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Zuellig RA, Cavallari G, Gerber P, Tschopp O, Spinas GA, Moritz W, Lehmann R. Improved physiological properties of gravity-enforced reassembled rat and human pancreatic pseudo-islets. J Tissue Eng Regen Med 2014; 11:109-120. [PMID: 24737702 DOI: 10.1002/term.1891] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2013] [Revised: 12/20/2013] [Accepted: 02/26/2014] [Indexed: 01/01/2023]
Abstract
Previously we demonstrated the superiority of small islets vs large islets in terms of function and survival after transplantation, and we generated reaggregated rat islets (pseudo-islets) of standardized small dimensions by the hanging-drop culture method (HDCM). The aim of this study was to generate human pseudo-islets by HDCM and to evaluate and compare the physiological properties of rat and human pseudo-islets. Isolated rat and human islets were dissociated into single cells and incubated for 6-14 days by HDCM. Newly formed pseudo-islets were analysed for dimensions, morphology, glucose-stimulated insulin secretion (GSIS) and total insulin content. The morphology of reaggregated human islets was similar to that of native islets, while rat pseudo-islets had a reduced content of α and δ cells. GSIS of small rat and human pseudo-islets (250 cells) was increased up to 4.0-fold (p < 0.01) and 2.5-fold (p < 0.001), respectively, when compared to their native counterparts. Human pseudo-islets showed a more pronounced first-phase insulin secretion as compared to intact islets. GSIS was inversely correlated to islet size, and small islets (250 cells) contained up to six-fold more insulin/cell than large islets (1500 cells). Tissue loss with this new technology could be reduced to 49.2 ± 1.5% in rat islets, as compared to the starting amount. With HDCM, pseudo-islets of standardized size with similar cellular composition and improved biological function can be generated, which compensates for tissue loss during production. Transplantation of small pseudo-islets may represent an attractive strategy to improve graft survival and function, due to better oxygen and nutrient supply during the phase of revascularization. Copyright © 2014 John Wiley & Sons, Ltd.
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Affiliation(s)
- R A Zuellig
- Division of Endocrinology, Diabetes and Clinical Nutrition, University Hospital Zurich, Switzerland
| | - G Cavallari
- Nephrology, Dialysis and Transplantation Unit (Stefoni), S.Orsola-Malpighi Hospital, University of Bologna, Italy
| | - P Gerber
- Division of Endocrinology, Diabetes and Clinical Nutrition, University Hospital Zurich, Switzerland
| | - O Tschopp
- Division of Endocrinology, Diabetes and Clinical Nutrition, University Hospital Zurich, Switzerland
| | - G A Spinas
- Division of Endocrinology, Diabetes and Clinical Nutrition, University Hospital Zurich, Switzerland
| | - W Moritz
- InSphero AG, Schlieren, Switzerland
| | - R Lehmann
- Division of Endocrinology, Diabetes and Clinical Nutrition, University Hospital Zurich, Switzerland
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Mueller KR, Martins KV, Murtaugh MP, Schuurman HJ, Papas KK. Manufacturing porcine islets: culture at 22 °C has no advantage above culture at 37 °C: a gene expression evaluation. Xenotransplantation 2013; 20:418-28. [PMID: 23941232 DOI: 10.1111/xen.12048] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2013] [Accepted: 07/16/2013] [Indexed: 11/28/2022]
Abstract
BACKGROUND The manufacturing process of islets includes a culture step which was originally introduced to ease the logistics of procedures in preparing the graft and transplant recipient. It has been suggested that culture at room temperature has an advantage over culture at 37 °C, in part by reducing immunogenicity via preferential elimination of contaminating cells (such as passenger leukocytes) within islets. We investigated this using islets isolated from pancreata of adult pigs. METHODS Porcine islets were isolated from three donors and cultured at 37 °C for 1 day, and then under three different conditions: 37 °C for 6 days (condition A); 22 °C for 6 days (condition B); or 22 °C for 5 days followed by 37 °C for 1 day (condition C). Recovery was assessed by DNA measurement, viability by oxygen consumption rate normalized for DNA (OCR/DNA), and gene expression by RT-PCR for a series of 9 lymphocyte markers, 11 lymphokines and chemokines, and 14 apoptotic and stress markers. RESULTS Post-culture islet recoveries were similar for the three culture conditions. Average OCR/DNA values were 129-159 nmol/min·mgDNA before culture, and 259-291, 204-212, and 207-228 nmol/min·mgDNA, respectively, for culture under conditions A, B, and C, respectively. Irrespective of culture condition, examined gene expression in all three series of lymphocyte markers, lymphokines and chemokines, and apoptotic and stress markers manifested a statistically significant decrease upon culture for 7 days. This decrease was most dramatic for condition A: in particular, most of lymphocyte markers showed a >10-fold reduction and also six markers in the lymphokine and chemokine series; these reductions are consistent with the elimination of immune cells present within islets during culture. The reduction was less for apoptotic and stress markers. For culture under condition B, the reduction in gene expression was less, and culture under condition C resulted in gene expression levels similar to those under condition A: this indicates that 24 h at 37 °C is sufficient to re-equilibrate gene expression levels from those in islets cultured at 22 °C to those in islets cultured at 37 °C. Results were consistent among the preparations from the three donors. CONCLUSIONS Culture of porcine islets at 37 °C provides benefits over culture at 22 °C with respect to OCR/DNA outcomes and reduced expression of genes encoding lymphocyte markers, lymphokines and chemokines, and markers for apoptosis and stress.
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Affiliation(s)
- Kate R Mueller
- Department of Surgery, Schulze Diabetes Institute, University of Minnesota, Minneapolis, MN, USA
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Kim YH, Wee YM, Choi MY, Lim DG, Kim SC, Han DJ. Interleukin (IL)-10 induced by CD11b(+) cells and IL-10-activated regulatory T cells play a role in immune modulation of mesenchymal stem cells in rat islet allografts. Mol Med 2011; 17:697-708. [PMID: 21365122 DOI: 10.2119/molmed.2010.00098] [Citation(s) in RCA: 52] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2010] [Accepted: 02/24/2011] [Indexed: 01/01/2023] Open
Abstract
Mesenchymal stem cells (MSCs) are suggested to be immune modulators because of their therapeutic potential in transplantation. In the present study, we evaluated the therapeutic potential of autologous MSCs for preventing graft rejection after allogeneic rat islet transplantation. We assessed the ability of MSCs to elicit an antiproliferative response in alloreactive lymphocytes and tested the immunosuppressive effect of MSCs in allogeneic islet transplantation. In islet allotransplantation, injection of autologous MSCs or a subtherapeutic dose of cyclosporine A (CsA; 5 mg/kg) alone did not prolong allograft survival. However, graft survival was attained for >100 d in 33% of autologous MSC-plus-CsA-treated recipients, indicating that graft acceptance was achieved in a subgroup of allograft recipients. Splenocytes from autologous MSC-plus-CsA-treated rats exhibited a reduced mixed lymphocyte reaction (MLR)-proliferative response to donor stimulators and increased interleukin (IL)-10 release. Interestingly, after excluding host CD11b(+) cells, splenic T cells from autologous MSC-plus-CsA-treated rats did not produce IL-10 or did not inhibit proliferative responses under the same conditions. The use of autologous MSC-plus-CsA downregulated immune responses, inducing donor-specific T-cell hyporesponsiveness by reducing the production of proinflammatory cytokines and inducing antiinflammatory cytokine production, especially that of IL-10, during the early posttransplantation period. T-regulatory cells made a contribution at a later phase. In conclusion, the combined use of autologous MSCs and low-dose CsA exerted a synergistic immunosuppressive effect in an islet allograft model, suggesting a role for autologous MSCs as an immune modulator.
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Affiliation(s)
- Yang-Hee Kim
- Department of Surgery, University of Ulsan College of Medicine, and Asan Medical Center, Seoul, Korea
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Daoud J, Rosenberg L, Tabrizian M. Pancreatic Islet Culture and Preservation Strategies: Advances, Challenges, and Future Outlook. Cell Transplant 2010; 19:1523-35. [DOI: 10.3727/096368910x515872] [Citation(s) in RCA: 49] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
Postisolation islet survival is a critical step for achieving successful and efficient islet transplantation. This involves the optimization of islet culture in order to prolong survival and functionality in vitro. Many studies have focused on different strategies to culture pancreatic islets in vitro through manipulation of culture media, surface modified substrates, and the use of various techniques such as encapsulation, embedding, scaffold, and bioreactor culture strategies. This review aims to present and discuss the different methodologies employed to optimize pancreatic islet culture in vitro as well as address their respective advantages and drawbacks.
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Affiliation(s)
- Jamal Daoud
- Department of Biomedical Engineering, Faculty of Medicine, McGill University, Montreal, QC, Canada
| | - Lawrence Rosenberg
- Department of Surgery, Faculty of Medicine, McGill University, Montreal, QC, Canada
| | - Maryam Tabrizian
- Department of Biomedical Engineering, Faculty of Medicine, McGill University, Montreal, QC, Canada
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Irani S, Mohseni Salehi Monfared S, Akbari-Kamrani M, Ostad S, Abdollahi M, Larijani B. Effect of Low-Level Laser Irradiation on In Vitro Function of Pancreatic Islets. Transplant Proc 2009; 41:4313-5. [DOI: 10.1016/j.transproceed.2009.09.065] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2009] [Revised: 05/03/2009] [Accepted: 09/15/2009] [Indexed: 12/01/2022]
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Wee YM, Lim DG, Kim YH, Kim JH, Kim SC, Yu E, Park MO, Choi MY, Park YH, Jang HJ, Cho EY, Cho MH, Han DJ. Cell Surface Modification by Activated Polyethylene Glycol Prevents Allosensitization after Islet Transplantation. Cell Transplant 2008; 17:1257-69. [DOI: 10.3727/096368908787236657] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
The necessity to transplant islet tissue without the need for immunosuppressant therapy has led to the development of materials for immune modulation. Pegylation makes islets antigenically silent, protecting them from the adsorption of foreign protein and thus avoiding immune injury. The aim of this study is to determine whether pegylation of islets prolongs islet survival and function both during tissue culture and posttransplantation. We used cyanuric chloride-activated methoxy-polyethylene glycol for cell surface modification. To detect the pegylation effect on splenocytes, we measured antibody binding inhibition and abrogation of lymphocyte proliferation. To detect the pegylation effect on islet grafts, we performed rodent islet transplantation. Islet viability and function were maintained after pegylation. Pegylated islets showed a 90% decrease in antibody binding and decreased lymphocyte proliferation in a mixed lymphocyte culture. However, when pegylated islets were transplanted, no prolongation of graft survival was observed. When a subtherapeutic dose of immunosuppressant was given at the time of transplantation of pegylated islets, islet graft survival was significantly prolonged. In addition, when rats were sensitized with donor splenocytes, graft survival was prolonged by pegylation. We observed that pegylation of islets, combined with a subtherapeutic dose of immunosuppressant, protects the graft from rejection. Prolonged graft survival in sensitized recipients showed that pegylation of islets shifted the pattern of rejection from an acute humoral response to a less aggressive cellular alloresponse.
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Affiliation(s)
- Yu-Mee Wee
- Department of Surgery, Ulsan University College of Medicine & Asan Medical Center, Seoul, 138-736, Korea
| | - Dong-Gyun Lim
- Department of Surgery, Ulsan University College of Medicine & Asan Medical Center, Seoul, 138-736, Korea
| | - Yang-Hee Kim
- Department of Surgery, Ulsan University College of Medicine & Asan Medical Center, Seoul, 138-736, Korea
| | - Jin-Hee Kim
- Department of Surgery, Ulsan University College of Medicine & Asan Medical Center, Seoul, 138-736, Korea
| | - Song-Cheol Kim
- Department of Surgery, Ulsan University College of Medicine & Asan Medical Center, Seoul, 138-736, Korea
| | - Eunsil Yu
- Department of Pathology, Ulsan University College of Medicine & Asan Medical Center, Seoul, 138-736, Korea
| | | | - Monica Young Choi
- Department of Surgery, Ulsan University College of Medicine & Asan Medical Center, Seoul, 138-736, Korea
| | - Youn-Hee Park
- Department of Surgery, Ulsan University College of Medicine & Asan Medical Center, Seoul, 138-736, Korea
| | - Hyuk-Jai Jang
- Department of Surgery, Ulsan University College of Medicine & Asan Medical Center, Seoul, 138-736, Korea
| | - Eun-Young Cho
- Department of Surgery, Ulsan University College of Medicine & Asan Medical Center, Seoul, 138-736, Korea
| | - Myung-Hwan Cho
- Department of Biological Science, Konkuk University, Seoul, 143-701, Korea
| | - Duck-Jong Han
- Department of Surgery, Ulsan University College of Medicine & Asan Medical Center, Seoul, 138-736, Korea
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Chen XB, Li YX, Jiao Y, Dong WP, Li G, Chen J, Tan JM. Influence of heme oxygenase-1 gene transfer on the viability and function of rat islets in in vitro culture. World J Gastroenterol 2007; 13:1053-9. [PMID: 17373739 PMCID: PMC4146867 DOI: 10.3748/wjg.v13.i7.1053] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the influence of heme oxygenase-1 (HO-1) gene transfer on the viability and function of cultured rat islets in vitro.
METHODS: Islets were isolated from the pancreata of Sprague-Dawley rats by intraductal collagenase digestion, and purified by discontinuous Ficoll density gradient centrifugation. Purified rat islets were transfected with adenoviral vectors containing human HO-1 gene (Ad-HO-1) or enhanced green fluorescent protein gene (Ad-EGFP), and then cultured for seven days. Transfection was confirmed by fluorescence microscopy and Western blot. Islet viability was evaluated by acridine orange/ propidium iodide fluorescent staining. Glucose-stimulated insulin release was detected using insulin radioimmunoassay kits and was used to assess the function of islets. Stimulation index (SI) was calculated by dividing the insulin release upon high glucose stimulation by the insulin release upon low glucose stimulation.
RESULTS: After seven days culture, the viability of cultured rat islets decreased significantly (92% ± 6% vs 52% ± 13%, P < 0.05), and glucose-stimulated insulin release also decreased significantly (6.47 ± 0.55 mIU/L/30IEQ vs 4.57 ± 0.40 mIU/L/30IEQ, 14.93 ± 1.17 mIU/L/30IEQ vs 9.63 ± 0.71 mIU/L/30IEQ, P < 0.05). Transfection of rat islets with adenoviral vectors at an MOI of 20 was efficient, and did not impair islet function. At 7 d post-transfection, the viability of Ad-HO-1 transfected islets was higher than that of control islets (71% ± 15% vs 52% ± 13%, P < 0.05). There was no significant difference in insulin release upon low glucose stimulation (2.8 mmol/L) among Ad-HO-1 transfected group, Ad-EGFP transfected group, and control group (P > 0.05), while when stimulated by high glucose (16.7 mmol/L) solution, insulin release in Ad-HO-1 transfected group was significantly higher than that in Ad-EGFP transfected group and control group, respectively (12.50 ± 2.17 mIU/L/30IEQ vs 8.87 ± 0.65 mIU/L/30IEQ; 12.50 ± 2.17 mIU/L/30IEQ vs 9.63 ± 0.71 mIU/L/30IEQ, P < 0.05). The SI of Ad-HO-1 transfected group was also significantly higher than that of Ad-EGFP transfected group and control group, respectively (2.21 ± 0.02 vs 2.08 ± 0.05; 2.21 ± 0.02 vs 2.11 ± 0.03, P < 0.05).
CONCLUSION: The viability and function of rat islets decrease over time in in vitro culture, and heme oxygenase-1 gene transfer could improve the viability and function of cultured rat islets.
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Affiliation(s)
- Xiao-Bo Chen
- Department of Renal Transplantation and Urology, the First People's Hospital, Shanghai Jiao Tong University, China
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