- Email: [email protected]
59. Hypothermic machine perfusion preservation of porcine pancreas facilitates islet isolation
Abstracts / Cryobiology 55 (2007) 324–378 traditionally frozen aortic valves (n = 4 animals/group) in the aortic position. Aortic insufficiency was assessed after implant and at 6 months, just prior to removal for pathology studies. Multiphoton imaging demonstrated branched elastic fibers and wavy bundles of collagen in almost all vitrified heart valve leaflets, cardiac muscle and aortic artery specimens. The SHG signals were similarly inducible within the ‘‘ 80 C’’ and ‘‘ 135 C’’ vitrified tissues demonstrating no statistically significant differences. In vivo vitrified heart valves stored at 80 C demonstrated a lower mean aortic insufficiency compared to both vitrified heart valves stored below 135 C and frozen valves. More significant was the gross pathology observation that all the frozen valves demonstrated structural deterioration, while all the vitrified valves stored at 80 C were free of structural deterioration (p < 0.05). In addition to avoidance of ice-induced matrix damage during freezing, the vitrification method permits storage at 80 C in mechanical storage freezers at potential clinical sites and transport to clinical sites on dry ice. In contrast, current American Association of Tissue Bank standards for cardiovascular tissues are storage below 135 C and transport below 100 C. We conclude that the ECM of heart valves is maintained by storage of vitrified samples at 80 C and in vivo studies in sheep are encouraging for future human applications. (Conflicts of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2007.10.060
58. 19F-MRI and headspace gas chromatography to determine penetration of perfluorocarbon in pancreas preserved by two-layer method for islet transplantation. Aditya Agrawal a, So Po-Wah b, Andy Penman c, Martin Press a, Steve Powis a, Barry Fuller a, Brian Davidson a, a Royal Free and University College Medical School, London, United Kingdom; b Biological Imaging Centre, Hammersmith Hospital, London, United Kingdom; c F2 Chemicals Ltd., Preston, United Kingdom The mechanism of action of the two-layer method remains unclear. Direct measurements of pO2 in the core of the pancreas by two separate investigators have yielded conflicting results [Matsumoto S et al. Transplantation 1996; 62(11): 1667-70 and Papas KK et al. Transplantation Proceedings 2005; 37(8): 3501-4]. A fundamental issue is to ascertain the likelihood of penetration of PFC into the substance of the pancreas during TLM cold storage. Segments of the pancreas (7.5 cm in length) were studied using a Varian Inova 9.4T MR spectrometer. Tissues were suspended in UW solution and an external standard of PFC was introduced for quantification. Four consecutive transverse images of 4mm thickness were obtained using a spin-echo sequence: repetition time (TR) of 1s, echo time (TE) of 20 ms, field of view (FOV) of 45 cm · 45 cm, matrix size of 256 · 12 and 4 averages. 19F MRI was then performed using the same parameters. For headspace gas chromatography 500 mg of pancreatic tissue was transferred to 50 ml capacity Pyrex vials containing 1g anhydrous magnesium sulphate. The bottles were placed in a microwave oven and heated for 90 sec at 200 C to volatilize the PFC. One ml of headspace was analysed in a gas chromatograph equipped with a flame ionisation detector. In a transverse 1H MRI image of the porcine pancreas after 24h storage in PFC, PFC was not detected by 1H MRI but was readily observed by 19F MRI. This is not surprising as PFC has no 1H for detection by 1H MRI. Using 19F MRI, the PFC standard can be observed, but no19F signals are detected from the pancreas, suggesting any PFC present in the pancreas is below the detection limit by the 19F MRI experiment. Using GC-MS, with headspace, the background control sample concentration of perfluorodecalin was 0.025 ppm w/w (0.013 nl/g) and the mean concentration in the test samples was 0.021 ± 0.013 ppm w/w (0.011 ± 0.006 nl/g), confirming that no PFC was retained in the test samples. The positive control was a 500 mg sample of porcine pancreas spiked with 0.1 ll of perfluorodecalin. The concentration of the spiked standard was 165.15 nl/g. We conclude that 19F MRI can be used in the setting of pancreas organ preservation, even though in the present work PFC was found not to penetrate the pancreas during TLM. The results from 19F MRI were confirmed by GC-MS. Other methods of perfusion during pancreas preservation (such as intraductal or intravas-
343
cular routes for PFC delivery) should be explored to improve oxygenation and organ viability. (Conflicts of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2007.10.061
59. Hypothermic machine perfusion preservation of porcine pancreas facilitates islet isolation. Michael J. Taylor a, Simona Baicu a, Brian Leman a, Elizabeth Greene a, Alma Vazquez a, John Brassil b, a Cell and Tissue Systems, N. Charleston, SC, USA; b Organ Recovery Systems, Des Plaines, IL, USA Implantation of functional islet cells is a potential cure for diabetes but the availability of high quality islets for transplantation is critical for success. Procurement of donor pancreata for islet isolation and transplantation is not yet widely practiced due to concerns about the effect of postmortem ischemia upon functional islet yields. Perfusion/preservation technology has had a major impact in circumventing ischemic injury in kidney transplantation. Here we applied this approach to the preservation and procurement of viable islets after hypothermic perfusion preservation of porcine pancreata (Px). Pancreata in anesthetized Domestic Yorkshire pigs (25-30 kg) were flushed in situ with cold Lactated Ringer’solution(2L) and surgically removed with intact pancreaticoduodenal arteries. The superior mesenteric artery and celiac trunk were cannulated and the splenic vessels and other arterial branches were ligated to allow uniform perfusion through the gland ex vivo. Pancreata were assigned to one of three experimental preservation treatment groups: G1) Controls - processed immediately with minimum cold ischemia (<1 h); G2) Static Cold Storage - flushed with cold UW-Viaspan and stored at 2-4 C for 24 h, and G3) Hypothermic Machine Perfusion (HMP) - perfused on a LifePort machine with KPS1 solution at 2-6 C and low pressure (10 mmHg) for 24 h. Islet isolation was then accomplished using standard techniques involving ductal distension of the gland with liberase enzyme, normothermic digestion and density gradient purification. Standard, accepted product release criteria were used including islet quantification and islet function using the glucose stimulated insulin secretion assay. Progressive edema during continuous hypothermic perfusion of organs is an intrinsic event that is usually restricted by optimizing perfusion media and conditions. In this study the measured degree of edema was 0%; 3.4 ± 0.5%; and 132 ± 25% for the groups respectively. Negative edema in G2 was due to the hypertonicity of UW-Viaspan during static cold storage. Islet yield expressed as Islet Equivalents (IEQ) was markedly different between the treatment groups: G1 = 1032 ± 253 IEQ/g (n = 6); G2 = 637 ± 119 IEQ/g (n = 8); G3 = 1964 ± 619 IEQ/g (n = 5). The increased yield in the HMP group over the static cold storage in UW-Viaspan was statistically significant (p < 0.05). Functionally, the islets from each experimental group were equivalent and not significantly different to controls (G1) with insulin stimulation indices of G1 = 3.3 ± 0.5(5); G2 = 2.30 ± 0.04(7); G3 = 3.1 ± 0.4(5). However, insulin content (ng/ml/IEQ) was different between the treatment groups with the highest insulin content in islets harvested from HMP Px (G3) = 12.2 ± 5.8(5) compared with G1 = 9.5 ± 4.2(6) and G2 = 4.8 ± 1.1(8) for controls and static cold storage respectively. The purity of the islet preps, measured as the ratio of insulin (from islets) to amylase (from exocrine cells), was: G1 = 28 ± 24%(6); G2 = 11 ± 2%(8); G3 = 20 ± 8%(5). Microscopic examination of the different preparations using Dithizone staining for islets showed a consistently more uniform digestion of the Px from G3 compared with G1 and G2, with greater separation of the tissue and less entrapped islets. We conclude that 24h of HMP is well tolerated leading to moderate edema but no loss of function of the harvested islets. The edema appears to aid in enzymatic digestion producing a greater yield and purity of islets compared with Px subjected to 24h of static cold storage. (Conflicts of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2007.10.062
58. 19F-MRI and headspace gas chromatography to determine penetration of perfluorocarbon in pancreas preserved by two-layer method for islet transplantation. Aditya Agrawal a, So Po-Wah b, Andy Penman c, Martin Press a, Steve Powis a, Barry Fuller a, Brian Davidson a, a Royal Free and University College Medical School, London, United Kingdom; b Biological Imaging Centre, Hammersmith Hospital, London, United Kingdom; c F2 Chemicals Ltd., Preston, United Kingdom The mechanism of action of the two-layer method remains unclear. Direct measurements of pO2 in the core of the pancreas by two separate investigators have yielded conflicting results [Matsumoto S et al. Transplantation 1996; 62(11): 1667-70 and Papas KK et al. Transplantation Proceedings 2005; 37(8): 3501-4]. A fundamental issue is to ascertain the likelihood of penetration of PFC into the substance of the pancreas during TLM cold storage. Segments of the pancreas (7.5 cm in length) were studied using a Varian Inova 9.4T MR spectrometer. Tissues were suspended in UW solution and an external standard of PFC was introduced for quantification. Four consecutive transverse images of 4mm thickness were obtained using a spin-echo sequence: repetition time (TR) of 1s, echo time (TE) of 20 ms, field of view (FOV) of 45 cm · 45 cm, matrix size of 256 · 12 and 4 averages. 19F MRI was then performed using the same parameters. For headspace gas chromatography 500 mg of pancreatic tissue was transferred to 50 ml capacity Pyrex vials containing 1g anhydrous magnesium sulphate. The bottles were placed in a microwave oven and heated for 90 sec at 200 C to volatilize the PFC. One ml of headspace was analysed in a gas chromatograph equipped with a flame ionisation detector. In a transverse 1H MRI image of the porcine pancreas after 24h storage in PFC, PFC was not detected by 1H MRI but was readily observed by 19F MRI. This is not surprising as PFC has no 1H for detection by 1H MRI. Using 19F MRI, the PFC standard can be observed, but no19F signals are detected from the pancreas, suggesting any PFC present in the pancreas is below the detection limit by the 19F MRI experiment. Using GC-MS, with headspace, the background control sample concentration of perfluorodecalin was 0.025 ppm w/w (0.013 nl/g) and the mean concentration in the test samples was 0.021 ± 0.013 ppm w/w (0.011 ± 0.006 nl/g), confirming that no PFC was retained in the test samples. The positive control was a 500 mg sample of porcine pancreas spiked with 0.1 ll of perfluorodecalin. The concentration of the spiked standard was 165.15 nl/g. We conclude that 19F MRI can be used in the setting of pancreas organ preservation, even though in the present work PFC was found not to penetrate the pancreas during TLM. The results from 19F MRI were confirmed by GC-MS. Other methods of perfusion during pancreas preservation (such as intraductal or intravas-
343
cular routes for PFC delivery) should be explored to improve oxygenation and organ viability. (Conflicts of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2007.10.061
59. Hypothermic machine perfusion preservation of porcine pancreas facilitates islet isolation. Michael J. Taylor a, Simona Baicu a, Brian Leman a, Elizabeth Greene a, Alma Vazquez a, John Brassil b, a Cell and Tissue Systems, N. Charleston, SC, USA; b Organ Recovery Systems, Des Plaines, IL, USA Implantation of functional islet cells is a potential cure for diabetes but the availability of high quality islets for transplantation is critical for success. Procurement of donor pancreata for islet isolation and transplantation is not yet widely practiced due to concerns about the effect of postmortem ischemia upon functional islet yields. Perfusion/preservation technology has had a major impact in circumventing ischemic injury in kidney transplantation. Here we applied this approach to the preservation and procurement of viable islets after hypothermic perfusion preservation of porcine pancreata (Px). Pancreata in anesthetized Domestic Yorkshire pigs (25-30 kg) were flushed in situ with cold Lactated Ringer’solution(2L) and surgically removed with intact pancreaticoduodenal arteries. The superior mesenteric artery and celiac trunk were cannulated and the splenic vessels and other arterial branches were ligated to allow uniform perfusion through the gland ex vivo. Pancreata were assigned to one of three experimental preservation treatment groups: G1) Controls - processed immediately with minimum cold ischemia (<1 h); G2) Static Cold Storage - flushed with cold UW-Viaspan and stored at 2-4 C for 24 h, and G3) Hypothermic Machine Perfusion (HMP) - perfused on a LifePort machine with KPS1 solution at 2-6 C and low pressure (10 mmHg) for 24 h. Islet isolation was then accomplished using standard techniques involving ductal distension of the gland with liberase enzyme, normothermic digestion and density gradient purification. Standard, accepted product release criteria were used including islet quantification and islet function using the glucose stimulated insulin secretion assay. Progressive edema during continuous hypothermic perfusion of organs is an intrinsic event that is usually restricted by optimizing perfusion media and conditions. In this study the measured degree of edema was 0%; 3.4 ± 0.5%; and 132 ± 25% for the groups respectively. Negative edema in G2 was due to the hypertonicity of UW-Viaspan during static cold storage. Islet yield expressed as Islet Equivalents (IEQ) was markedly different between the treatment groups: G1 = 1032 ± 253 IEQ/g (n = 6); G2 = 637 ± 119 IEQ/g (n = 8); G3 = 1964 ± 619 IEQ/g (n = 5). The increased yield in the HMP group over the static cold storage in UW-Viaspan was statistically significant (p < 0.05). Functionally, the islets from each experimental group were equivalent and not significantly different to controls (G1) with insulin stimulation indices of G1 = 3.3 ± 0.5(5); G2 = 2.30 ± 0.04(7); G3 = 3.1 ± 0.4(5). However, insulin content (ng/ml/IEQ) was different between the treatment groups with the highest insulin content in islets harvested from HMP Px (G3) = 12.2 ± 5.8(5) compared with G1 = 9.5 ± 4.2(6) and G2 = 4.8 ± 1.1(8) for controls and static cold storage respectively. The purity of the islet preps, measured as the ratio of insulin (from islets) to amylase (from exocrine cells), was: G1 = 28 ± 24%(6); G2 = 11 ± 2%(8); G3 = 20 ± 8%(5). Microscopic examination of the different preparations using Dithizone staining for islets showed a consistently more uniform digestion of the Px from G3 compared with G1 and G2, with greater separation of the tissue and less entrapped islets. We conclude that 24h of HMP is well tolerated leading to moderate edema but no loss of function of the harvested islets. The edema appears to aid in enzymatic digestion producing a greater yield and purity of islets compared with Px subjected to 24h of static cold storage. (Conflicts of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2007.10.062