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Degraded Collagenase Deteriorates Islet Viability
Degraded Collagenase Deteriorates Islet Viability H. Brandhorst, N. Raemsch-Guenther, C. Raemsch, O. Friedrich, M. Kurfuerst, O. Korsgren, and D. Brandhorst ABSTRACT Objective. The utilization of purified enzyme blends consisting of collagenase class I (CI) and II (CII) and neutral protease is an essential step for clinical islet isolation. Previous studies suggested that the use of enzyme lots containing degraded CI reduced islet release from human pancreata. The present study sought to assess the effect of degraded collagenase on islet function in vitro and posttransplantation. Materials and Methods. Crude collagenase was chromatographically separated into CI, CII, and a mixture of degraded CI and CII isomers. Subsequently, classes were recombined to obtain a CII/CI ratio of 0.5. Rat islets were isolated utilizing neutral protease and 20 units of recombined collagenase containing either intact (Ci) or degraded isomers (Cd). Results. Digestion time was reduced utilizing Cd (P ⬍ .001). The highest islet yield and lowest islet fragmentation were obtained with Ci (P ⬍ .01). Utilization of Cd corresponded to a reduction in viability and in vitro function (NS). Islet transplantation reversed hyperglycemia in diabetic nude mice, but revealed an absence of weight gain in recipients receiving islets isolated using Cd (P ⬍ .01). Conclusion. This study suggested that islet function posttransplantation is affected by degraded collagenase isomers. This finding has to be considered for the purification process of collagenase.
T
HE UTILIZATION of purified enzyme blends consisting of collagenase class I (CI) and II (CII) and neutral protease is an essential step within the current standard procedure for clinical islet isolation. Nevertheless, these enzyme blends are not completely characterized. Previous studies suggested that the use of enzyme lots containing degraded CI reduced islet release from human pancreata.1 The present study sought to assess the effect of degraded collagenase on in vitro and posttransplantation islet function.
RESULTS
MATERIALS AND METHODS
Compared with Ci (19.0 ⫾ 0.3, mean ⫾ SEM), digestion time was significantly reduced utilizing Cd (13.3 ⫾ 0.2; P ⬍ .001). Increased islet yield per pancreas (2085 ⫾ 161 vs 1740 ⫾ 73 IE; P ⬍ .01) and reduced fragmentation (0.44 ⫾ 0.05 vs 0.64 ⫾ 0.03; P ⬍ .01) were achieved with Ci compared with Cd. Utilization of Cd corresponded to a reduction in viability and in vitro function (NS). Islet transplantation reversed hyperglycemia in all diabetic recipients, but revealed an absence of weight gain or reduction in body weight among mice that had received islets isolated by means of Cd (P ⬍ .01).
Crude collagenase was chromatographically separated into CI, CII, and a mixture of small molecular weight CI and CII isomers. Subsequently, classes were recombined to obtain a CII/CI ratio of 0.5. Rat islets were isolated by a standard procedure utilizing neutral protease and 20 units of PZ-U recombined collagenase containing either intact (Ci) or degraded isomers (Cd) (Serva Electophoresis GmbH, Heidelberg, Germany).2,3 Quality assessment included yield on islet equivalents (IE), fragmentation index (islet number/IE), viability, insulin release during static glucose incubation, and posttransplantation function in diabetic nude mice.
From the Department of Clinical Immunology, Uppsala University Hospital, Uppsala, Sweden (H.B., O.K., D.B.); Serva Electrophoresis GmbH, Heidelberg, Germany (N.R.-G.); and Nordmark Arzneimittel GmbH & Co KG, Jetersen, Germany (C.R., O.F., M.K.). Address reprint requests to Daniel Brandhorst, PhD, Department of Clinical Immunology, Uppsala University Hospital, Dag Hammarskjölds väg 20, SE-75185 Uppsala, Sweden. E-mail: [email protected]
0041-1345/08/$–see front matter doi:10.1016/j.transproceed.2008.01.013 370
© 2008 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 40, 370 –371 (2008)
DEGRADED COLLAGENASE REDUCES ISLET FUNCTION
371
DISCUSSION
REFERENCES
Previous studies suggested that the use of Liberase lots containing degraded collagenase deteriorated islet release from human donor pancreata.1 Moreover, it was demonstrated that components of Liberase HI blend can penetrate into the cytoplasma of human and murine islet cells resulting in decreased function in vitro and posttransplantation.4 A cytotoxic effect of Liberase was also demonstrated in isolated rat islets.5 It is still unknown whether this toxicity was induced by degradation of collagenase or neutral proteases such as thermolysin. The present study suggested that degraded collagenase reduced posttransplantation islet function, a finding that has to be considered for the purification process of collagenase.
1. Barnett MJ, Zhai X, LeGatt DF, et al: Quantitative assessment of collagenase blends for human islet isolation. Transplantation 80:723, 2005 2. Brandhorst D, Huettler S, Alt A, et al: Adjustment of the ratio between collagenase class II and I improves islet isolation outcome. Transplant Proc 37:3450, 2005 3. Jahr H, Hussmann B, Eckhardt T, et al: Successful single donor islet allotransplantation in the streptozotocin diabetes rat model. Cell Transplant 11:513, 2002 4. Balamurugan AN, He J, Guo F, et al: Harmful delayed effects of exogenous isolation enzymes on isolated human islets: relevance to clinical transplantation. Am J Transplant 5:2671, 2005 5. Vargas F, Julian JF, Liamazares JF, et al: Engraftment of islets obtained by collagenase and Liberase in diabetic rats: a comparative study. Pancreas 23:406, 2001
T
HE UTILIZATION of purified enzyme blends consisting of collagenase class I (CI) and II (CII) and neutral protease is an essential step within the current standard procedure for clinical islet isolation. Nevertheless, these enzyme blends are not completely characterized. Previous studies suggested that the use of enzyme lots containing degraded CI reduced islet release from human pancreata.1 The present study sought to assess the effect of degraded collagenase on in vitro and posttransplantation islet function.
RESULTS
MATERIALS AND METHODS
Compared with Ci (19.0 ⫾ 0.3, mean ⫾ SEM), digestion time was significantly reduced utilizing Cd (13.3 ⫾ 0.2; P ⬍ .001). Increased islet yield per pancreas (2085 ⫾ 161 vs 1740 ⫾ 73 IE; P ⬍ .01) and reduced fragmentation (0.44 ⫾ 0.05 vs 0.64 ⫾ 0.03; P ⬍ .01) were achieved with Ci compared with Cd. Utilization of Cd corresponded to a reduction in viability and in vitro function (NS). Islet transplantation reversed hyperglycemia in all diabetic recipients, but revealed an absence of weight gain or reduction in body weight among mice that had received islets isolated by means of Cd (P ⬍ .01).
Crude collagenase was chromatographically separated into CI, CII, and a mixture of small molecular weight CI and CII isomers. Subsequently, classes were recombined to obtain a CII/CI ratio of 0.5. Rat islets were isolated by a standard procedure utilizing neutral protease and 20 units of PZ-U recombined collagenase containing either intact (Ci) or degraded isomers (Cd) (Serva Electophoresis GmbH, Heidelberg, Germany).2,3 Quality assessment included yield on islet equivalents (IE), fragmentation index (islet number/IE), viability, insulin release during static glucose incubation, and posttransplantation function in diabetic nude mice.
From the Department of Clinical Immunology, Uppsala University Hospital, Uppsala, Sweden (H.B., O.K., D.B.); Serva Electrophoresis GmbH, Heidelberg, Germany (N.R.-G.); and Nordmark Arzneimittel GmbH & Co KG, Jetersen, Germany (C.R., O.F., M.K.). Address reprint requests to Daniel Brandhorst, PhD, Department of Clinical Immunology, Uppsala University Hospital, Dag Hammarskjölds väg 20, SE-75185 Uppsala, Sweden. E-mail: [email protected]
0041-1345/08/$–see front matter doi:10.1016/j.transproceed.2008.01.013 370
© 2008 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 40, 370 –371 (2008)
DEGRADED COLLAGENASE REDUCES ISLET FUNCTION
371
DISCUSSION
REFERENCES
Previous studies suggested that the use of Liberase lots containing degraded collagenase deteriorated islet release from human donor pancreata.1 Moreover, it was demonstrated that components of Liberase HI blend can penetrate into the cytoplasma of human and murine islet cells resulting in decreased function in vitro and posttransplantation.4 A cytotoxic effect of Liberase was also demonstrated in isolated rat islets.5 It is still unknown whether this toxicity was induced by degradation of collagenase or neutral proteases such as thermolysin. The present study suggested that degraded collagenase reduced posttransplantation islet function, a finding that has to be considered for the purification process of collagenase.
1. Barnett MJ, Zhai X, LeGatt DF, et al: Quantitative assessment of collagenase blends for human islet isolation. Transplantation 80:723, 2005 2. Brandhorst D, Huettler S, Alt A, et al: Adjustment of the ratio between collagenase class II and I improves islet isolation outcome. Transplant Proc 37:3450, 2005 3. Jahr H, Hussmann B, Eckhardt T, et al: Successful single donor islet allotransplantation in the streptozotocin diabetes rat model. Cell Transplant 11:513, 2002 4. Balamurugan AN, He J, Guo F, et al: Harmful delayed effects of exogenous isolation enzymes on isolated human islets: relevance to clinical transplantation. Am J Transplant 5:2671, 2005 5. Vargas F, Julian JF, Liamazares JF, et al: Engraftment of islets obtained by collagenase and Liberase in diabetic rats: a comparative study. Pancreas 23:406, 2001