and the original TLM with similar results. Postculture islet yields were 23,877 ± 3619 IE/g in the static TLM, 21,895 ± 3742 IE/g in the original TLM, and 6773 ± 735 IE/g in UW. In the human model, both the static TLM and the original TLM significantly increased islet yield compared with UW with postculture islet yields of 2659 ± 549 IE/g in the static TLM, 2244 ± 557 IE/g in the original TLM, and 1293 ± 451 IE/g in UW. Nonhuman primate and human pancreata stored in the static TLM, immediately upon procurement, yield isolated islets of a substantially higher quantity than when pancreata are stored in UW. Thus, the use of the static TLM should replace the use of UW for storage of pancreata during transport prior to islet isolation.
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Affiliation(s)
- Shinichi Matsumoto
- Puget Sound Blood Center/Northwest Tissue Center, 921 Terry Avenue, Seattle, WA 98104
- University of Washington Medical Center, Department of Surgery, Division of Transplantation, 1959 N.E. Pacific Street, Seattle, WA 98195
| | - Theodore H. Rigley
- University of Nebraska Medical Center, Department of Surgery, Section of Transplantation, 983285 Nebraska Medical Center, Omaha, NE 68498-3285
| | - Sabrina A. Qualley
- Puget Sound Blood Center/Northwest Tissue Center, 921 Terry Avenue, Seattle, WA 98104
| | - Yoshikazu Kuroda
- Department of Gastroenterological Surgery, Graduate School of Medical Sciences, Kobe University, 7-5-2 Kusunoki-Cho Chuo-Ku Kobe, Japan 650
| | - Jo Anna Reems
- Puget Sound Blood Center/Northwest Tissue Center, 921 Terry Avenue, Seattle, WA 98104
- University of Washington Medical Center, Department of Hematology, Division of Medicine, 1959 N.E. Pacific Street, Seattle, WA 98195
| | - R. Brian Stevens
- University of Nebraska Medical Center, Department of Surgery, Section of Transplantation, 983285 Nebraska Medical Center, Omaha, NE 68498-3285
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Chen RL, James RFL. Characterization of an Important Enzymatic Component in Collagenase that is Essential for the Effective Digestion of the Human and Porcine Pancreas. Cell Transplant 2017. [DOI: 10.3727/000000001783986189] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Affiliation(s)
- Ruo L. Chen
- Department of Surgery, University of Leicester, RKB, Royal Infirmary, Leicester LE2 7LX, UK
| | - Roger F. L. James
- Department of Surgery, University of Leicester, RKB, Royal Infirmary, Leicester LE2 7LX, UK
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Brandhorst D, Brandhorst H, Johnson PRV. Enzyme Development for Human Islet Isolation: Five Decades of Progress or Stagnation? Rev Diabet Stud 2017. [PMID: 28632819 DOI: 10.1900/rds.2017.14.22] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/25/2023] Open
Abstract
In comparison to procedures used for the separation of individual cell types from other organs, the process of human pancreatic islet isolation aims to digest the pancreatic exocrine matrix completely without dispersing the individual cells within the endocrine cell cluster. This objective is unique within the field of tissue separation, and outlines the challenge of islet isolation to balance two opposing priorities. Although significant progress has been made in the characterization and production of enzyme blends for islet isolation, there are still numerous areas which require improvement. The ultimate goal of enzyme production, namely the routine production of a consistent and standardized enzyme blend, has still not been realized. This seems to be mainly the result of a lack of detailed knowledge regarding the structure of the pancreatic extracellular matrix and the synergistic interplay between collagenase and different supplementary proteases during the degradation of the extracellular matrix. Furthermore, the activation of intrinsic proteolytic enzymes produced by the pancreatic acinar cells, also impacts on the chance of a successful outcome of human islet isolation. This overview discusses the challenges of pancreatic enzymatic digestion during human islet isolation, and outlines the developments in this field over the past 5 decades.
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Affiliation(s)
- Daniel Brandhorst
- Nuffield Department of Surgical Sciences, University of Oxford, United Kingdom
| | - Heide Brandhorst
- Nuffield Department of Surgical Sciences, University of Oxford, United Kingdom
| | - Paul R V Johnson
- Nuffield Department of Surgical Sciences, University of Oxford, United Kingdom
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27
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Park S, Lee DY. The anterior chamber of the eye as a site for pancreatic islet transplantation. J IND ENG CHEM 2017. [DOI: 10.1016/j.jiec.2017.02.027] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
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Abstract
Type 1 diabetes (T1D) patients who receive pancreatic islet transplant experience significant improvement in their quality-of-life. This comes primarily through improved control of blood sugar levels, restored awareness of hypoglycemia, and prevention of serious and potentially life-threatening diabetes-associated complications, such as kidney failure, heart and vascular disease, stroke, nerve damage, and blindness. Therefore, beta cell replacement through transplantation of isolated islets is an important option in the treatment of T1D. However, lasting success of this promising therapy depends on durable survival and efficacy of the transplanted islets, which are directly influenced by the islet isolation procedures. Thus, isolating pancreatic islets with consistent and reliable quality is critical in the clinical application of islet transplantation.Quality of isolated islets is important in pre-clinical studies as well, as efforts to advance and improve clinical outcomes of islet transplant therapy have relied heavily on animal models ranging from rodents, to pigs, to nonhuman primates. As a result, pancreatic islets have been isolated from these and other species and used in a variety of in vitro or in vivo applications for this and other research purposes. Protocols for islet isolation have been somewhat similar across species, especially, in mammals. However, given the increasing evidence about the distinct structural and functional features of human and mouse islets, using similar methods of islet isolation may contribute to inconsistencies in the islet quality, immunogenicity, and experimental outcomes. This may also contribute to the discrepancies commonly observed between pre-clinical findings and clinical outcomes. Therefore, it is prudent to consider the particular features of pancreatic islets from different species when optimizing islet isolation protocols.In this chapter, we explore the structural and functional features of pancreatic islets from mice, pigs, nonhuman primates, and humans because of their prevalent use in nonclinical, preclinical, and clinical applications.
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Kesseli SJ, Wagar M, Jung MK, Smith KD, Lin YK, Walsh RM, Hatipoglu B, Freeman ML, Pruett TL, Beilman GJ, Sutherland DER, Dunn TB, Axelrod DA, Chaidarun SS, Stevens TK, Bellin M, Gardner TB. Long-Term Glycemic Control in Adult Patients Undergoing Remote vs. Local Total Pancreatectomy With Islet Autotransplantation. Am J Gastroenterol 2017; 112:643-649. [PMID: 28169284 DOI: 10.1038/ajg.2017.14] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/02/2016] [Accepted: 01/04/2017] [Indexed: 12/11/2022]
Abstract
OBJECTIVES Total pancreatectomy with islet autotransplantation (TPIAT) is increasingly performed with remote islet cell processing and preparation, i.e., with islet cell isolation performed remotely from the primary surgical site at an appropriately equipped islet isolation facility. We aimed to determine whether TPIAT using remote islet isolation results in comparable long-term glycemic outcomes compared with TPIAT performed with standard local isolation. METHODS We performed a retrospective cohort study of adult patients who underwent TPIAT at three tertiary care centers from 2010 to 2013. Two centers performed remote isolation and one performed local isolation. Explanted pancreata in the remote cohort were transported ∼130 miles to and from islet isolation facilities. The primary outcome was insulin independence 1 year following transplant. RESULTS Baseline characteristics were similar between groups except the remote cohort had higher preoperative hemoglobin A1c (HbA1c; 5.43 vs. 5.25, P=0.02) and there were more females in the local cohort (58% vs. 76%, P=0.049). At 1 year, 27% of remote and 32% of local patients were insulin independent (P=0.48). Remote patients experienced a greater drop in fasting c-peptide (-1.66 vs. -0.64, P=0.006) and a greater rise in HbA1c (1.65 vs. 0.99, P=0.014) at 1-year follow-up. A preoperative c-peptide >2.7 (odds ratio (OR) 4.4, 95% confidence interval (CI) 1.6-14.3) and >3,000 islet equivalents/kg (OR 11.0, 95% CI 3.2-37.3) were associated with one-year insulin independence in the local group. CONCLUSIONS At 1 year after TPIAT, patients undergoing remote surgery have equivalent rates of long-term insulin independence compared with patients undergoing TPIAT locally, but metabolic control is superior with local isolation.
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Affiliation(s)
- Samuel J Kesseli
- Section of Gastroenterology and Hepatology, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire, USA
| | - Matthew Wagar
- Section of Endocrinology, University of Minnesota Medical Center, Minneapolis, Minnesota, USA
| | - Min K Jung
- Section of Gastroenterology and Hepatology, Digestive Disease Institute, Cleveland Clinic Foundation, Cleveland, Ohio, USA
| | - Kerrington D Smith
- Section of General Surgery, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire, USA
| | - Yu Kuei Lin
- Department of Endocrinology, Endocrinology and Metabolism Institute, Cleveland Clinic Foundation, Cleveland, Ohio, USA
| | - R Matthew Walsh
- Department of General Surgery, Digestive Disease Institute, Cleveland Clinic Foundation, Cleveland, Ohio, USA
| | - Betul Hatipoglu
- Section of Gastroenterology and Hepatology, Digestive Disease Institute, Cleveland Clinic Foundation, Cleveland, Ohio, USA
| | - Martin L Freeman
- Section of Gastroenterology, University of Minnesota Medical Center, Minneapolis, Minnesota, USA
| | - Timothy L Pruett
- Deparment of Surgery, University of Minnesota Medical Center, Minneapolis, Minnesota, USA
| | - Gregory J Beilman
- Deparment of Surgery, University of Minnesota Medical Center, Minneapolis, Minnesota, USA
| | - David E R Sutherland
- Deparment of Surgery, University of Minnesota Medical Center, Minneapolis, Minnesota, USA
| | - Ty B Dunn
- Deparment of Surgery, University of Minnesota Medical Center, Minneapolis, Minnesota, USA
| | - David A Axelrod
- Section of Transplant Surgery, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire, USA
| | - Sushela S Chaidarun
- Section of Endocrinology, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire, USA
| | - Tyler K Stevens
- Section of Gastroenterology and Hepatology, Digestive Disease Institute, Cleveland Clinic Foundation, Cleveland, Ohio, USA
| | - Melena Bellin
- Section of Endocrinology, University of Minnesota Medical Center, Minneapolis, Minnesota, USA
| | - Timothy B Gardner
- Section of Gastroenterology and Hepatology, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire, USA
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Al-Abdullah IH, Bagramyan K, Bilbao S, Qi M, Kalkum M. Fluorogenic Peptide Substrate for Quantification of Bacterial Enzyme Activities. Sci Rep 2017; 7:44321. [PMID: 28287171 PMCID: PMC5347087 DOI: 10.1038/srep44321] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2016] [Accepted: 02/06/2017] [Indexed: 12/18/2022] Open
Abstract
A novel peptide substrate (A G G P L G P P G P G G) was developed for quantifying the activities of bacterial enzymes using a highly sensitive Fluorescence Resonance Energy Transfer (FRET) based assay. The peptide substrate was cleaved by collagenase class I, II, Liberase MTF C/T, collagenase NB1, and thermolysin/neutral protease, which was significantly enhanced in the presence of CaCl2. However, the activities of these enzymes were significantly decreased in the presence of ZnSO4 or ZnCl2. Collagenase I, II, Liberase MTF C/T, thermolysin/neutral protease share similar cleavage sites, L↓G and P↓G. However, collagenase NB1 cleaves the peptide substrate at G↓P and P↓L, in addition to P↓G. The enzyme activity is pH dependent, within a range of 6.8 to 7.5, but was significantly diminished at pH 8.0. Interestingly, the peptide substrate was not cleaved by endogenous pancreatic protease such as trypsin, chymotrypsin, and elastase. In conclusion, the novel peptide substrate is collagenase, thermolysin/neutral protease specific and can be applied to quantify enzyme activities from different microbes. Furthermore, the assay can be used for fine-tuning reaction mixtures of various agents to enhance the overall activity of a cocktail of multiple enzymes and achieve optimal organ/tissue digestion, while protecting the integrity of the target cells.
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Affiliation(s)
- Ismail H. Al-Abdullah
- Department of Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research Institute, Beckman Research Institute of the City of Hope, USA
| | - Karine Bagramyan
- Department of Molecular Immunology, Beckman Research Institute of the City of Hope, USA
| | - Shiela Bilbao
- Department of Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research Institute, Beckman Research Institute of the City of Hope, USA
| | - Meirigeng Qi
- Department of Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research Institute, Beckman Research Institute of the City of Hope, USA
| | - Markus Kalkum
- Department of Molecular Immunology, Beckman Research Institute of the City of Hope, USA
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31
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Bartlett DC, Newsome PN. A Modified Protocol for the Isolation of Primary Human Hepatocytes with Improved Viability and Function from Normal and Diseased Human Liver. Methods Mol Biol 2017; 1506:61-73. [PMID: 27830545 DOI: 10.1007/978-1-4939-6506-9_4] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
Successful hepatocyte isolation is critical for continued development of cellular transplantation. However, most tissue available for research is from diseased liver and the results of hepatocyte isolation from such tissue are inferior compared to normal tissue. Here we describe a modified method, combining the use of Liberase and N-acetylcysteine (NAC), for the isolation of primary human hepatocytes with high viability from normal and diseased liver.
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Affiliation(s)
- David C Bartlett
- National Institute for Health Research (NIHR) Biomedical Research Unit and Centre for Liver Research, University of Birmingham, Birmingham, UK. .,The Liver Unit, Queen Elizabeth Hospital Birmingham, Birmingham, UK.
| | - Philip N Newsome
- National Institute for Health Research (NIHR) Biomedical Research Unit and Centre for Liver Research, University of Birmingham, Birmingham, UK. .,The Liver Unit, Queen Elizabeth Hospital Birmingham, Birmingham, UK.
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32
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Kim JW, You YH, Ham DS, Yang HK, Yoon KH. The Paradoxical Effects of AMPK on Insulin Gene Expression and Glucose-Induced Insulin Secretion. J Cell Biochem 2016; 117:239-46. [PMID: 26105159 DOI: 10.1002/jcb.25271] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2015] [Accepted: 06/19/2015] [Indexed: 12/23/2022]
Abstract
The activation of AMP-activated protein kinase (AMPK) is known to repress the expression of the insulin gene and glucose-stimulated insulin secretion (GSIS). However, the mechanisms by which this occurs, as well as the effects of AMPK activation on glucolipotoxicity-induced β-cell dysfunction, have not been elucidated. To investigate the effects of 5-amino-4-imidazolecarboxamide ribonucleotide (AICAR) and peroxisome proliferator-activated receptorγ-coactivator-1α (PGC-1α) on β-cell-specific genes under glucolipotoxic conditions, we performed real-time PCR and measured insulin secretion by primary islets. To study these effects in vivo, we administered AICAR for 10 days (1 mg/g body weight) to 90% pancreatectomized hyperglycemic mice. The exposure of isolated rat and human islets to glucolipotoxic conditions and the overexpression of PGC-1α suppressed insulin and NEUROD1 mRNA expression. However, the expression of these genes was preserved by AICAR treatment and by PGC-1α inhibition. Exposure of isolated islets to glucolipotoxic conditions for 3 days decreased GSIS, which was also well maintained by AICAR treatment and by PGC-1α inhibition. The administration of AICAR to 90% pancreatectomized hyperglycemic mice improved glucose tolerance and insulin secretion. These results indicate that treatment of islets with an AMPK agonist under glucolipotoxic conditions protects against glucolipotoxicity-induced β-cell dysfunction. A better understanding of the functions of molecules such as PGC-1α and AMPK, which play key roles in intracellular fuel regulation, could herald a new era for the treatment of patients with type 2 diabetes mellitus by providing protection against glucolipotoxicity.
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Affiliation(s)
- Ji-Won Kim
- Department of Endocrinology & Metabolism, The Catholic University of Korea, Seoul, 137-040, Korea
| | - Young-Hye You
- Department of Endocrinology & Metabolism, The Catholic University of Korea, Seoul, 137-040, Korea
| | - Dong-Sik Ham
- Department of Endocrinology & Metabolism, The Catholic University of Korea, Seoul, 137-040, Korea
| | - Hae Kyung Yang
- Department of Endocrinology & Metabolism, The Catholic University of Korea, Seoul, 137-040, Korea
| | - Kun-Ho Yoon
- Department of Endocrinology & Metabolism, The Catholic University of Korea, Seoul, 137-040, Korea
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Abstract
Although experimental islet isolation and transplantation has continued for over 25 years, the results of human islet transplantation in patients with type 1 diabetes were disappointing until June 2000 when the University of Alberta, Edmonton, Canada reported 100% insulin independence in a cohort of seven patients. The study introduced several innovations now under evaluation worldwide. In the United Kingdom, an Islet Transplant Consortium has been established to co-ordinate clinical trials in several centres. The Canadian results have renewed hope of establishing islet transplantation as a treatment for diabetes while highlighting the need to identify plentiful sources of insulin secreting tissue and alternatives to current immunosuppressive therapies.
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Affiliation(s)
| | | | - Richard Downing
- The Islet Research Laboratory, Worcestershire Clinical Research Unit, Worcestershire Acute Hospitals NHS Trust, Worcester Royal Infirmary, Newtown Road, Worcester, WR5 1HN,
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Oh YS, Seo EH, Lee YS, Cho SC, Jung HS, Park SC, Jun HS. Increase of Calcium Sensing Receptor Expression Is Related to Compensatory Insulin Secretion during Aging in Mice. PLoS One 2016; 11:e0159689. [PMID: 27441644 PMCID: PMC4956240 DOI: 10.1371/journal.pone.0159689] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2016] [Accepted: 07/05/2016] [Indexed: 02/06/2023] Open
Abstract
Type 2 diabetes is caused by both insulin resistance and relative insulin deficiency. To investigate age-related changes in glucose metabolism and development of type 2 diabetes, we compared glucose homeostasis in different groups of C57BL/6J mice ranging in age from 4 months to 20 months (4, 8, 12, 16 and 20 months). Interestingly, we observed that non-fasting glucose levels were not significantly changed, but glucose tolerance gradually increased by 20 months of age, whereas insulin sensitivity declined with age. We found that the size of islets and glucose-stimulated insulin secretion increased with aging. However, mRNA expression of pancreatic and duodenal homeobox 1 and granuphilin was decreased in islets of older mice compared with that of 4-month-old mice. Serum calcium (Ca2+) levels were significantly decreased at 12, 20 and 28 months of age compared with 4 months and calcium sensing receptor (CaSR) mRNA expression in the islets significantly increased with age. An extracellular calcium depletion agent upregulated CaSR mRNA expression and consequently enhanced insulin secretion in INS-1 cells and mouse islets. In conclusion, we suggest that decreased Ca2+ levels and increased CaSR expression might be involved in increased insulin secretion to compensate for insulin resistance in aged mice.
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Affiliation(s)
- Yoon Sin Oh
- Lee Gil Ya Cancer and Diabetes Institute, Gachon University, Incheon, Korea
- Gachon Medical Research Institute, Gil Hospital, Incheon, Korea
| | - Eun-Hui Seo
- Lee Gil Ya Cancer and Diabetes Institute, Gachon University, Incheon, Korea
| | - Young-Sun Lee
- Lee Gil Ya Cancer and Diabetes Institute, Gachon University, Incheon, Korea
| | - Sung Chun Cho
- Lee Gil Ya Cancer and Diabetes Institute, Gachon University, Incheon, Korea
| | - Hye Seung Jung
- Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Korea
| | - Sang Chul Park
- Lee Gil Ya Cancer and Diabetes Institute, Gachon University, Incheon, Korea
| | - Hee-Sook Jun
- Lee Gil Ya Cancer and Diabetes Institute, Gachon University, Incheon, Korea
- Gachon Medical Research Institute, Gil Hospital, Incheon, Korea
- College of Pharmacy and Gachon Institute of Pharmaceutical Science, Gachon University, Incheon, Korea
- * E-mail:
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Truong W, Shapiro AMJ. Progress in islet transplantation in patients with type 1 diabetes mellitus. ACTA ACUST UNITED AC 2016; 5:147-58. [PMID: 16677057 DOI: 10.2165/00024677-200605030-00003] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/02/2022]
Abstract
More than 500 patients with type 1 diabetes mellitus have now received islet transplants at over 50 institutions worldwide in the past 5 years. Rates of insulin independence at 1 year with current protocols are impressive. However, inexorable decay of islet function over time indicates that there are many opportunities for improvement. Improved control of glycosylated hemoglobin and reduced risk of recurrent hypoglycemia are seen as important benefits of islet transplantation, irrespective of the status regarding insulin independence. For the use of islet transplantation to expand it is essential that the donor-to-recipient ratio be reliably reduced to 1 : 1. Enormous opportunities lie ahead for the development of successful living donor islet transplantation, single donor protocols, improved engraftment, islet proliferation in vitro and in the recipient, alternative islet sources, and novel tolerizing drugs. With these emerging opportunities, islet transplantation may expand to include more patients with type 1 diabetes, including children, and will not be restricted to the most unstable forms of the disease, as it is today.
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Affiliation(s)
- Wayne Truong
- Department of Surgery, Faculty of Medicine, University of Alberta, Edmonton, Alberta, Canada
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Hawthorne WJ, Williams L, Chew YV. Clinical Islet Isolation. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2016; 938:89-122. [PMID: 27586424 DOI: 10.1007/978-3-319-39824-2_7] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
The overarching success of islet transplantation relies on the success in the laboratory to isolate the islets. This chapter focuses on the processes of human islet cell isolation and the ways to optimally provide islet cells for transplantation. The major improvements in regards to the choice of enzyme type, way the digested pancreas tissue is handled to best separate islets from the acinar and surrounding tissues, the various methods of purification of the islets, their subsequent culture and quality assurance to improve outcomes to culminate in safe and effective islet transplantation will be discussed. After decades of improvements, islet cell isolation and transplantation now clearly offer a safe, effective and feasible therapeutic treatment option for an increasing number of patients suffering from type 1 diabetes specifically for those with severe hypoglycaemic unawareness.
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Affiliation(s)
- Wayne J Hawthorne
- National Pancreas and Islet Transplant Laboratories, The Westmead Institute for Medical Research, Westmead, NSW, 2145, Australia. .,Department of Surgery, Westmead Clinical School, Westmead Hospital, University of Sydney, Westmead, NSW, 2145, Australia.
| | - Lindy Williams
- National Pancreas and Islet Transplant Laboratories, The Westmead Institute for Medical Research, Westmead, NSW, 2145, Australia
| | - Yi Vee Chew
- National Pancreas and Islet Transplant Laboratories, The Westmead Institute for Medical Research, Westmead, NSW, 2145, Australia
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Identifying Effective Enzyme Activity Targets for Recombinant Class I and Class II Collagenase for Successful Human Islet Isolation. Transplant Direct 2015; 2:e54. [PMID: 27500247 DOI: 10.1097/txd.0000000000000563] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2015] [Accepted: 11/17/2015] [Indexed: 11/25/2022] Open
Abstract
UNLABELLED Isolation following a good manufacturing practice-compliant, human islet product requires development of a robust islet isolation procedure where effective limits of key reagents are known. The enzymes used for islet isolation are critical but little is known about the doses of class I and class II collagenase required for successful islet isolation. METHODS We used a factorial approach to evaluate the effect of high and low target activities of recombinant class I (rC1) and class II (rC2) collagenase on human islet yield. Consequently, 4 different enzyme formulations with divergent C1:C2 collagenase mass ratios were assessed, each supplemented with the same dose of neutral protease. Both split pancreas and whole pancreas models were used to test enzyme targets (n = 20). Islet yield/g pancreas was compared with historical enzymes (n = 42). RESULTS Varying the Wunsch (rC2) and collagen degradation activity (CDA, rC1) target dose, and consequently the C1:C2 mass ratio, had no significant effect on tissue digestion. Digestions using higher doses of Wunsch and CDA resulted in comparable islet yields to those obtained with 60% and 50% of those activities, respectively. Factorial analysis revealed no significant main effect of Wunsch activity or CDA for any parameter measured. Aggregate results from 4 different collagenase formulations gave 44% higher islet yield (>5000 islet equivalents/g) in the body/tail of the pancreas (n = 12) when compared with those from the same segment using a standard natural collagenase/protease mixture (n = 6). Additionally, islet yields greater than 5000 islet equivalents/g pancreas were also obtained in whole human pancreas. CONCLUSIONS A broader C1:C2 ratio can be used for human islet isolation than has been used in the past. Recombinant collagenase is an effective replacement for the natural enzyme and we have determined that high islet yield can be obtained even with low doses of rC1:rC2, which is beneficial for the survival of islets.
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Quiskamp N, Bruin JE, Kieffer TJ. Differentiation of human pluripotent stem cells into β-cells: Potential and challenges. Best Pract Res Clin Endocrinol Metab 2015; 29:833-47. [PMID: 26696513 DOI: 10.1016/j.beem.2015.10.011] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) hold great potential as the basis for cell-based therapies of degenerative diseases, including diabetes. Current insulin-based therapies for diabetes do not prevent hyperglycaemia or the associated long-term organ damage. While transplantation of pancreatic islets can achieve insulin independence and improved glycemic control, it is limited by donor tissue scarcity, challenges of purifying islets from the pancreas, and the need for immunosuppression to prevent rejection of transplants. Large-scale production of β-cells from stem cells is a promising alternative. Recent years have seen considerable progress in the optimization of in vitro differentiation protocols to direct hESCs/iPSCs into mature insulin-secreting β-cells and clinical trials are now under way to test the safety and efficiency of hESC-derived pancreatic progenitor cells in patients with type 1 diabetes. Here, we discuss key milestones leading up to these trials in addition to recent developments and challenges for hESC/iPSC-based diabetes therapies and disease modeling.
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Affiliation(s)
- Nina Quiskamp
- Laboratory of Molecular and Cellular Medicine, Department of Cellular & Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, BC, Canada.
| | - Jennifer E Bruin
- Laboratory of Molecular and Cellular Medicine, Department of Cellular & Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, BC, Canada.
| | - Timothy J Kieffer
- Laboratory of Molecular and Cellular Medicine, Department of Cellular & Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, BC, Canada; Department of Surgery, University of British Columbia, Vancouver, BC, Canada.
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Lablanche S, Borot S, Wojtusciszyn A, Bayle F, Tétaz R, Badet L, Thivolet C, Morelon E, Frimat L, Penfornis A, Kessler L, Brault C, Colin C, Tauveron I, Bosco D, Berney T, Benhamou PY. Five-Year Metabolic, Functional, and Safety Results of Patients With Type 1 Diabetes Transplanted With Allogenic Islets Within the Swiss-French GRAGIL Network. Diabetes Care 2015; 38:1714-22. [PMID: 26068866 DOI: 10.2337/dc15-0094] [Citation(s) in RCA: 89] [Impact Index Per Article: 8.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/15/2015] [Accepted: 05/25/2015] [Indexed: 02/03/2023]
Abstract
OBJECTIVE To describe the 5-year outcomes of islet transplantation within the Swiss-French GRAGIL Network. RESEARCH DESIGN AND METHODS Retrospective analysis of all subjects enrolled in the GRAGIL-1c and GRAGIL-2 islet transplantation trials. Parameters related to metabolic control, graft function, and safety outcomes were studied. RESULTS Forty-four patients received islet transplantation (islet transplantation alone [ITA] 24 patients [54.5%], islet after kidney [IAK] transplantation 20 patients [45.5%]) between September 2003 and April 2010. Recipients received a total islet mass of 9,715.75 ± 3,444.40 IEQ/kg. Thirty-four patients completed a 5-year follow-up, and 10 patients completed a 4-year follow-up. At 1, 4, and 5 years after islet transplantation, respectively, 83%, 67%, and 58% of the ITA recipients and 80%, 70%, and 60% of the IAK transplant recipients reached HbA1c under 7% (53 mmol/mol) and were free of severe hypoglycemia, while none of the ITA recipients and only 10% of the IAK transplant recipients met this composite criterion at the preinfusion stage. Thirty-three of 44 patients (75%) experienced insulin independence during the entire follow-up period, with a median duration of insulin independence of 19.25 months (interquartile range 2-58). Twenty-nine of 44 recipients (66%) exhibited at least one adverse event; 18 of 55 adverse events (33%) were possibly related to immunosuppression; and complications related to the islet infusion (n = 84) occurred in 10 recipients (11.9%). CONCLUSIONS In a large cohort with a 5-year follow-up and in a multicenter network setting, islet transplantation was safe and efficient in restoring good and lasting glycemic control and preventing severe hypoglycemia in patients with type 1 diabetes.
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Affiliation(s)
- Sandrine Lablanche
- Department of Endocrinology, Pôle DigiDune, Grenoble University Hospital, Joseph Fourier University, Grenoble, France
| | - Sophie Borot
- Centre Hospitalier Universitaire Jean Minjoz, Service d'Endocrinologie-Métabolisme et Diabétologie-Nutrition, Besançon, France
| | - Anne Wojtusciszyn
- Centre Hospitalier de Montpellier, Pôle Rein Hypertension Métabolisme, Service d'Endocrinologie, Montpellier, France
| | - Francois Bayle
- Department of Nephrology, Pôle DigiDune, Grenoble University Hospital, Joseph Fourier University, Grenoble, France
| | - Rachel Tétaz
- Department of Nephrology, Pôle DigiDune, Grenoble University Hospital, Joseph Fourier University, Grenoble, France
| | - Lionel Badet
- Hospices Civils de Lyon, Service d'Urologie et de Chirurgie de la Transplantation, Pôle Chirurgie, Lyon, France
| | - Charles Thivolet
- Hospices Civils de Lyon, Service d'Endocrinologie Diabète Nutrition, Lyon, France
| | - Emmanuel Morelon
- Hospices Civils de Lyon, Service de Néphrologie Médecine de la Transplantation et Immunologie Clinique, Lyon, France
| | - Luc Frimat
- Centre Hospitalier Universitaire de Nancy, Service de Néphrologie, Nancy, France
| | - Alfred Penfornis
- Service d'Endocrinologie, Diabétologie et Maladies Métaboliques, Centre Hospitalier Sud-Francilien, Corbeil-Essonnes, France
| | - Laurence Kessler
- Hôpitaux Universitaires de Strasbourg, Service d'Endocrinologie Diabète et Maladies Métaboliques, Pôle NUDE, Strasbourg, France
| | - Coralie Brault
- Hospices Civils de Lyon, Pôle Information Médicale Evaluation Recherche, and Université de Lyon, EA Santé-Individu-Société 4129, Lyon, France
| | - Cyrille Colin
- Hospices Civils de Lyon, Pôle Information Médicale Evaluation Recherche, and Université de Lyon, EA Santé-Individu-Société 4129, Lyon, France
| | - Igor Tauveron
- CHU de Clermont-Ferrand, Service Endocrinologie-Diabète-Maladies Métaboliques, Clermont Ferrand Université, Clermont-Ferrand, France
| | - Domenico Bosco
- Departement of Surgery, Islet Isolation, and Transplantation Center, Geneva University Hospitals, Geneva, Switzerland
| | - Thierry Berney
- Departement of Surgery, Islet Isolation, and Transplantation Center, Geneva University Hospitals, Geneva, Switzerland
| | - Pierre-Yves Benhamou
- Department of Endocrinology, Pôle DigiDune, Grenoble University Hospital, Joseph Fourier University, Grenoble, France
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Specific and redundant roles of PKBα/AKT1 and PKBβ/AKT2 in human pancreatic islets. Exp Cell Res 2015; 338:82-8. [PMID: 26318486 DOI: 10.1016/j.yexcr.2015.08.008] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2015] [Revised: 07/16/2015] [Accepted: 08/20/2015] [Indexed: 01/09/2023]
Abstract
Protein kinase Bα (PKBα)/AKT1 and PKBβ/AKT2 are required for normal peripheral insulin action but their role in pancreatic β cells remains enigmatic as indicated by the relatively mild islet phenotype of mice with deficiency for either one of these two isoforms. In this study we have analysed proliferation, apoptosis, β cell size and glucose-stimulated insulin secretion in human islets overexpressing either PKBα or PKBβ. Our results reveal redundant and specific functions. Overexpression of either isoform resulted in increased β cell size, but insulin production and secretion remained unchanged. Proliferation and apoptosis of β cells were only significantly stimulated and inhibited, respectively, by PKBα/AKT1. Importantly, overexpression of PKBα/AKT1 in dissociated islets increased the ratio of β cells to non-β cells. These results confirm our previous findings obtained with rodent islets and strongly indicate that PKBα/AKT1 can regulate β cell mass also in human islets.
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The Choice of Enzyme for Human Pancreas Digestion is a Critical Factor for Increasing the Success of Islet Isolation. Transplant Direct 2015; 1. [PMID: 26146662 DOI: 10.1097/txd.0000000000000522] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023] Open
Abstract
BACKGROUND We evaluated three commercially available enzymes for pancreatic digestion by comparing key parameters during the islet isolation process, as well as islet quality post-isolation. METHODS Retrospectively compared and analyzed islet isolations from pancreata using three different enzyme groups: Liberase HI (n=63), Collagenase NB1/Neutral Protease (NP) (n=43), and Liberase Mammalian Tissue Free Collagenase/Thermolysin (MTF C/T) (n=115). A standardized islet isolation and purification method was used. Islet quality assessment was carried out using islet count, viability, in vitro glucose-stimulated insulin secretion (GSIS), glucose-stimulated oxygen consumption rate (ΔOCR), and in vivo transplantation model in mice. RESULTS Donor characteristics were not significantly different among the three enzyme groups used in terms of age, sex, hospital stay duration, cause of death, body mass index (BMI), hemoglobin A1c (HbA1c), cold ischemia time (CIT), and pancreas weight. Digestion efficacy (percentage of digested tissue by weight) was significantly higher in the Liberase MTF C/T group (73.5 ± 1.5 %) when compared to the Liberase HI group (63.6 ± 2.3 %) (p<0.001) and the Collagenase NB1/NP group (61.7 ± 2.9%) (p<0.001). The stimulation index for GSIS was significantly higher in the Liberase MTF C/T group (5.3 ± 0.5) as compared to the Liberase HI (2.9 ± 0.2) (p<0.0001) and the Collagenase NB1/NP (3.6 ± 2.9) (p=0.012) groups. Furthermore, the Liberase MTF C/T enzymes showed the highest success rate of transplantation in diabetic NOD Scid mice (65%), which was significantly higher than the Liberase HI (42%, p=0.001) and the Collagenase NB1/NP enzymes (41%, p<0.001). CONCLUSIONS Liberase MTF C/T is superior to Liberase HI and Collagenase NB1/NP in terms of digestion efficacy and glucose-stimulated insulin secretion in vitro. Moreover, Liberase MTF C/T had a significantly higher success rate of transplantation in diabetic NOD Scid mice compared to Liberase HI and Collagenase NB1/NP enzymes.
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Rheinheimer J, Bauer AC, Silveiro SP, Estivalet AAF, Bouças AP, Rosa AR, Souza BMD, Oliveira FSD, Cruz LA, Brondani LA, Azevedo MJ, Lemos NE, Carlessi R, Assmann TS, Gross JL, Leitão CB, Crispim D. Human pancreatic islet transplantation: an update and description of the establishment of a pancreatic islet isolation laboratory. ARCHIVES OF ENDOCRINOLOGY AND METABOLISM 2015; 59:161-70. [PMID: 25993680 DOI: 10.1590/2359-3997000000030] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/06/2014] [Accepted: 02/23/2015] [Indexed: 11/22/2022]
Abstract
Type 1 diabetes mellitus (T1DM) is associated with chronic complications that lead to high morbidity and mortality rates in young adults of productive age. Intensive insulin therapy has been able to reduce the likelihood of the development of chronic diabetes complications. However, this treatment is still associated with an increased incidence of hypoglycemia. In patients with "brittle T1DM", who have severe hypoglycemia without adrenergic symptoms (hypoglycemia unawareness), islet transplantation may be a therapeutic option to restore both insulin secretion and hypoglycemic perception. The Edmonton group demonstrated that most patients who received islet infusions from more than one donor and were treated with steroid-free immunosuppressive drugs displayed a considerable decline in the initial insulin independence rates at eight years following the transplantation, but showed permanent C-peptide secretion, which facilitated glycemic control and protected patients against hypoglycemic episodes. Recently, data published by the Collaborative Islet Transplant Registry (CITR) has revealed that approximately 50% of the patients who undergo islet transplantation are insulin independent after a 3-year follow-up. Therefore, islet transplantation is able to successfully decrease plasma glucose and HbA1c levels, the occurrence of severe hypoglycemia, and improve patient quality of life. The goal of this paper was to review the human islet isolation and transplantation processes, and to describe the establishment of a human islet isolation laboratory at the Endocrine Division of the Hospital de Clínicas de Porto Alegre - Rio Grande do Sul, Brazil.
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Affiliation(s)
- Jakeline Rheinheimer
- Laboratory of Human Pancreatic Islet Biology, Endocrinology Division, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil
| | - Andrea C Bauer
- Laboratory of Human Pancreatic Islet Biology, Endocrinology Division, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil
| | - Sandra P Silveiro
- Laboratory of Human Pancreatic Islet Biology, Endocrinology Division, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil
| | - Aline A F Estivalet
- Laboratory of Human Pancreatic Islet Biology, Endocrinology Division, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil
| | - Ana P Bouças
- Laboratory of Human Pancreatic Islet Biology, Endocrinology Division, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil
| | - Annelise R Rosa
- Laboratory of Human Pancreatic Islet Biology, Endocrinology Division, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil
| | - Bianca M de Souza
- Laboratory of Human Pancreatic Islet Biology, Endocrinology Division, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil
| | - Fernanda S de Oliveira
- Laboratory of Human Pancreatic Islet Biology, Endocrinology Division, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil
| | - Lavínia A Cruz
- Laboratory of Human Pancreatic Islet Biology, Endocrinology Division, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil
| | - Letícia A Brondani
- Laboratory of Human Pancreatic Islet Biology, Endocrinology Division, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil
| | - Mirela J Azevedo
- Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil
| | - Natália E Lemos
- Laboratory of Human Pancreatic Islet Biology, Endocrinology Division, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil
| | - Rodrigo Carlessi
- Laboratory of Human Pancreatic Islet Biology, Endocrinology Division, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil
| | - Taís S Assmann
- Laboratory of Human Pancreatic Islet Biology, Endocrinology Division, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil
| | - Jorge L Gross
- Laboratory of Human Pancreatic Islet Biology, Endocrinology Division, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil
| | - Cristiane B Leitão
- Laboratory of Human Pancreatic Islet Biology, Endocrinology Division, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil
| | - Daisy Crispim
- Laboratory of Human Pancreatic Islet Biology, Endocrinology Division, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil
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Kesseli SJ, Smith KA, Gardner TB. Total pancreatectomy with islet autologous transplantation: the cure for chronic pancreatitis? Clin Transl Gastroenterol 2015; 6:e73. [PMID: 25630865 PMCID: PMC4418411 DOI: 10.1038/ctg.2015.2] [Citation(s) in RCA: 40] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/23/2014] [Revised: 12/18/2014] [Accepted: 01/02/2015] [Indexed: 12/30/2022] Open
Abstract
Chronic pancreatitis (CP) is a debilitating disease that leads to varying degrees
of pancreatic endocrine and exocrine dysfunction. One of the most difficult
symptoms of CP is severe abdominal pain, which is often challenging to control
with available analgesics and therapies. In the last decade, total
pancreatectomy with autologous islet cell transplantation has emerged as a
promising treatment for the refractory pain of CP and is currently performed at
approximately a dozen centers in the United States. While total pancreatectomy
is not a new procedure, the endocrine function-preserving autologous islet cell
isolation and re-implantation have made the prospect of total pancreatectomy
more acceptable to patients and clinicians. This review will focus on the
current status of total pancreatectomy with autologous islet cell transplant
including patient selection, technical considerations, and outcomes. As the
procedure is performed at an increasing number of centers, this review will
highlight opportunities for quality improvement and outcome optimization.
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Affiliation(s)
- Samuel J Kesseli
- Dartmouth Geisel School of Medicine, Hanover, New Hampshire, USA
| | - Kerrington A Smith
- Section of General Surgery, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire, USA
| | - Timothy B Gardner
- Section of Gastroenterology and Hepatology, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire, USA
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Ahearn AJ, Parekh JR, Posselt AM. Islet transplantation for Type 1 diabetes: where are we now? Expert Rev Clin Immunol 2014; 11:59-68. [DOI: 10.1586/1744666x.2015.978291] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
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Taylor MJ, Baicu SC. Nonenzymatic Cryogenic Isolation of Therapeutic Cells: Novel Approach for Enzyme-Free Isolation of Pancreatic Islets Using In Situ Cryopreservation of Islets and Concurrent Selective Freeze Destruction of Acinar Tissue. Cell Transplant 2014; 23:1365-79. [DOI: 10.3727/096368913x672055] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022] Open
Abstract
Cell-based therapies, which all involve processes for procurement and reimplantation of living cells, currently rely upon expensive, inconsistent, and even toxic enzyme digestion processes. A prime example is the preparation of isolated pancreatic islets for the treatment of type 1 diabetes by transplantation. To avoid the inherent pitfalls of these enzymatic methods, we have conceptualized an alternative approach based on the hypothesis that cryobiological techniques can be used for differential freeze destruction of the pancreas (Px) to release islets that are selectively cryopreserved in situ. Pancreata were procured from juvenile pigs using approved procedures. The concept of cryoisolation is based on differential processing of the pancreas in five stages: 1) infiltrating islets in situ preferentially with a cryoprotectant (CPA) cocktail via antegrade perfusion of the major arteries; 2) retrograde ductal infusion of water to distend the acinar; 3) freezing the entire Px solid to lt; −160°C for storage in liquid nitrogen; 4) mechanically crushing and pulverizing the frozen Px into small fragments; 5) thawing the frozen fragments, filtering, and washing to remove the CPA. Finally, the filtered effluent (cryoisolate) was stained with dithizone for identification of intact islets and with Syto 13/PI for fluorescence viability testing and glucose-stimulated insulin release assessment. As predicted, the cryoisolate contained small fragments of residual tissue comprising an amorphous mass of acinar tissue with largely intact and viable (>90%) embedded islets. Islets were typically larger (range 50–500 μm diameter) than their counterparts isolated from juvenile pigs using conventional enzyme digestion techniques. Functionally, the islets from replicate cryoisolates responded to a glucose challenge with a mean stimulation index = 3.3 ± 0.7. An enzyme-free method of islet isolation relying on in situ cryopreservation of islets with simultaneous freeze destruction of acinar tissue is feasible and proposed as a new and novel method that avoids the problems associated with conventional collagenase digestion methods.
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Affiliation(s)
- Michael J. Taylor
- Cell and Tissue Systems, Inc., N. Charleston, SC, USA
- Carnegie Mellon University, Pittsburgh, PA, USA
| | - Simona C. Baicu
- Cell and Tissue Systems, Inc., N. Charleston, SC, USA
- Ocular and Tissue Recovery Operations, LifePoint, Inc., Charleston, SC, USA
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Morphological changes of post-isolation of caprine pancreatic islet. In Vitro Cell Dev Biol Anim 2014; 51:113-20. [DOI: 10.1007/s11626-014-9821-7] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2014] [Accepted: 09/10/2014] [Indexed: 01/04/2023]
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Hilling DE, Bouwman E, Terpstra OT, Marang-Van De Mheen PJ. Effects of Donor-, Pancreas-, and Isolation-Related Variables on Human Islet Isolation Outcome: A Systematic Review. Cell Transplant 2014; 23:921-8. [DOI: 10.3727/096368913x666412] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
Different factors have been reported to influence islet isolation outcome, but their importance varies between studies and are hampered by the small sample sizes in most studies. The purpose of this study was to perform a systematic review to assess the impact of donor-, pancreas-, and isolation-related variables on successful human islet isolation outcome. PubMed, Embase, and Web of Science were searched electronically in April 2009. All studies reporting on donor-, pancreas-, and isolation-related factors relating to prepurification and postpurification islet isolation yield and proportion of successful islet isolations were selected. Seventy-four retrospective studies had sufficient data and were included in the analyses. Higher pre- and postpurification islet yields and a higher proportion of successful islet isolations were obtained when pancreata were preserved with the two-layer method rather than University of Wisconsin solution in donors with shorter cold ischemia times (CITs) [1 h longer CIT resulted in an average decline of prepurification and postpurification yields and proportion of successful isolations of 59 islet equivalents (IEQs)/g, 54 IEQs/g, and 21%, respectively]. Higher prepurification yields and higher percentage of successful islet isolations were found in younger donors with higher body mass index. Lower yields were found in donation after brain death donors compared to donation after cardiac death donors. Higher postpurification yields were found for isolation with Serva collagenase. This review identified donor-, pancreas-, and isolation-related factors that influence islet isolation yield. Standardized reports of these factors in all future studies may improve the power and identify additional factors and thereby contribute to improving islet isolation yield.
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Affiliation(s)
- Denise E. Hilling
- Department of Surgery, Leiden University Medical Center, Leiden, the Netherlands
| | - Eelco Bouwman
- Department of Surgery, Leiden University Medical Center, Leiden, the Netherlands
| | - Onno T. Terpstra
- Department of Surgery, Leiden University Medical Center, Leiden, the Netherlands
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Neuman JC, Truchan NA, Joseph JW, Kimple ME. A method for mouse pancreatic islet isolation and intracellular cAMP determination. J Vis Exp 2014:e50374. [PMID: 24998772 DOI: 10.3791/50374] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023] Open
Abstract
Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The β-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the β-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on β-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased β-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3(H)]-thymidine incorporation, protein abundance, and mRNA expression.
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Affiliation(s)
- Joshua C Neuman
- Department of Nutrional Sciences, University of Wisconsin-Madison
| | - Nathan A Truchan
- Department of Medicine, Division of Endocrinology, Diabetes, and Metabolism, University of Wisconsin-Madison
| | | | - Michelle E Kimple
- Department of Medicine, Division of Endocrinology, Diabetes, and Metabolism, University of Wisconsin-Madison;
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Vrabelova D, Adin C, Gilor C, Rajab A. Pancreatic islet transplantation: from dogs to humans and back again. Vet Surg 2014; 43:631-41. [PMID: 24909456 DOI: 10.1111/j.1532-950x.2014.12224.x] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2013] [Accepted: 03/01/2014] [Indexed: 02/06/2023]
Abstract
Pancreatic islet transplantation is a cell-based therapy that provides a potential cure for type 1 diabetes mellitus. After the introduction of an automated method for islet isolation and steroid-free immunosuppressive protocols, reversal of diabetes by islet transplantation is now performed at major human medical centers around the world. Despite extensive use of animal models in islet transplantation research, practical concerns have slowed the introduction of the technique into clinical veterinary practice and only a small number of studies have reported results of transplantation in dogs with spontaneously occurring diabetes mellitus; however, recent advances in islet isolation and encapsulation may make it possible to perform islet transplantation without immunosuppression in companion animals. This review summarizes experimental and clinical studies of pancreatic islet transplantation in dogs, including future directions for cell therapy in animals with naturally occurring disease.
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Affiliation(s)
- Daniela Vrabelova
- Department of Veterinary Clinical Sciences, Ohio State University, Columbus, Ohio
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Figliuzzi M, Bonandrini B, Silvani S, Remuzzi A. Mesenchymal stem cells help pancreatic islet transplantation to control type 1 diabetes. World J Stem Cells 2014; 6:163-172. [PMID: 24772243 PMCID: PMC3999774 DOI: 10.4252/wjsc.v6.i2.163] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/29/2013] [Revised: 12/20/2013] [Accepted: 03/04/2014] [Indexed: 02/06/2023] Open
Abstract
Islet cell transplantation has therapeutic potential to treat type 1 diabetes, which is characterized by autoimmune destruction of insulin-producing pancreatic islet β cells. It represents a minimal invasive approach for β cell replacement, but long-term blood control is still largely unachievable. This phenomenon can be attributed to the lack of islet vasculature and hypoxic environment in the immediate post-transplantation period that contributes to the acute loss of islets by ischemia. Moreover, graft failures continue to occur because of immunological rejection, despite the use of potent immunosuppressive agents. Mesenchymal stem cells (MSCs) have the potential to enhance islet transplantation by suppressing inflammatory damage and immune mediated rejection. In this review we discuss the impact of MSCs on islet transplantation and focus on the potential role of MSCs in protecting islet grafts from early graft failure and from autoimmune attack.
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