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New Class of Oxygen Carriers Improves Islet Isolation From Long-Term Stored Rat Pancreata
New Class of Oxygen Carriers Improves Islet Isolation From LongTerm Stored Rat Pancreata H. Brandhorst, B. Muehling, H. Yamaya, J. Henriksnaes, P.O. Carlsson, O. Korsgren, and D. Brandhorst ABSTRACT Objective. Pancreas shipment is frequently associated with prolonged ischemia deteriorating islet graft function. The strategy to prevent ischemic damage utilizing perfluorodecalin (PFD) for human pancreas oxygenation does not seem to improve isolation outcome. The present study investigated the efficiency of perfluorohexyloctane (F6H8), a hyperoxygen carrier characterized by low specific density (1.33 g/cm3) and lipophilic qualities, to facilitate islet isolation from long-term stored rat pancreata. Materials and Methods. Prior to islet isolation, pancreata were intraductally flushed in situ with Kyoto solution (KS) and stored for 24 hours in KS, oxygenated PFD, or F6H8. Results. Islet isolation performed after 24-hour storage in KS failed completely. The intrapancreatic pO2 in PFD- and F6H8-incubated pancreata was almost the same. In correspondence, the ATP content and viability of isolated islets were similar as well. In contrast, islet yield and in vitro function were significantly reduced after storage in PFD compared with F6H8. Conclusion. This study suggested that islet isolation performed after long-term pancreas preservation can be significantly improved utilizing semifluorinated alkanes as oxygen carriers.
P
ANCREAS SHIPMENT is frequently associated with prolonged ischemia deteriorating islet graft function. The strategy to prevent ischemic damage utilizing perfluorodecalin (PFD) for human pancreas oxygenation does not seem to improve isolation outcome.1,2 Herein we investigated the efficiency of perfluorohexyloctane (F6H8), a semifluorinated alkane with oxygen-dissolving capacity, to facilitate islet isolation from long-term stored rat pancreata. MATERIALS AND METHODS Pancreata obtained from Lewis rats were preserved with Kyoto solution (KS; kindly provided by Prof Nakamura, Kyoto University, Kyoto, Japan), since KS seems to have a lower inhibitory effect on collagenase compared with University of Wisconsin (UW) solution.3 Organs were intraductally flushed with KS and stored for 24 hours in KS (n ⫽ 3), preoxygenated PFD (n ⫽ 10), or F6H8 (n ⫽ 13) (Novaliq GmbH, Heidelberg, Germany).4 Islets were isolated by a standard procedure utilizing collagenase NB1 and neutral protease (Serva Electrophoresis GmbH, Heidelberg, Germany).
RESULTS
Islet isolation performed after 24-hour storage in KS failed completely. The intrapancreatic pO2 did not differ between © 2008 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 40, 393–394 (2008)
PFD- and F6H8-incubated pancreata. The ATP content of freshly isolated islets was slightly higher in F6H8- compared with PFD-oxygenated organs (1.39 ⫾ 0.22 vs 0.98 ⫾ 0.18 pg/islet equivalents [IE], mean ⫾ SEM; NS). Islet yield was significantly reduced after storage in PFD compared with F6H8 (1140 ⫾ 103 vs 1516 ⫾ 63 IE/pancreas; P ⬍ .01). In contrast to viability, which was almost the same in both experimental groups (⬎90%), the glucose stimulation index was significantly decreased after PFD utilization when compared with F6H8 storage (1.25 ⫾ 0.5 vs 1.71 ⫾ 0.14; P ⬍ .05).
From the Departments of Clinical Immunology (H.B., H.Y., O.K., D.B.) and Medical Cell Biology (J.H., P.O.C.), Uppsala University Hospital, Uppsala, Sweden, and Novaliq GmbH, Heidelberg, Germany (B.M.). This work was supported by JDRF grant 1-2007-198 (D.B.). 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.2008.01.012 393
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BRANDHORST, MUEHLING, YAMAYA ET AL
DISCUSSION
REFERENCES
Recent studies in large populations of human donor pancreata indicated that the utilization of oxygenated PFD does not seem to improve islet isolation or posttransplantation function compared with UW storage.1,2 It rather seems that the physical characteristics of PFD prevent oxygen penetration into the core of cold-stored pancreata and may even damage the tissue by enormous gravity. We hypothesized that a significant improvement could be reached by the introduction of biocompatible oxygen carriers. F6H8, used in the present study, has nearly the same oxygen solubility as PFD but is characterized by a lower specific density (1.33 vs 1.93 g/cm3) and lipophilic qualities that can provide oxygen release into the core of ischemic donor pancreata.5 The present study suggested that islet isolation from long-term stored pancreata can be significantly improved utilizing F6H8 as oxygen carrier.
1. Kin T, Mirbolooki M, Salehi P, et al: Islet isolation and transplantation outcomes of pancreas preserved with University of Wisconsin solution versus two-layer method using preoxygenated perfluorocarbon. Transplantation 82:1286, 2006. 2. Caballero-Corbalán J, Eich T, Lundgren T, et al: No beneficial effect of two-layer storage compared with UW-storage on human islet isolation and transplantation. Transplantation 84:864, 2007. 3. Noguchi H, Ueda M, Nakai Y, et al: Modified two-layer preservation method (M-Kyoto/PFC) improves islet yields in islet isolation. Am J Transplant 6:496, 2006. 4. Brandhorst D, Iken M, Brendel MD, et al: Successful pancreas preservation by a perfluorocarbon-based one-layer method for subsequent pig islet isolation. Transplantation 79:433, 2005. 5. Hoerauf H, Kobuch K, Dresp J, et al: Combined use of partially fluorinated alkanes, perfluorocarbon liquids and silicone oil: an experimental study. Graefes Arch Clin Exp Ophthalmol 239:373, 2001.
P
ANCREAS SHIPMENT is frequently associated with prolonged ischemia deteriorating islet graft function. The strategy to prevent ischemic damage utilizing perfluorodecalin (PFD) for human pancreas oxygenation does not seem to improve isolation outcome.1,2 Herein we investigated the efficiency of perfluorohexyloctane (F6H8), a semifluorinated alkane with oxygen-dissolving capacity, to facilitate islet isolation from long-term stored rat pancreata. MATERIALS AND METHODS Pancreata obtained from Lewis rats were preserved with Kyoto solution (KS; kindly provided by Prof Nakamura, Kyoto University, Kyoto, Japan), since KS seems to have a lower inhibitory effect on collagenase compared with University of Wisconsin (UW) solution.3 Organs were intraductally flushed with KS and stored for 24 hours in KS (n ⫽ 3), preoxygenated PFD (n ⫽ 10), or F6H8 (n ⫽ 13) (Novaliq GmbH, Heidelberg, Germany).4 Islets were isolated by a standard procedure utilizing collagenase NB1 and neutral protease (Serva Electrophoresis GmbH, Heidelberg, Germany).
RESULTS
Islet isolation performed after 24-hour storage in KS failed completely. The intrapancreatic pO2 did not differ between © 2008 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 40, 393–394 (2008)
PFD- and F6H8-incubated pancreata. The ATP content of freshly isolated islets was slightly higher in F6H8- compared with PFD-oxygenated organs (1.39 ⫾ 0.22 vs 0.98 ⫾ 0.18 pg/islet equivalents [IE], mean ⫾ SEM; NS). Islet yield was significantly reduced after storage in PFD compared with F6H8 (1140 ⫾ 103 vs 1516 ⫾ 63 IE/pancreas; P ⬍ .01). In contrast to viability, which was almost the same in both experimental groups (⬎90%), the glucose stimulation index was significantly decreased after PFD utilization when compared with F6H8 storage (1.25 ⫾ 0.5 vs 1.71 ⫾ 0.14; P ⬍ .05).
From the Departments of Clinical Immunology (H.B., H.Y., O.K., D.B.) and Medical Cell Biology (J.H., P.O.C.), Uppsala University Hospital, Uppsala, Sweden, and Novaliq GmbH, Heidelberg, Germany (B.M.). This work was supported by JDRF grant 1-2007-198 (D.B.). 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.2008.01.012 393
394
BRANDHORST, MUEHLING, YAMAYA ET AL
DISCUSSION
REFERENCES
Recent studies in large populations of human donor pancreata indicated that the utilization of oxygenated PFD does not seem to improve islet isolation or posttransplantation function compared with UW storage.1,2 It rather seems that the physical characteristics of PFD prevent oxygen penetration into the core of cold-stored pancreata and may even damage the tissue by enormous gravity. We hypothesized that a significant improvement could be reached by the introduction of biocompatible oxygen carriers. F6H8, used in the present study, has nearly the same oxygen solubility as PFD but is characterized by a lower specific density (1.33 vs 1.93 g/cm3) and lipophilic qualities that can provide oxygen release into the core of ischemic donor pancreata.5 The present study suggested that islet isolation from long-term stored pancreata can be significantly improved utilizing F6H8 as oxygen carrier.
1. Kin T, Mirbolooki M, Salehi P, et al: Islet isolation and transplantation outcomes of pancreas preserved with University of Wisconsin solution versus two-layer method using preoxygenated perfluorocarbon. Transplantation 82:1286, 2006. 2. Caballero-Corbalán J, Eich T, Lundgren T, et al: No beneficial effect of two-layer storage compared with UW-storage on human islet isolation and transplantation. Transplantation 84:864, 2007. 3. Noguchi H, Ueda M, Nakai Y, et al: Modified two-layer preservation method (M-Kyoto/PFC) improves islet yields in islet isolation. Am J Transplant 6:496, 2006. 4. Brandhorst D, Iken M, Brendel MD, et al: Successful pancreas preservation by a perfluorocarbon-based one-layer method for subsequent pig islet isolation. Transplantation 79:433, 2005. 5. Hoerauf H, Kobuch K, Dresp J, et al: Combined use of partially fluorinated alkanes, perfluorocarbon liquids and silicone oil: an experimental study. Graefes Arch Clin Exp Ophthalmol 239:373, 2001.