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68. Organ perfusion with cryoprotectants
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Abstracts / Cryobiology 61 (2010) 362–408 Conflict of interest: None declared. Source of funding: None declared.
doi:10.1016/j.cryobiol.2010.10.070
67. Oxygen persufflation of the pancreas prior to islet isolation. Klearchos K. Papas, Department of Surgery, University of Minnesota Medical School, Minneapolis, MN, USA Islet transplantation (ITx) is emerging as an attractive treatment option for qualified patients with type-1 diabetes. The success of ITx (insulin independence) is strongly influenced by the number of transplanted viable islet equivalents (IE) per kilogram recipient body weight. Reduction in islet viability occurs at multiple pancreas and islet processing steps, especially those exposing islets to hypoxia. Islets express very low levels of LDHa, the enzyme needed to generate energy with anaerobic glycolysis and are therefore particularly sensitive to hypoxia. Current static cold preservation protocols, including the state-of-the-art two layer method (TLM), do not prevent exposure of the majority of the human pancreas to hypoxia or even anoxia. This highlights the need for the development of improved methods for pancreas oxygenation during preservation. In this context we explore arterial oxygen persufflation (PSF), as an effective method of oxygen delivery to porcine and human pancreata, and report on its ability to improve pancreas preservation and ultimately islet isolation outcomes relative to the TLM. Human pancreata and paired lobes from porcine-pancreata were preserved up to 24 h with PSF or TLM. PSF was performed by pumping 20 cc/min 40%-oxygen humidified gas to the superior mesenteric artery and celiac trunk averaging pressures of 10– 20 mmHg. Homogeneity of PSF was assessed by NMR imaging, visualizing negative contrast associated with the presence of gas in the vasculature. ATP and the ATP to inorganic phosphate ratio (ATP:Pi) were non-invasively measured by 31P-NMR spectroscopy at 1.5 T. Porcine islets were isolated following 6- or 24-hour preservation, cultured for 2 days, and assessed by islet counts, morphology, DNA content, oxygen consumption rate normalized to DNA (OCR/DNA), and the diabetic-nude mouse bioassay (2000 IE transplanted per mouse). NMR imaging demonstrated that pancreatic tissue was homogeneously persufflated. ATP was undetectable in porcine or human pancreata stored on TLM, while substantial levels of ATP were detected when porcine and human pancreata were persufflated. ATP:Pi remained elevated throughout PSF, with minimal-gradual decline over time observed. In some organs, PSF was stopped resulting in an immediate decline in ATP:Pi until ATP levels were undetectable. Restoration of PSF partially restored ATP:Pi. On average the persufflated lobes resulted in higher islet yield and post-culture recovery than paired lobes stored with the TLM, but the differences were not statistically significant. Islets isolated from persufflated porcine lobes exhibited higher morphology score (P < 0.05) and viability (OCR/DNA, P < 0.05) when compared with those isolated from paired TLM lobes. Importantly, islet yield, viability, post culture recovery and cure rates in the diabetic-nude mouse bioassay with islets isolated from lobes persufflated for 24 h were similar to those obtained with islets isolated from lobes shortly after procurement. It is concluded that PSF increases ATP levels and viable islet yield relative to TLM and may extend pancreas preservation time to 24 h without compromising viable islet yield. PSF can potentially improve the logistics and cost-effectiveness of organ allocation and preservation, while enhancing islet isolation and transplantation outcomes. Conflict of interest: None declared. Source of funding: National Institutes of Health (National Institute of Diabetes and Digestive and Kidney Diseases), grant number: R43 DK070400. doi:10.1016/j.cryobiol.2010.10.071
68. Organ perfusion with cryoprotectants. Gregory M. Fahy, 21st Century Medicine Inc., Fontana, CA, USA The perfusion of organs with cryoprotectants entails many unique requirements and considerations not encountered with other perfusion processes. Initial attempts at organ cryoprotection demonstrated the importance of controlling osmotic damage and the limits of organ tolerance of abrupt osmotic shifts resulting from step changes in concentration. The realization that very high concentrations of cryoprotectants might be required made it clear that apparatus capable of introducing graded concentration changes and thus avoiding osmotic issues would be needed, and the first designs of relatively sophisticated cryoprotectant perfusion devices were published in the early 1970s. The first well-described methods for gradual addition and removal of cryoprotectants were published by Pegg et al. in 1977 and 1978 and showed for the first time that up to 3 M glycerol could be added to rabbit kidneys and removed with acceptable damage following transplantation. In the early 1980s, Adem and Harness applied computerization to cryoprotectant perfusion of rat hearts, but most studies have utilized rabbit kidneys, so the present review will
focus on that particular model organ. In the mid 1980s, my lab introduced a fully computerized system that was based on a gravity-driven gradient former approach, a sensor loop separate from the organ perfusion loop to minimize time lags between the gradient former and the organ, the ability to change concentrations either slowly or abruptly and to recirculate or perfuse open circuit as needed, real-time sensing, control, display, recording, and processing of perfusion variables, and automated priming and cleaning. This system was used to prepare rabbit kidneys for manual perfusion of an ambient pressure (8.4 M) vitrification solution at 24 °C in 1995, with subsequent life support by these kidneys after transplantation. In 1998, a similar system was built that added automated cooling to and warming from 22 °C without interruption of organ perfusion. Careful use of this system allowed rabbit kidneys perfused with an ultra-stable 9.3 M vitrification solution (M22) to support life after transplantation with reversible damage comparable to the damage seen after 3 M glycerol perfusion in 1978, and in 2009 it was reported that a kidney vitrified with M22 was able to support life indefinitely after transplantation. The success of organ cryoprotectant perfusion for vitrification is now known to depend on the exact degree of equilibration of the organ with the vitrification solution. In the case of the rabbit kidney, urine concentrations equal to 95% of the concentration of M22 are diagnostic of tissue concentrations sufficient to escape devitrification, and the degree of urine equilibration is related to the urine flow rate, which in turn is inversely proportional to the viscosity of the perfusate. Many unpublished studies have shown that it is important to minimize viscosity during the early stages of perfusion to avoid the development of increasing lags in tissue equilibration, and that perfusing lower concentrations for longer times to level accumulated lags can allow the kidney to attain target concentrations after shorter durations of perfusion with the final vitrification solution itself. Interestingly, histological examination of the surviving vitrified kidney showed that medullary damage attributed to devitrification was confined to one side of the kidney, suggesting a role also for renal position and gravity in medullary equilibration. Conflict of interest: None. Source of funding: 21st Century Medicine Inc.
doi:10.1016/j.cryobiol.2010.10.072
69. Evidence for the involvement of ERK in cold-induced cell injury. Victoria Laszlo, Ursula Rauen *, Institut für Physiologische Chemie, Universitätsklinikum Essen, 45122 Essen, Germany Hypothermic injury/cold-induced apoptosis, mediated by an increase in cellular chelatable iron ions followed by formation of reactive oxygen species and mitochondrial injury, occurs in various cell types and constitutes part of the preservation injury occurring to organs in transplantation medicine. Mitogen-activated protein kinases (MAPKs) have, among other factors, been suggested to play a role in preservation injury; however, their role in cold-induced cell injury is unclear. Therefore, we here studied whether one (or more) of the three main MAPK pathways is involved in cold-induced injury of rat liver endothelial cells and primary rat hepatocytes. Rat hepatocytes and liver endothelial cells were incubated at 4 °C in Krebs– Henseleit buffer or in University of Wisconsin (UW) solution, followed by rewarming in cell culture medium with heat-inactivated FCS. Activation of the MAPKs JNK, ERK and p38 was assessed by Western blot, lethal cell injury by LDH release and mitochondrial membrane potential by laser scanning microscopy using the fluorescent indicator TMRM. Hypothermia decreased the phosphorylation of ERK and JNK during cold incubation itself in both cell types, but triggered a pronounced activation of both MAPKs during rewarming. The phosphorylation status of p38, in contrast, was increased during cold incubation and moderately attenuated by rewarming. Inhibition of MEK and thus of ERK phosphorylation by PD 98059 or U0126 (with the negative control U0124) strongly protected the cells against cold-induced injury (e.g., 17 ± 6% vs. 62 ± 22% LDH release in primary hepatocytes and 0 ± 0% vs. 84 ± 4% in liver endothelial cells after 24 h cold incubation in UW solution plus rewarming). One of the two p38 inhibitors applied provided partial protection (that could also be due to unspecific effects on lipid peroxidation), whereas the other one did not provide any protection. The inhibition of JNK had no effect on the survival of cold-exposed hepatocytes. The cold-induced activation of ERK appeared to be largely independent of the cellular iron and calcium homeostasis, of Rho, of the EGF receptor or the PKC pathway. The protective effect of ERK did not appear to be mediated by an action of ERK on PARP, p53, Akt, or Bcl-2, but to sensitivation of the mitochondria to iron-/ROS-dependent injury: inhibition of MEK and thus of ERK phosphorylation prevented the loss of mitochondrial membrane potential in both cell types during cold incubation/rewarming – and thus inhibited the mitochondrial injury that can also be prevented by iron chelators. These results suggest that ERK, usually considered to exert primarily anti-apoptotic and/or protective effects, contributes to cold-induced injury of isolated liver cells, most likely by a ’sensitizing’ effect on mitochondria.
Abstracts / Cryobiology 61 (2010) 362–408 Conflict of interest: None declared. Source of funding: None declared.
doi:10.1016/j.cryobiol.2010.10.070
67. Oxygen persufflation of the pancreas prior to islet isolation. Klearchos K. Papas, Department of Surgery, University of Minnesota Medical School, Minneapolis, MN, USA Islet transplantation (ITx) is emerging as an attractive treatment option for qualified patients with type-1 diabetes. The success of ITx (insulin independence) is strongly influenced by the number of transplanted viable islet equivalents (IE) per kilogram recipient body weight. Reduction in islet viability occurs at multiple pancreas and islet processing steps, especially those exposing islets to hypoxia. Islets express very low levels of LDHa, the enzyme needed to generate energy with anaerobic glycolysis and are therefore particularly sensitive to hypoxia. Current static cold preservation protocols, including the state-of-the-art two layer method (TLM), do not prevent exposure of the majority of the human pancreas to hypoxia or even anoxia. This highlights the need for the development of improved methods for pancreas oxygenation during preservation. In this context we explore arterial oxygen persufflation (PSF), as an effective method of oxygen delivery to porcine and human pancreata, and report on its ability to improve pancreas preservation and ultimately islet isolation outcomes relative to the TLM. Human pancreata and paired lobes from porcine-pancreata were preserved up to 24 h with PSF or TLM. PSF was performed by pumping 20 cc/min 40%-oxygen humidified gas to the superior mesenteric artery and celiac trunk averaging pressures of 10– 20 mmHg. Homogeneity of PSF was assessed by NMR imaging, visualizing negative contrast associated with the presence of gas in the vasculature. ATP and the ATP to inorganic phosphate ratio (ATP:Pi) were non-invasively measured by 31P-NMR spectroscopy at 1.5 T. Porcine islets were isolated following 6- or 24-hour preservation, cultured for 2 days, and assessed by islet counts, morphology, DNA content, oxygen consumption rate normalized to DNA (OCR/DNA), and the diabetic-nude mouse bioassay (2000 IE transplanted per mouse). NMR imaging demonstrated that pancreatic tissue was homogeneously persufflated. ATP was undetectable in porcine or human pancreata stored on TLM, while substantial levels of ATP were detected when porcine and human pancreata were persufflated. ATP:Pi remained elevated throughout PSF, with minimal-gradual decline over time observed. In some organs, PSF was stopped resulting in an immediate decline in ATP:Pi until ATP levels were undetectable. Restoration of PSF partially restored ATP:Pi. On average the persufflated lobes resulted in higher islet yield and post-culture recovery than paired lobes stored with the TLM, but the differences were not statistically significant. Islets isolated from persufflated porcine lobes exhibited higher morphology score (P < 0.05) and viability (OCR/DNA, P < 0.05) when compared with those isolated from paired TLM lobes. Importantly, islet yield, viability, post culture recovery and cure rates in the diabetic-nude mouse bioassay with islets isolated from lobes persufflated for 24 h were similar to those obtained with islets isolated from lobes shortly after procurement. It is concluded that PSF increases ATP levels and viable islet yield relative to TLM and may extend pancreas preservation time to 24 h without compromising viable islet yield. PSF can potentially improve the logistics and cost-effectiveness of organ allocation and preservation, while enhancing islet isolation and transplantation outcomes. Conflict of interest: None declared. Source of funding: National Institutes of Health (National Institute of Diabetes and Digestive and Kidney Diseases), grant number: R43 DK070400. doi:10.1016/j.cryobiol.2010.10.071
68. Organ perfusion with cryoprotectants. Gregory M. Fahy, 21st Century Medicine Inc., Fontana, CA, USA The perfusion of organs with cryoprotectants entails many unique requirements and considerations not encountered with other perfusion processes. Initial attempts at organ cryoprotection demonstrated the importance of controlling osmotic damage and the limits of organ tolerance of abrupt osmotic shifts resulting from step changes in concentration. The realization that very high concentrations of cryoprotectants might be required made it clear that apparatus capable of introducing graded concentration changes and thus avoiding osmotic issues would be needed, and the first designs of relatively sophisticated cryoprotectant perfusion devices were published in the early 1970s. The first well-described methods for gradual addition and removal of cryoprotectants were published by Pegg et al. in 1977 and 1978 and showed for the first time that up to 3 M glycerol could be added to rabbit kidneys and removed with acceptable damage following transplantation. In the early 1980s, Adem and Harness applied computerization to cryoprotectant perfusion of rat hearts, but most studies have utilized rabbit kidneys, so the present review will
focus on that particular model organ. In the mid 1980s, my lab introduced a fully computerized system that was based on a gravity-driven gradient former approach, a sensor loop separate from the organ perfusion loop to minimize time lags between the gradient former and the organ, the ability to change concentrations either slowly or abruptly and to recirculate or perfuse open circuit as needed, real-time sensing, control, display, recording, and processing of perfusion variables, and automated priming and cleaning. This system was used to prepare rabbit kidneys for manual perfusion of an ambient pressure (8.4 M) vitrification solution at 24 °C in 1995, with subsequent life support by these kidneys after transplantation. In 1998, a similar system was built that added automated cooling to and warming from 22 °C without interruption of organ perfusion. Careful use of this system allowed rabbit kidneys perfused with an ultra-stable 9.3 M vitrification solution (M22) to support life after transplantation with reversible damage comparable to the damage seen after 3 M glycerol perfusion in 1978, and in 2009 it was reported that a kidney vitrified with M22 was able to support life indefinitely after transplantation. The success of organ cryoprotectant perfusion for vitrification is now known to depend on the exact degree of equilibration of the organ with the vitrification solution. In the case of the rabbit kidney, urine concentrations equal to 95% of the concentration of M22 are diagnostic of tissue concentrations sufficient to escape devitrification, and the degree of urine equilibration is related to the urine flow rate, which in turn is inversely proportional to the viscosity of the perfusate. Many unpublished studies have shown that it is important to minimize viscosity during the early stages of perfusion to avoid the development of increasing lags in tissue equilibration, and that perfusing lower concentrations for longer times to level accumulated lags can allow the kidney to attain target concentrations after shorter durations of perfusion with the final vitrification solution itself. Interestingly, histological examination of the surviving vitrified kidney showed that medullary damage attributed to devitrification was confined to one side of the kidney, suggesting a role also for renal position and gravity in medullary equilibration. Conflict of interest: None. Source of funding: 21st Century Medicine Inc.
doi:10.1016/j.cryobiol.2010.10.072
69. Evidence for the involvement of ERK in cold-induced cell injury. Victoria Laszlo, Ursula Rauen *, Institut für Physiologische Chemie, Universitätsklinikum Essen, 45122 Essen, Germany Hypothermic injury/cold-induced apoptosis, mediated by an increase in cellular chelatable iron ions followed by formation of reactive oxygen species and mitochondrial injury, occurs in various cell types and constitutes part of the preservation injury occurring to organs in transplantation medicine. Mitogen-activated protein kinases (MAPKs) have, among other factors, been suggested to play a role in preservation injury; however, their role in cold-induced cell injury is unclear. Therefore, we here studied whether one (or more) of the three main MAPK pathways is involved in cold-induced injury of rat liver endothelial cells and primary rat hepatocytes. Rat hepatocytes and liver endothelial cells were incubated at 4 °C in Krebs– Henseleit buffer or in University of Wisconsin (UW) solution, followed by rewarming in cell culture medium with heat-inactivated FCS. Activation of the MAPKs JNK, ERK and p38 was assessed by Western blot, lethal cell injury by LDH release and mitochondrial membrane potential by laser scanning microscopy using the fluorescent indicator TMRM. Hypothermia decreased the phosphorylation of ERK and JNK during cold incubation itself in both cell types, but triggered a pronounced activation of both MAPKs during rewarming. The phosphorylation status of p38, in contrast, was increased during cold incubation and moderately attenuated by rewarming. Inhibition of MEK and thus of ERK phosphorylation by PD 98059 or U0126 (with the negative control U0124) strongly protected the cells against cold-induced injury (e.g., 17 ± 6% vs. 62 ± 22% LDH release in primary hepatocytes and 0 ± 0% vs. 84 ± 4% in liver endothelial cells after 24 h cold incubation in UW solution plus rewarming). One of the two p38 inhibitors applied provided partial protection (that could also be due to unspecific effects on lipid peroxidation), whereas the other one did not provide any protection. The inhibition of JNK had no effect on the survival of cold-exposed hepatocytes. The cold-induced activation of ERK appeared to be largely independent of the cellular iron and calcium homeostasis, of Rho, of the EGF receptor or the PKC pathway. The protective effect of ERK did not appear to be mediated by an action of ERK on PARP, p53, Akt, or Bcl-2, but to sensitivation of the mitochondria to iron-/ROS-dependent injury: inhibition of MEK and thus of ERK phosphorylation prevented the loss of mitochondrial membrane potential in both cell types during cold incubation/rewarming – and thus inhibited the mitochondrial injury that can also be prevented by iron chelators. These results suggest that ERK, usually considered to exert primarily anti-apoptotic and/or protective effects, contributes to cold-induced injury of isolated liver cells, most likely by a ’sensitizing’ effect on mitochondria.