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perfluorochemical) preservation method on clinical grade pancreata prior to islet isolation and transplantation
The Effect of Two-Layer (University of Wisconsin Solution/Perfluorochemical) Preservation Method on Clinical Grade Pancreata Prior to Islet Isolation and Transplantation S. Matsumoto, G. Zhang, S. Qualley, J. Clever, Y. Tombrello, D.M. Strong, and J.A. Reems ABSTRACT Background. Research-grade pancreata preserved by the two-layer method (TLM) compared to organs stored with University of Wisconsin (UW) solution prior to islet isolation result in significantly better islet yields. However, it is unknown whether the TLM improves islet yields from pancreata that meet the criteria for the selection of clinical-grade organs. Methods. Six clinical-grade pancreata were preserved for 4.8 ⫾ 0.5 hour in UW and three clinical-grade pancreata were preserved by the TLM for 11.7 ⫾ 2.0 hour. The local team procured all pancreata. All donors were hemodynamically stable without norepinephrine usage and length of hospitalization was less than 96 hour. Causes of death were either head trauma or cerebrovascular accident. Islets were isolated and evaluated according to the Edmonton protocol. Results. The TLM as compared to UW resulted in a significant increase in islet yields (3415 ⫾ 227 vs 2006 ⫾ 337 IE/g pancreas, P ⬍ .03). The quality of islets as assessed by visual score was significantly better in the TLM group (8.7 ⫾ 0.2 vs 7.3 ⫾ 0.3, P ⬍ .02) but other parameters (viability, survival rate after culture, insulin content, stimulation index) were similar between the two groups. We transplanted all three islet preparations in the TLM group but only two of six preparations from the UW group. Conclusion. Compared to UW, exposure of pancreata to the TLM resulted in greater islet yields and extended preservation times with clinical grade pancreas. Pancreata should be preserved by the TLM prior to islet isolation even for donors that meet clinical grade organ selection criteria. The Human Islet Transplantation in Seattle (HITS) Consortium is supported in part by a grant from the Juvenile Diabetes Research Foundation International. The HITS consortium is an islet transplant program involving the University of Washington, Pacific Northwest Research Institute, the Puget Sound Blood Center, Fred Hutchinson Cancer Research Center, Swedish Hospital, and the Virginia Mason Research Center.
T
HE UNIVERSITY OF ALBERTA group showed the substantial improvement of allogeneic islet transplantation for the treatment of type I diabetes.1 While this represents a milestone for islet transplantation, it is important to improve the efficacy of islet isolation. Recently we have demonstrated that the two-layer method (TLM) of pancreas preservation prior to islet isolation significantly improved islet yield.2 In those studies we used researchgrade human pancreata that had already been declined for both clinical whole pancreas and islet transplantation. Usually islets are isolated from research-grade pancreata; however, if islet transplantation becomes as reliable as pancreas
transplantation, many clinical-grade pancreata would be directly allocated to islet transplantation. Indeed, for the clinical islet transplantation we are able to isolate islets
From the Islet and Cell Processing Laboratory (ICPL) at Puget Sound Blood Center/Northwest Tissue Center (S.M., G.Z., S.O., J.C., Y.T., D.M.S., J.A.R.) University of Washington Department of Surgery, Division of Transplantation (S.M.) Address reprint requests to Shinichi Matsumoto, MD, PhD, Kyoto University Hospital Transplant Unit, 54 Kawara-cho Shogoin, Sakyo-ku Kyoto 606-8507, Japan. E-mail: shinichi@kuhp. kyoto-u.ac.jp
© 2004 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/04/$–see front matter doi:10.1016/j.transproceed.2004.04.012
Transplantation Proceedings, 36, 1037–1039 (2004)
1037
1038
MATSUMOTO, ZHANG, QUALLEY ET AL Table 1. Outcome of Clinical Islet Isolation
UW TLM P value
Yield (IE/g)
Score
Viability (%)
Purity (%)
Pellet size (mL)
Transplant
2006 ⫾ 337 3415 ⫾ 227 ⬍.03
7.3 ⫾ 0.3 8.7 ⫾ 0.2 ⬍.02
92.2 ⫾ 3.0 97.7 ⫾ 0.7 .25
44 ⫾ 6 43 ⫾ 9 .94
2.7 ⫾ 0.3 3.5 ⫾ 0.3 .13
2/6 3/3 .17
from clinical-grade pancreata if the donor is older than 35 years old in our northwest region (UNOS region 6). Since we are able to isolate islets from the best pancreas, we might not need the TLM prior to islet isolation. Indeed the efficacy of the TLM with clinical-grade pancreata before clinical whole pancreas transplantation was not as clear as canine experiments, since the pancreata were in good condition even with UW storage.3 In this study, we compared the TLM to UW for clinical-grade human pancreas preservation prior to islet isolation. MATERIALS AND METHODS Pancreas Procurement and Preservation From April 2002 to September 2002, nine pancreata were distributed to the Islet and Cell Processing Laboratory (ICPL) at the Northwest Tissue Center. Suitable donors for islet isolation were determined based on Edmonton’s experiences.1 All donors were hemodynamically stable as assessed by creatinine, transaminases levels, and use of vasopressor. Hospitalization period was less than 96 hours. Our local team performed all procurements. Causes of death were either head trauma or cerebrovascular accident. Cold preservation time was restricted up to 12 hours with the UW and up to 18 hours with the TLM. At the time of procurement, the pancreas was either immediately placed in chilled UW (n ⫽ 6) (ViaSpan, DuPont Pharma) or preserved by oxygen-charged static TLM (n ⫽ 3).4
group had significant longer preservation period (P ⬍ .004) due to different time limitation. Between the two groups there were no significant differences about donor-related variables such as mean age, BMI, peak blood glucose, amylase, and creatinine levels or isolation-related variables such as pancreas weight, time length of the digestion, and the amount of undigested tissue. Islet yields were significantly higher in the TLM group (P ⬍ .03; Table 1). As indicated by the visual score the quality of the islets were significantly higher in the TLM group (P ⬍ .02; Table 1). Viability, purity, and pellet size were not significantly different between the two groups (Table 1). Additionally, there were no significant differences between the two groups comparing stimulation index after glucose challenge test, survival rate after culture, and insulin content (data not shown). In terms of sterility all islet preparations showed negative gram stain and acceptable endotoxin levels. All preparations qualified for viability and packed cell volume. One preparation from the UW group had unacceptable morphological score and another preparation from this group had unacceptable purity for transplantation. Only two of six preparations (33%) in the UW group had high enough islet yields for transplantation but all (three of three) preparations (100%) in the TLM group had high enough islet yields for transplantation (Table 1).
Islet Isolation Islet isolation and evaluation was conducted as previously described2 in accordance to the Edmonton protocol.1
Criteria for Transplantation Transplantable islets were determined based on the Edmonton protocol with some modification.1 The original Edmonton protocol required more than 4000 IE/kg recipient body weight for transplantation. Our protocol required more than 5000 IE/kg when pancreata were preserved in UW and more than 3000 IE/kg when pancreata were preserved by the TLM. Morphological score must be more than 7.0.2 Viability must be more than 70%, purity of islets must be more than 30%, and packed tissue volume must be less than 10 mL.1 In addition, for the sterility testing, the supernatant from the final product must have a negative gram stain, and the endotoxin level must be less than 5 EU/kg recipient body weight. Islet preparations that qualified for these criteria were transplanted as soon as possible without additional culture.1 All results are expressed as mean ⫾ SEM. All statistical analyses were performed using Stat View 5.0.1 (SAS Institute Inc, Cary, NC, USA). P values less than .05 were considered significant.
RESULTS
Average preservation time was 4.8 ⫾ 0.5 hours in the UW group and 11.7 ⫾ 2.0 hours in the TLM group. The TLM
DISCUSSION
The TLM oxygenates pancreata and activates metabolism to generate ATP5,6 and leads to resuscitation of ischemically damaged pancreata.7 Research-grade pancreata often suffered warm ischemic injury due to hypotensive episodes during prolonged hospitalization, usage of vasopressor, and unstable hemodynamic status. The TLM is the most useful when pancreata suffered such type of damage due to its resuscitation effect.8 Recently the University of Miami group demonstrated that the TLM of human pancreas preservation prior to islet isolation significantly improved the islet yield with older donor ages.9 The University of Alberta group also demonstrated that the additional TLM of pancreas preservation prior to islet isolation significantly improved islet yield when pancreata were already preserved in UW solution for relatively long periods.10 However, it is not clear whether the TLM is also beneficial when pancreata are in good condition prior to islet isolation. In this study the TLM prior to islet isolation clearly improved islet yield and morphological score compared to UW storage with clinical-grade pancreata. In addition, it revealed that the main hurdle of clinical islet transplantation is to obtain a high enough islet yield. Since the TLM increased the islet
TWO-LAYER PRESERVATION METHOD
yield, it will directly improve the success rate of transplantation. In the canine model, when pancreata were preserved for 3 hours by the UW or the TLM, the islet yields were similar but the cure rate after islet transplantation was significantly better in the TLM group.11 Based on such data it was decided that minimum islet yield for transplantation in the TLM group would be 3000 IE/kg recipient body weight instead of the original Edmonton’s protocol 4000 IE/kg. With these criteria, we were able to transplant all cases when pancreata were preserved by the TLM in contrast to only 33% when pancreata were preserved in the UW. However, the adequate minimum number for transplantation is unknown and further examination is necessary to decide the minimum acceptable islet yield for transplantation. In conclusion, the TLM significantly improved islet yield and morphological score compared to UW storage using clinical-grade pancreata. Pancreata should be preserved by the TLM prior to islet isolation even when donors are in good condition. ACKNOWLEDGMENT This work was funded in part by the Juvenile Diabetes Research Foundation International (Human Islet Transplant in Seattle). The Human Islet Transplantation in Seattle (HITS) Consortium is supported in part by a grant from the Juvenile Diabetes Research Foundation International. The HITS consortium is an islet trans-
1039 plant program involving the University of Washington, Pacific Northwest Research Institute, the Puget Sound Blood Center, Fred Hutchinson Cancer Research Center, Swedish Hospital, and the Virginia Mason Research Center. The authors thank Lynn Sundborg, Erik Olson, and Sharon Kaplan (ICPL) for technical support, and Laura Schreiber for editorial assistance.
REFERENCES 1. Shapiro A, Lakey J, Ryan E, et al: N Engl J Med 343:230, 2000 2. Matsumoto S, Qualley S, Goel S, et al: Transplantation 74:1414, 2002 3. Matsumoto S, Kandaswamy R, Sutherland D, et al: Transplantation 70:771, 2000 4. Matsumoto S, Rigley T, Qualley S, et al: Cell Transplant 11:769, 2002 5. Matsumoto S, Fujino Y, Suzuki Y, et al: Pancreas 20:411, 2000 6. Matsumoto S, Kuroda Y, Hamano M, et al: Transplantation 62:1667, 1996 7. Kuroda Y, Morita A, Fujino Y, et al: Transplantation 55:227, 1993 8. Matsumoto S, Kuroda Y: Transplantation 74:1804, 2002 9. Fraker C, Alejandro R, Ricordi C: Transplantation 74:1811, 2002 10. Lakey J, Tsujimura T, Shapiro A, Kuroda Y: Transplantation 74:1809, 2002 11. Tanioka Y, Sutherland D, Kuroda Y, et al: Surgery 122:435, 1997
T
HE UNIVERSITY OF ALBERTA group showed the substantial improvement of allogeneic islet transplantation for the treatment of type I diabetes.1 While this represents a milestone for islet transplantation, it is important to improve the efficacy of islet isolation. Recently we have demonstrated that the two-layer method (TLM) of pancreas preservation prior to islet isolation significantly improved islet yield.2 In those studies we used researchgrade human pancreata that had already been declined for both clinical whole pancreas and islet transplantation. Usually islets are isolated from research-grade pancreata; however, if islet transplantation becomes as reliable as pancreas
transplantation, many clinical-grade pancreata would be directly allocated to islet transplantation. Indeed, for the clinical islet transplantation we are able to isolate islets
From the Islet and Cell Processing Laboratory (ICPL) at Puget Sound Blood Center/Northwest Tissue Center (S.M., G.Z., S.O., J.C., Y.T., D.M.S., J.A.R.) University of Washington Department of Surgery, Division of Transplantation (S.M.) Address reprint requests to Shinichi Matsumoto, MD, PhD, Kyoto University Hospital Transplant Unit, 54 Kawara-cho Shogoin, Sakyo-ku Kyoto 606-8507, Japan. E-mail: shinichi@kuhp. kyoto-u.ac.jp
© 2004 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/04/$–see front matter doi:10.1016/j.transproceed.2004.04.012
Transplantation Proceedings, 36, 1037–1039 (2004)
1037
1038
MATSUMOTO, ZHANG, QUALLEY ET AL Table 1. Outcome of Clinical Islet Isolation
UW TLM P value
Yield (IE/g)
Score
Viability (%)
Purity (%)
Pellet size (mL)
Transplant
2006 ⫾ 337 3415 ⫾ 227 ⬍.03
7.3 ⫾ 0.3 8.7 ⫾ 0.2 ⬍.02
92.2 ⫾ 3.0 97.7 ⫾ 0.7 .25
44 ⫾ 6 43 ⫾ 9 .94
2.7 ⫾ 0.3 3.5 ⫾ 0.3 .13
2/6 3/3 .17
from clinical-grade pancreata if the donor is older than 35 years old in our northwest region (UNOS region 6). Since we are able to isolate islets from the best pancreas, we might not need the TLM prior to islet isolation. Indeed the efficacy of the TLM with clinical-grade pancreata before clinical whole pancreas transplantation was not as clear as canine experiments, since the pancreata were in good condition even with UW storage.3 In this study, we compared the TLM to UW for clinical-grade human pancreas preservation prior to islet isolation. MATERIALS AND METHODS Pancreas Procurement and Preservation From April 2002 to September 2002, nine pancreata were distributed to the Islet and Cell Processing Laboratory (ICPL) at the Northwest Tissue Center. Suitable donors for islet isolation were determined based on Edmonton’s experiences.1 All donors were hemodynamically stable as assessed by creatinine, transaminases levels, and use of vasopressor. Hospitalization period was less than 96 hours. Our local team performed all procurements. Causes of death were either head trauma or cerebrovascular accident. Cold preservation time was restricted up to 12 hours with the UW and up to 18 hours with the TLM. At the time of procurement, the pancreas was either immediately placed in chilled UW (n ⫽ 6) (ViaSpan, DuPont Pharma) or preserved by oxygen-charged static TLM (n ⫽ 3).4
group had significant longer preservation period (P ⬍ .004) due to different time limitation. Between the two groups there were no significant differences about donor-related variables such as mean age, BMI, peak blood glucose, amylase, and creatinine levels or isolation-related variables such as pancreas weight, time length of the digestion, and the amount of undigested tissue. Islet yields were significantly higher in the TLM group (P ⬍ .03; Table 1). As indicated by the visual score the quality of the islets were significantly higher in the TLM group (P ⬍ .02; Table 1). Viability, purity, and pellet size were not significantly different between the two groups (Table 1). Additionally, there were no significant differences between the two groups comparing stimulation index after glucose challenge test, survival rate after culture, and insulin content (data not shown). In terms of sterility all islet preparations showed negative gram stain and acceptable endotoxin levels. All preparations qualified for viability and packed cell volume. One preparation from the UW group had unacceptable morphological score and another preparation from this group had unacceptable purity for transplantation. Only two of six preparations (33%) in the UW group had high enough islet yields for transplantation but all (three of three) preparations (100%) in the TLM group had high enough islet yields for transplantation (Table 1).
Islet Isolation Islet isolation and evaluation was conducted as previously described2 in accordance to the Edmonton protocol.1
Criteria for Transplantation Transplantable islets were determined based on the Edmonton protocol with some modification.1 The original Edmonton protocol required more than 4000 IE/kg recipient body weight for transplantation. Our protocol required more than 5000 IE/kg when pancreata were preserved in UW and more than 3000 IE/kg when pancreata were preserved by the TLM. Morphological score must be more than 7.0.2 Viability must be more than 70%, purity of islets must be more than 30%, and packed tissue volume must be less than 10 mL.1 In addition, for the sterility testing, the supernatant from the final product must have a negative gram stain, and the endotoxin level must be less than 5 EU/kg recipient body weight. Islet preparations that qualified for these criteria were transplanted as soon as possible without additional culture.1 All results are expressed as mean ⫾ SEM. All statistical analyses were performed using Stat View 5.0.1 (SAS Institute Inc, Cary, NC, USA). P values less than .05 were considered significant.
RESULTS
Average preservation time was 4.8 ⫾ 0.5 hours in the UW group and 11.7 ⫾ 2.0 hours in the TLM group. The TLM
DISCUSSION
The TLM oxygenates pancreata and activates metabolism to generate ATP5,6 and leads to resuscitation of ischemically damaged pancreata.7 Research-grade pancreata often suffered warm ischemic injury due to hypotensive episodes during prolonged hospitalization, usage of vasopressor, and unstable hemodynamic status. The TLM is the most useful when pancreata suffered such type of damage due to its resuscitation effect.8 Recently the University of Miami group demonstrated that the TLM of human pancreas preservation prior to islet isolation significantly improved the islet yield with older donor ages.9 The University of Alberta group also demonstrated that the additional TLM of pancreas preservation prior to islet isolation significantly improved islet yield when pancreata were already preserved in UW solution for relatively long periods.10 However, it is not clear whether the TLM is also beneficial when pancreata are in good condition prior to islet isolation. In this study the TLM prior to islet isolation clearly improved islet yield and morphological score compared to UW storage with clinical-grade pancreata. In addition, it revealed that the main hurdle of clinical islet transplantation is to obtain a high enough islet yield. Since the TLM increased the islet
TWO-LAYER PRESERVATION METHOD
yield, it will directly improve the success rate of transplantation. In the canine model, when pancreata were preserved for 3 hours by the UW or the TLM, the islet yields were similar but the cure rate after islet transplantation was significantly better in the TLM group.11 Based on such data it was decided that minimum islet yield for transplantation in the TLM group would be 3000 IE/kg recipient body weight instead of the original Edmonton’s protocol 4000 IE/kg. With these criteria, we were able to transplant all cases when pancreata were preserved by the TLM in contrast to only 33% when pancreata were preserved in the UW. However, the adequate minimum number for transplantation is unknown and further examination is necessary to decide the minimum acceptable islet yield for transplantation. In conclusion, the TLM significantly improved islet yield and morphological score compared to UW storage using clinical-grade pancreata. Pancreata should be preserved by the TLM prior to islet isolation even when donors are in good condition. ACKNOWLEDGMENT This work was funded in part by the Juvenile Diabetes Research Foundation International (Human Islet Transplant in Seattle). The Human Islet Transplantation in Seattle (HITS) Consortium is supported in part by a grant from the Juvenile Diabetes Research Foundation International. The HITS consortium is an islet trans-
1039 plant program involving the University of Washington, Pacific Northwest Research Institute, the Puget Sound Blood Center, Fred Hutchinson Cancer Research Center, Swedish Hospital, and the Virginia Mason Research Center. The authors thank Lynn Sundborg, Erik Olson, and Sharon Kaplan (ICPL) for technical support, and Laura Schreiber for editorial assistance.
REFERENCES 1. Shapiro A, Lakey J, Ryan E, et al: N Engl J Med 343:230, 2000 2. Matsumoto S, Qualley S, Goel S, et al: Transplantation 74:1414, 2002 3. Matsumoto S, Kandaswamy R, Sutherland D, et al: Transplantation 70:771, 2000 4. Matsumoto S, Rigley T, Qualley S, et al: Cell Transplant 11:769, 2002 5. Matsumoto S, Fujino Y, Suzuki Y, et al: Pancreas 20:411, 2000 6. Matsumoto S, Kuroda Y, Hamano M, et al: Transplantation 62:1667, 1996 7. Kuroda Y, Morita A, Fujino Y, et al: Transplantation 55:227, 1993 8. Matsumoto S, Kuroda Y: Transplantation 74:1804, 2002 9. Fraker C, Alejandro R, Ricordi C: Transplantation 74:1811, 2002 10. Lakey J, Tsujimura T, Shapiro A, Kuroda Y: Transplantation 74:1809, 2002 11. Tanioka Y, Sutherland D, Kuroda Y, et al: Surgery 122:435, 1997