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Improved Method of Human Islet Isolation for Young Donors
Improved Method of Human Islet Isolation for Young Donors M. Shimoda, H. Noguchi, B. Naziruddin, Y. Fujita, D. Chujo, M. Takita, H. Peng, Y. Tamura, G.S. Olsen, K. Sugimoto, T. Itoh, N. Onaca, M.F. Levy, P.A. Grayburn, and S. Matsumoto ABSTRACT Background. Although islet transplantation using young donors is more effective than older donors, islet isolation from young donor is notoriously difficult. This may relate to islet ontogeny and collagen composition in the young pancreas. Therefore, we examined whether a high concentration of collagenase could improve the separation of islets from exocrine tissues resulting in an high islet yield. Methods. We used six human pancreata from brain-dead donors of less than 30 years old. Islet isolation was performed based on the Edmonton protocol with modifications. All pancreata were digested with Collagenase NB1 Premium Grade (Serva). The pancreas was expanded by injecting either 200 mL of cold collagenase solution (2.5 mg/mL, standard group, n ⫽ 3) or 100 mL of solution (5 mg/mL, new group, n ⫽ 3) in a controlled manner under low pressure for 5 minutes. Then the pressure was raised for another 5 minutes. The following procedure and evaluation were performed based on the Edmonton protocol. Results. Phase II time in the new group was significantly shorter than the standard group. The ratio of embedded islets in the new group was significantly lower than the standard group. The postpurification islet equivalents per pancreas weight (IEQ/g) and the recovery rate in the new group were higher than the standard group, but not significantly. There was no significant difference in the postpurification purity, viability, and final tissue volume. Conclusion. Our simple modification with an initially concentrated collagenase preparation using a syringe significantly improved the ratio of embedded islets, resulting in a higher yield from young donors. LTHOUGH ISLET TRANSPLANTATION is a promising treatment for type 1 diabetes,1–5 its success relies heavily on the quality of the donor pancreas and upon an optimal isolation. Islet transplantation using young donors is more effective than using older donors; however, islet isolation from young donors is notoriously difficult.6 –11 Islets in young human donor pancreata are surrounded, even after isolation and purification, by layers of exocrine cells (embedded islets). Embedded islets are difficult to separate from acinar cells with current purification methods using density gradient centrifugation. This problem may be due to the peculiar characteristics of young pancreata such as islet ontogeny and collagen composition,12 so the current collagenases, which are suitable for isolation from older donor pancreata, may not effectively degrade collagens in a young pancreas. Therefore, we studied whether highly concentrated collagenase could separate islets from exocrine tissues to improve islet yield after purification.
A
METHODS We used six human pancreata from brain-dead donors less than 30 years old, which were procured from either the Southwest Trans-
From the Division of Cardiology, Department of Internal Medicine (M.S., P.A.G.), Baylor University Medical Center, Baylor Heart and Vascular Institute, Dallas, Texas, USA; Baylor Research Institute Fort-Worth Campus (M.S., H.N., M.T., K.S., T.I., S.M., Y.F.), Islet Cell Laboratory, Texas, USA; Baylor Institute for Immunology Research (D.C.), Dallas, Texas, USA; Baylor Regional Transplant Institute (B.N., Y.T., G.S.O., N.O., M.F.L.), Dallas and Fort-Worth, Texas, USA; and Institute of Biomedical Studies (B.N., H.P.), Baylor University, Waco, Texas, USA. This study was partially supported by All Saints Health Foundation. Address reprint requests to Shinichi Matsumoto, MD, PhD, Baylor Research Institute Fort Worth Campus, Islet Cell Laboratory, 1400 8th Ave, Fort Worth, TX 76104. E-mail: shinichm@ baylorhealth.edu
0041-1345/10/$–see front matter doi:10.1016/j.transproceed.2010.05.094
© 2010 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
2024
Transplantation Proceedings, 42, 2024 –2026 (2010)
IMPROVED HUMAN ISLET ISOLATION
2025
plant Alliance (Dallas, Tex, USA) or LifeGift (Fort Worth, Tex, USA) between 2008 and 2009. They were allocated to the standard or the new method groups. The islets were isolated based on the Edmonton protocol with modifications.1,13–16 All pancreata were digested with 1 vial of Collagenase NB1 Premium Grade and two vials of Neutral Protease (Serva). Different concentrations of collagenases were used in the perfusion step in the two groups. In the standard group (n ⫽ 3), the pancreas was expanded by injecting 200 mL of cold collagenase solution (2.5 mg/mL) under a lowpressure, controlled manner (60 – 80 mm Hg) for 5 minutes. Then the pressure was raised to 160 to 180 mm Hg for another 5 minutes. In contrast, in the new method (n ⫽ 3), the expansion was first performed with approximately the same volume of pancreas weight in grams of cold collagenase solution (approximately 100 mL, 5 mg/mL) using a syringe in a pressure-controlled manner (60 – 80 mm Hg) for 5 minutes followed by adding perfusion solution to 200 mL and performing a high-pressure step in the same condition as the standard one. We used a syringe for the initial perfusion step of the new method, because 100 mL of solution was not enough to fulfill the pump circuit. The following procedure and evaluation were performed based on the Edmonton protocol. For the statistical analysis, we considered P values less than .05 as significant in this study.
RESULTS
The donor and isolation variables are shown in Table 1 There was no significant difference in the average of used collagenase activity, donor age, pancreas weight, phase I time, and undigested tissue volume. In contrast, phase II time in the new group was significantly shorter than standard group: new: 37 ⫾ 1 versus standard: 52 ⫾ 4 (P ⬍ .05). Importantly, the percentage of embedded islets in the digests of the new group was significantly lower than the standard group: new: 54% ⫾ 4%, versus standard: 83% ⫾ 5% (P ⬍ .05). The postpurification islet equivalents per pancreas weight (IEQ/g) and the recovery rate in the new group were higher than the standard group, although they Table 1. Donor and Isolation Variables of Two Groups Standard Method (n ⫽ 3)
Donor age (y) BMI Cold ischemic time (min) Pancreas weight (g) Collagenase activity (PZU/vial) Phase I Phase II Undigested tissue % Trapped islet Prepurification IEQ/g Postpurification IEQ/g Postpurification purity Postpurification viability Recovery rate (%) Final pellet (mL) Stimulation index
New Method (n ⫽ 3)
P Value
23.7 ⫾ 0.7 25.0 ⫾ 0.2 203 ⫾ 6 80 ⫾ 10 2430 ⫾ 71
21.3 ⫾ 1.5 32.6 ⫾ 2.6 166 ⫾ 48 86 ⫾ 17 2428 ⫾ 13
.320 .071 .560 .836 .989
14 ⫾ 1 52 ⫾ 4 7⫾4 83 ⫾ 5 7802 ⫾ 832 3901 ⫾ 637 47.3 ⫾ 2.9 99.1 ⫾ 0.3 50.3 ⫾ 7.2 11.3 ⫾ 1.0 8.1 ⫾ 2.5
17 ⫾ 2 37 ⫾ 1 4⫾1 54 ⫾ 4 9558 ⫾ 1921 6475 ⫾ 1538 49.3 ⫾ 2.7 98.0 ⫾ 0.6 66.8 ⫾ 3.9 7.3 ⫾ 1.0 13.7 ⫾ 6.3
.286 .029 .577 .019 .531 .275 .704 .328 .175 .156 .539
All values are expressed by the average ⫾ standard errors of the mean. BMI, body mass index; PZU, ; IEQ/g, islet equivalent per pancreas weight.
did not reach statistical significance (IEQ/g, new: 6475 ⫾ 1538, standard: 3901 ⫾ 637; recovery rate, new: 66.8% ⫾ 3.9%, standard: 50.3% ⫾ 7.2%, respectively). The stimulation index of isolated islets in the new group was also higher than the standard group, but not significantly (new: 13.7 ⫾ 6.3, standard: 8.1 ⫾ 2.5). There was no significant difference between the two groups in the postpurification purity, viability, and final tissue volume. DISCUSSION
In consideration of the donor shortage, it is crucial to make more human donors accessible for islet transplantation. In clinical transplantation, pancreata obtained from young donors showed good performance in adult recipients,17 encouraging improved isolation of islets from young donors. However, a major obstacle to utilizing young donors is the technical inability to obtain “free” islets, which are not surrounded by exocrine cells, which makes the purification step extremely difficult. This problem may be due to the anatomic and physiological features of young pancreas.6,12 Many efforts have been made to overcome the difficulty, but so far few successful results have been reported.6,12 In this study, we hypothesized that the current collagenases used for islet isolation were not sufficient to digest the collagens of the young pancreas; therefore, a high concentration of collagenase might improve the digestion. For this purpose, in our new method, we infused collagenase at almost double the concentration compared with the standard method. Our data showed that this method enabled us to significantly improve the islet separation from exocrine cells. It strongly indicated that higher collagenase concentrations can more effectively digest collagens of the young pancreas. In contrast, a highly dense collagenase might harm the islets. However, this study showed that the islet viability using the new method was not different from the standard outcome. Moreover, an in vitro functional assay (stimulation index) showed even better results with the new method (Table 1). However, considering that more than half of the digested islets were embedded even in the new method group there is a requirement for further study and modification. In conclusion, a simple modification of the perfusion method using a concentrated collagenase with a syringe may reduce the degree of embedded islets and increase the yield after purification. REFERENCES 1. Shapiro AM, Lakey JR, Ryan EA, et al: Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 343:230, 2000 2. Robertson RP: Islet transplantation as a treatment for diabetes—a work in progress. N Engl J Med 350:694, 2004 3. Matsumoto S, Okitsu T, Iwanaga Y, et al: Insulin independence after living-donor distal pancreatectomy and islet allotransplantation. Lancet 365:1642, 2005 4. Matsumoto S, Okitsu T, Iwanaga Y, et al: Successful islet transplantation from nonheartbeating donor pancreata using modified Ricordi islet isolation method. Transplantation 82:460, 2006
2026 5. Ryan EA, Paty BW, Senior PA, et al: Five-year follow-up after clinical islet transplantation. Diabetes 54:2060, 2005 6. Lakey JR, Warnock GL, Rajotte RV, et al: Variables in organ donors that affect the recovery of human islets of Langerhans. Transplantation 61:1047, 1996 7. Hanley SC, Paraskevas S, Rosenberg L: Donor and isolation variables predicting human islet isolation success. Transplantation 85:950, 2008 8. Socci C, Davalli AM, Vignali A, et al: A significant increase of islet yield by early injection of collagenase into the pancreatic duct of young donors. Transplantation 55:661, 1993 9. Ricordi C: Islet transplantation: a brave new world. Diabetes 52:1595, 2003 10. Ricordi C, Alejandro R, Zeng Y, et al: Human islet isolation and purification from pediatric-age donors. Transplant Proc 23: 783, 1991 11. Socci C, Davalli AM, Vignali A, et al: A significant increase of islet yield by early injection of collagenase into the pancreatic duct of young donors. Transplantation 55:661, 1993
SHIMODA, NOGUCHI, NAZIRUDDIN ET AL 12. Balamurugan AN, Chang Y, Bertera S, et al: Suitability of human juvenile pancreatic islets for clinical use. Diabetologia 49:1845, 2006 13. Matsumoto S, Noguchi H, Naziruddin B, et al: Improvement of pancreatic islet cell isolation for transplantation. Proc (Bayl Univ Med Cent) 20:357, 2007 14. Noguchi H, Ikemoto T, Naziruddin B, et al: Iodixanolcontrolled density gradient during islet purification improves recovery rate in human islet isolation. Transplantation 87:1629, 2009 15. Ikemoto T, Noguchi H, Shimoda M, et al: Islet cell transplantation for the treatment of type 1 diabetes in the USA. J Hepatobiliary Pancreat Surg 16:118, 2009 16. Matsumoto S, Noguchi H, Hatanaka N, et al: Estimation of donor usability for islet transplantation in the United States with the Kyoto islet isolation method. Cell Transplant 18:549, 2009 17. Buggenhout A, Hoang AD, Hut F, et al: Pediatric en bloc dual kidney–pancreas transplantation into an adult recipient: a simplified technique. Benefits of the en bloc kidney–pancreas transplantation technique in pediatric donors. Am J Transplant 4:663, 2004
A
METHODS We used six human pancreata from brain-dead donors less than 30 years old, which were procured from either the Southwest Trans-
From the Division of Cardiology, Department of Internal Medicine (M.S., P.A.G.), Baylor University Medical Center, Baylor Heart and Vascular Institute, Dallas, Texas, USA; Baylor Research Institute Fort-Worth Campus (M.S., H.N., M.T., K.S., T.I., S.M., Y.F.), Islet Cell Laboratory, Texas, USA; Baylor Institute for Immunology Research (D.C.), Dallas, Texas, USA; Baylor Regional Transplant Institute (B.N., Y.T., G.S.O., N.O., M.F.L.), Dallas and Fort-Worth, Texas, USA; and Institute of Biomedical Studies (B.N., H.P.), Baylor University, Waco, Texas, USA. This study was partially supported by All Saints Health Foundation. Address reprint requests to Shinichi Matsumoto, MD, PhD, Baylor Research Institute Fort Worth Campus, Islet Cell Laboratory, 1400 8th Ave, Fort Worth, TX 76104. E-mail: shinichm@ baylorhealth.edu
0041-1345/10/$–see front matter doi:10.1016/j.transproceed.2010.05.094
© 2010 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
2024
Transplantation Proceedings, 42, 2024 –2026 (2010)
IMPROVED HUMAN ISLET ISOLATION
2025
plant Alliance (Dallas, Tex, USA) or LifeGift (Fort Worth, Tex, USA) between 2008 and 2009. They were allocated to the standard or the new method groups. The islets were isolated based on the Edmonton protocol with modifications.1,13–16 All pancreata were digested with 1 vial of Collagenase NB1 Premium Grade and two vials of Neutral Protease (Serva). Different concentrations of collagenases were used in the perfusion step in the two groups. In the standard group (n ⫽ 3), the pancreas was expanded by injecting 200 mL of cold collagenase solution (2.5 mg/mL) under a lowpressure, controlled manner (60 – 80 mm Hg) for 5 minutes. Then the pressure was raised to 160 to 180 mm Hg for another 5 minutes. In contrast, in the new method (n ⫽ 3), the expansion was first performed with approximately the same volume of pancreas weight in grams of cold collagenase solution (approximately 100 mL, 5 mg/mL) using a syringe in a pressure-controlled manner (60 – 80 mm Hg) for 5 minutes followed by adding perfusion solution to 200 mL and performing a high-pressure step in the same condition as the standard one. We used a syringe for the initial perfusion step of the new method, because 100 mL of solution was not enough to fulfill the pump circuit. The following procedure and evaluation were performed based on the Edmonton protocol. For the statistical analysis, we considered P values less than .05 as significant in this study.
RESULTS
The donor and isolation variables are shown in Table 1 There was no significant difference in the average of used collagenase activity, donor age, pancreas weight, phase I time, and undigested tissue volume. In contrast, phase II time in the new group was significantly shorter than standard group: new: 37 ⫾ 1 versus standard: 52 ⫾ 4 (P ⬍ .05). Importantly, the percentage of embedded islets in the digests of the new group was significantly lower than the standard group: new: 54% ⫾ 4%, versus standard: 83% ⫾ 5% (P ⬍ .05). The postpurification islet equivalents per pancreas weight (IEQ/g) and the recovery rate in the new group were higher than the standard group, although they Table 1. Donor and Isolation Variables of Two Groups Standard Method (n ⫽ 3)
Donor age (y) BMI Cold ischemic time (min) Pancreas weight (g) Collagenase activity (PZU/vial) Phase I Phase II Undigested tissue % Trapped islet Prepurification IEQ/g Postpurification IEQ/g Postpurification purity Postpurification viability Recovery rate (%) Final pellet (mL) Stimulation index
New Method (n ⫽ 3)
P Value
23.7 ⫾ 0.7 25.0 ⫾ 0.2 203 ⫾ 6 80 ⫾ 10 2430 ⫾ 71
21.3 ⫾ 1.5 32.6 ⫾ 2.6 166 ⫾ 48 86 ⫾ 17 2428 ⫾ 13
.320 .071 .560 .836 .989
14 ⫾ 1 52 ⫾ 4 7⫾4 83 ⫾ 5 7802 ⫾ 832 3901 ⫾ 637 47.3 ⫾ 2.9 99.1 ⫾ 0.3 50.3 ⫾ 7.2 11.3 ⫾ 1.0 8.1 ⫾ 2.5
17 ⫾ 2 37 ⫾ 1 4⫾1 54 ⫾ 4 9558 ⫾ 1921 6475 ⫾ 1538 49.3 ⫾ 2.7 98.0 ⫾ 0.6 66.8 ⫾ 3.9 7.3 ⫾ 1.0 13.7 ⫾ 6.3
.286 .029 .577 .019 .531 .275 .704 .328 .175 .156 .539
All values are expressed by the average ⫾ standard errors of the mean. BMI, body mass index; PZU, ; IEQ/g, islet equivalent per pancreas weight.
did not reach statistical significance (IEQ/g, new: 6475 ⫾ 1538, standard: 3901 ⫾ 637; recovery rate, new: 66.8% ⫾ 3.9%, standard: 50.3% ⫾ 7.2%, respectively). The stimulation index of isolated islets in the new group was also higher than the standard group, but not significantly (new: 13.7 ⫾ 6.3, standard: 8.1 ⫾ 2.5). There was no significant difference between the two groups in the postpurification purity, viability, and final tissue volume. DISCUSSION
In consideration of the donor shortage, it is crucial to make more human donors accessible for islet transplantation. In clinical transplantation, pancreata obtained from young donors showed good performance in adult recipients,17 encouraging improved isolation of islets from young donors. However, a major obstacle to utilizing young donors is the technical inability to obtain “free” islets, which are not surrounded by exocrine cells, which makes the purification step extremely difficult. This problem may be due to the anatomic and physiological features of young pancreas.6,12 Many efforts have been made to overcome the difficulty, but so far few successful results have been reported.6,12 In this study, we hypothesized that the current collagenases used for islet isolation were not sufficient to digest the collagens of the young pancreas; therefore, a high concentration of collagenase might improve the digestion. For this purpose, in our new method, we infused collagenase at almost double the concentration compared with the standard method. Our data showed that this method enabled us to significantly improve the islet separation from exocrine cells. It strongly indicated that higher collagenase concentrations can more effectively digest collagens of the young pancreas. In contrast, a highly dense collagenase might harm the islets. However, this study showed that the islet viability using the new method was not different from the standard outcome. Moreover, an in vitro functional assay (stimulation index) showed even better results with the new method (Table 1). However, considering that more than half of the digested islets were embedded even in the new method group there is a requirement for further study and modification. In conclusion, a simple modification of the perfusion method using a concentrated collagenase with a syringe may reduce the degree of embedded islets and increase the yield after purification. REFERENCES 1. Shapiro AM, Lakey JR, Ryan EA, et al: Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 343:230, 2000 2. Robertson RP: Islet transplantation as a treatment for diabetes—a work in progress. N Engl J Med 350:694, 2004 3. Matsumoto S, Okitsu T, Iwanaga Y, et al: Insulin independence after living-donor distal pancreatectomy and islet allotransplantation. Lancet 365:1642, 2005 4. Matsumoto S, Okitsu T, Iwanaga Y, et al: Successful islet transplantation from nonheartbeating donor pancreata using modified Ricordi islet isolation method. Transplantation 82:460, 2006
2026 5. Ryan EA, Paty BW, Senior PA, et al: Five-year follow-up after clinical islet transplantation. Diabetes 54:2060, 2005 6. Lakey JR, Warnock GL, Rajotte RV, et al: Variables in organ donors that affect the recovery of human islets of Langerhans. Transplantation 61:1047, 1996 7. Hanley SC, Paraskevas S, Rosenberg L: Donor and isolation variables predicting human islet isolation success. Transplantation 85:950, 2008 8. Socci C, Davalli AM, Vignali A, et al: A significant increase of islet yield by early injection of collagenase into the pancreatic duct of young donors. Transplantation 55:661, 1993 9. Ricordi C: Islet transplantation: a brave new world. Diabetes 52:1595, 2003 10. Ricordi C, Alejandro R, Zeng Y, et al: Human islet isolation and purification from pediatric-age donors. Transplant Proc 23: 783, 1991 11. Socci C, Davalli AM, Vignali A, et al: A significant increase of islet yield by early injection of collagenase into the pancreatic duct of young donors. Transplantation 55:661, 1993
SHIMODA, NOGUCHI, NAZIRUDDIN ET AL 12. Balamurugan AN, Chang Y, Bertera S, et al: Suitability of human juvenile pancreatic islets for clinical use. Diabetologia 49:1845, 2006 13. Matsumoto S, Noguchi H, Naziruddin B, et al: Improvement of pancreatic islet cell isolation for transplantation. Proc (Bayl Univ Med Cent) 20:357, 2007 14. Noguchi H, Ikemoto T, Naziruddin B, et al: Iodixanolcontrolled density gradient during islet purification improves recovery rate in human islet isolation. Transplantation 87:1629, 2009 15. Ikemoto T, Noguchi H, Shimoda M, et al: Islet cell transplantation for the treatment of type 1 diabetes in the USA. J Hepatobiliary Pancreat Surg 16:118, 2009 16. Matsumoto S, Noguchi H, Hatanaka N, et al: Estimation of donor usability for islet transplantation in the United States with the Kyoto islet isolation method. Cell Transplant 18:549, 2009 17. Buggenhout A, Hoang AD, Hut F, et al: Pediatric en bloc dual kidney–pancreas transplantation into an adult recipient: a simplified technique. Benefits of the en bloc kidney–pancreas transplantation technique in pediatric donors. Am J Transplant 4:663, 2004