76. Cryopreservation: A design specification for a novel bioreactor

July 23, 2017 | Autor: Mayur Patel | Categoria: Cryobiology, Medical Physiology, Biochemistry and cell biology
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Abstracts / Cryobiology 57 (2008) 315–340

Andrew A. Gage b,c, John G. Baust a, a Institute of Biomedical Technology, Binghamton University, Binghamton, NY, USA, b Cell Preservation Services, Inc., Owego, NY, USA, c Department of Surgery, SUNY Buffalo, Buffalo, NY, USA Investigations into the molecular-based responses of cancerous cells following cold exposure have led to the discovery of delayed-onset, apoptotic cell death occurring 6–24 h post-thaw and located in the periphery of the cryolesion. The apoptotic pathway typically attributed to this delayed death is the intrinsic/mitochondrialmediated pathway, and is characterized by a loss of mitochondrial potential, release of cytochrome c, and activation of caspase-9. More recent studies, however, have shown that at lower temperatures (< 20 °C) a rapid, programmed cell death response occurs and likely progresses through the extrinsic/membrane-mediated apoptotic pathway. This report provides an initial approach to deciphering the molecular signals triggering these rapid-onset apoptotic events. Human prostate cancer cells (PC3) were frozen at temperatures of 60, 30, and 15 °C. Flow cytometery and Western blot analyses indicate that following exposure to ultra low temperatures ( 60 °C) apoptotic death occurs within 30 min, while cells frozen to elevated subfreezing temperatures ( 15 °C) show no signs of apoptosis until 3–6 h post-thaw. Additionally, activation of caspase-8 by 30 min post-thaw and delayed (>90 min) depolarization of mitochondria in samples frozen to 60 °C were found, implying that the extrinsic apoptotic pathway is responsible for rapid-onset apoptosis. Using immunofluorescent and Western blot analyses, investigations into upstream events triggering rapid apoptosis have identified alterations in cellular attachments, integrin signaling, and cytoskeletal damage as likely initiators. Furthermore, by employing a tissue engineered prostate equivalent we have shown that cellular attachments are more involved in the overall survival/death signaling cascades in cells grown 3-dimensionally than those grown in 2-dimensional monolayers. Our investigations have identified the importance of cellular attachments following cryosurgery and their involvement in inducing extrinsic apoptotic pathways. By better understanding focal adhesion and cytoskeletal signaling following cryosurgery, novel targets and or adjunctive agents may be identified that will improve cryosurgical outcome. (Conflicts of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2008.10.073

73. Influence of temperature profile and CPA distribution on cryopreservation of native tissue. Nicola Hofmann, Huan Sun, Inga Bernemann, Birgit Glasmacher, Institute of Multiphase Processes and Centre for Biomedical Engineering, Leibniz Universitaet Hannover, Hannover, Germany Effective cryopreservation of tissue poses a specific challenge for biomedical applications. Controlled freezing and thawing methods are required for successful long-term storage of native tissue. The temperature distribution and CPA (cryoprotective agent) gradients are important in such three dimensional geometries. In order to optimise these freezing parameters, samples of porcine muscle tissue with different sizes (thickness 40 mm, Ø 5 mm, resp. Ø 9 mm) were investigated. Modified 1.8 mL cryovials (NalgeneÒ) were used for controlled rate freezing of the samples. Three two-step freezing programs were investigated (B1 in the range of 4 °C to 30 °C and B2 from 30 °C to 80 °C: 1:3 K/min, 2:2 K/min, 5:10 K/min). In addition various CPA concentrations (2.5, 7.5, 10% v/v Me2SO) and different ways of CPA application were tested. The samples were thawed in an adapted, standardised rewarming equipment at 20 °C. Temperature profiles were taken at defined positions within the samples and thereby the cooling rates determined in the tissue matrix. The measured temperature gradient during freezing had a maximum difference of 5 K from the border area to the centre of the tissue. Cell viability after cryopreservation was determined by using an MTT-assay (survival rate measurement) and a Necrosis/ Apoptosis assay (AnnexinV staining). The MTT-assay showed cell survival rates up to 80% for porcine muscle tissue. The investigation of the stained tissue slices allowed conclusions of cell death and survival depended on temperature profile and CPA distribution in the sample. As determined in previous studies, our results demonstrated again that an optimised freezing protocol allows cryopreservation of different biological material. Even tissue samples with larger dimensions reached high cell survival rates when Me2SO was injected in the centre of the sample balancing the inversely directed temperature gradient. (Conflicts of interest: None declared. Source of funding: The project was supported by the Cluster of Excellence ‘‘REBIRTH” (DFG).) doi:10.1016/j.cryobiol.2008.10.074

74. Effects of glycerol, sucrose and trehalose on cryopreserved human dermis against gamma irradiation damage. Wendell Sun, LifeCell Corporation, Branchburg, NJ, USA Glycerol, sucrose and trehalose are common cryoprotectants for cell and tissue preservation, and are present in some formulations for gamma irradiation. This study compared their protective effects on cryopreserved human dermis against gamma

irradiation. Acellular human dermis (AHD) was produced from donated cadaver skin tissue via de-cellularization. Skin epidermis was removed by incubating in 1 M NaCl, and cellular components were removed with 2% sodium deoxycholate. AHD was then washed in phosphate-buffered saline to remove detergent residues. The effects of glycerol, sucrose and trehalose were compared using a base citrate/phosphate buffer (pH 6.0). Glycerol, sucrose, trehalose were dissolved in the base buffer. AHD was cryopreserved in different solutions, and gamma-irradiated at a dose of 13 kGy at 80 °C. Gamma irradiation damage in AHD was assessed using histological evaluation (10% formalin fixation and hematoxylin-eosin stain). Histological slides were examined for freezing and gamma irradiation damage based on hole formation, collagen damage, and the change of the papillary to reticular layer transition. Structural change was scored from 0 (least) to 10 (highest). The data shows that freezing did not alter AHD structure, but gamma irradiation resulted in significant structural damage including the formation of large holes, collagen breaks and separation, as well as the loss of dermis tissue histology. When used alone, trehalose provided better protection against gamma irradiation damage than did glycerol and sucrose. When used together, the glycerol/trehalose combination provide best tissue protection. The study was repeated with five human skin donor lots, and it was observed that the donor-todonor variation was significant in AHD damage upon gamma irradiation. For less sensitive donor tissue lots, glycerol, sucrose or trehalose alone were able to provide good protection. However, for more sensitive donor tissue lots, only the glycerol/trehalose combination was able to provide sufficient structural protection. (Conflicts of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2008.10.075

75. Modified calcium uptake in cryopreserved human saphenous veins. Else MüllerSchweinitzer, Martin T. R. Grapow, Friedrich S. Eckstein, University Hospital Basel, CH-4031 Basel, Switzerland Previous studies have shown that cryopreservation of human internal mammary arteries enhances Ca2+ influx. The present experiments suggest that in human saphenous veins (HSV) cryoinjury is also associated with changes in internal Ca2+ content. HSV were investigated in vitro either unfrozen within 12 h or after cryopreservation and storage at 196 °C in a cryomedium containing 1.8 M Me2SO and 0.1 M sucrose as cryoprotective agents. Freezing and thawing were performed slowly at mean rates of 0.6 °C/min and 15 °C/min, respectively. HSV samples loaded with fluorescent dyes were investigated using a confocal microscope (LSM510). Functional studies were performed in organ bath experiments. It was found that experiments with HEDAF-stained HSV revealed identical fluorescence emission in unfrozen and Me2SO-sucrose-protected cryopreserved veins, whereas significantly stronger fluorescence emission in HSV frozen without any cryoprotectant additive indicated marked membrane breakage. Functional studies revealed similar inhibitory activities of both 1,4dihydropyridine derivatives nifedipine and the Ca2+channel blocking ( )-(R) enantiomer of 202–791 in unfrozen and cryopreserved HSV. By contrast, the Ca2+ channel activating (+)-(S) enantiomer of 202–791 proved to be 10 times less effective at enhancing responses to CaCl2 when tested after cryopreservation. To detect photochemically cytosolic calcium [Ca2+]iHSV were loaded with Fluo-3AM. Basal fluorescence was significantly stronger in cryopreserved than in unfrozen HSV (P < 0.05). Incubation with (+)-(S) 202–791 (100 nM) increased fluorescence in both unfrozen and cryopreserved HSV significantly (P < 0.05). KCl (60 mM)-stimulation without and in combination with (+)-(S) 202–791 increased fluorescence significantly (P < 0.05) in unfrozen but not in cryopreserved HSV. In conclusion, cryopreservation did not induce enhanced cell breakage but modified Ca2+ influx of HSV by causing Ca2+ overload of the smooth muscle cells. (Conflicts of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2008.10.076

76. Cryopreservation: A design specification for a novel bioreactor. Mei Niu, Mayur Patel, Robin Coger, University of North Carolina—Charlotte, Charlotte, NC, USA A novel radial flow bioreactor (RFB) has been designed. It supports a cellular space that (1) is adaptable for the support of a range of tissue equivalents; (2) minimizes the cell-nutrient transport barrier; (3) maintains a uniform microenvironment for the cells; (4) minimizes the cells’ exposure to shear forces; and (5) can be easily cryopreserved. In the current study, we coupled experiment with predictive modeling to demonstrate the ability of the cellular space of our novel RFB to be safely cryopreserved when supporting cultured rat primary hepatocytes. Development and optimization of the cryoprotocol relied on evaluating the post-thaw cell viability of the hepatocytes when cryopreserved in various Me2SO solutions. The results suggest that the cellular space of the RFB can be effectively used for the safe storage of liver tissue equivalents. Specifically, hepatocytes were successfully cryopreserved with a viability of up to 74.3 ± 5.3% using the applied cryoprotocol. Future studies will include optimization of the cryoprotrocol, as well as post-thaw evaluation of key cellular func-

Abstracts / Cryobiology 57 (2008) 315–340 tions, such as albumin secretion, ammonia clearance, and cytochrome P450. (Conflicts of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2008.10.077

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promising results in a first transplantation model. It might be superior to current preservation solutions and is therefore now being assessed in other small and large animal models of organ transplantation. (Conflicts of interest: U. Rauen and H de Groot are consultants for Dr Franz Köhler Chemie GmbH Alsbach-Hähnlein, Germany. Source of funding: None declared.) doi:10.1016/j.cryobiol.2008.10.079

Contributed papers 12. Hypothermic preservation of tissues and organs

77. Intravital confocal/multiphoton imaging of liver function and injury after hepatic ischemia/reperfusion and liver transplantation. Zhi Zhong, Tom P. Theruvath, Venkat K. Ramshesh, Hasibur Rehman, John J. Lemasters, Medical University of South Carolina, Charleston, SC, USA

79. Involvement of membrane induced apoptosis in cold storage failure. William L. Corwin a,b, John M. Baust a,b, Robert G. Van Buskirk a,b, John G. Baust a, a Institute of Biomedical Technology, Binghamton University, Binghamton, NY, USA, b Cell Preservation Services, Inc., Owego, NY, USA

Ischemia/reperfusion (I/R) causes many pathophysiological changes that contribute to the development of liver injury. Monitoring these pathophysiological processes in vivo has been difficult. Recent developments in intravital confocal/multiphoton microscopy using introduced fluorophores and fluorescent protein-expressing mice provide novel approaches to visualize pathophysiological processes such as mitochondrial depolarization, onset of the mitochondrial permeability transition (MPT), disturbances of microcirculation, inflammation, and cell death in living animals. Confocal/multiphoton microscopy achieves much improved axial resolution compared to widefield microscopy. Red/infrared excitation used in multiphoton microscopy allows deeper tissue penetration with less photodamage and more efficient collection of fluorescence in a scattering environment. Using these novel imaging technologies, we documented many pathophysiological disturbances after hepatic I/R. Mitochondrial polarization, cell death, and the MPT were assessed using rhodamine 123 (Rh123), propidium iodide (PI), and calcein, respectively. In sham-operated mice, green Rh123 fluorescence was punctate in virtually all hepatocytes, indicating mitochondrial polarization. Two hours after warm I/R, mitochondria depolarized in >70% of cells, but cell death in almost all these hepatocytes had not yet occurred. Entry of calcein into mitochondria indicated MPT onset in vivo. NIM811, a specific inhibitor of the MPT, and ethyl 3,4-dihyroxybenzoate, a prolyl hydroxylase inhibitor that upregulates HIF-1a, prevented MPT onset, mitochondrial depolarization and liver injury after I/R. NIM811, minocycline, and ischemic preconditioning also protected against MPT onset and liver graft failure after full-size and small-for-size rat liver transplantation, as again documented by intravital confocal/multiphoton microscopy. After transplantation, blood flow velocity and sinusoidal diameter revealed by intravital microscopy of fluorescein-labeled erythrocytes markedly decreased in eNOS-deficient liver grafts compared to wild-type grafts. Such microcirculatory disturbances were associated with more severe graft injury, indicating that donor eNOS attenuates storage/reperfusion injury partially by improving microcirculation. Taken together, these examples show that intravital confocal/multiphoton imaging is a powerful new technology to study alteration of liver function after I/R. (Conflicts of interest: None declared. Source of funding: None declared.)

Numerous studies have described the involvement and control of molecularbased cell death during hypothermic storage. One of the initial reports detailed the benefits of the inclusion of a free-radical scavenger as well as protease inhibitor in hypothermic storage media. Since then, numerous reports have expanded on this concept, yet few have emerged examining the specific stress/death pathways involved in system failure following hypothermic exposure. Accordingly, we examined the stress pathways activated during hypothermic storage and their role in cellular demise with a focus on membrane-induced apoptosis (MIA). We hypothesized that MIA is activated due to membrane alterations, which occur during cold storage, including reduced fluidity, protein aggregation, lipid peroxidation, and others. Human lung fibroblasts (IMR-90) were stored for 1–3 days at 4 °C in media or ViaSpan. Viability was assessed 1–3 days post-storage using the metabolic indicator alamarBlueTM. Levels of apoptosis and necrosis were assessed via flow cytometery and fluorescent microscopy using the VibrantÒ apoptosis assay at 1, 4, 8 and 24 h post-storage. Western Blots were performed to evaluate MIA pathway members including Fas, FADD, Caspase 8 as well as non-MIA proteins, AIF and Caspase 9. Target inhibition studies were conducted to evaluate the overall effect of pathway activation. Sample storage for 2 days resulted in a significant decline in viability in all conditions with ViaSpan yielding the best outcome (26% ± 5.5%). Flow cytometry demonstrated both apoptosis (5%) and necrosis (20%) were initiated post-storage. Western blot analysis revealed a significant alteration in MIA associated proteins, Fas and Caspase 8. Targeted inhibition of Caspase 8, Fas, and AIF resulted in significant improvements in overall outcome (2.3, 1.9, and 2.1-fold, respectively). The data demonstrate the significant role that the membrane associated (extrinsic) pathway plays during hypothermic storage and that a further understanding of the activation of this pathway may prove beneficial. (Conflicts of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2008.10.080

doi:10.1016/j.cryobiol.2008.10.078

78. Custodiol-N—A new, mechanism-based organ preservation solution. Ursula Rauen a, K. Wu b, Oliver Witzke b, Herbert Groot de a, a Institut für Physiologische Chemie, Universitätsklinikum Essen, Essen, Germany, b Klinik für Nieren- und Hochdruckkrankheiten, Universitätsklinikum Essen, Essen, Germany Preservation injury to an organ graft is composed of injury triggered by hypoxia, injury triggered by hypothermia, a certain toxicity of preservation solutions and, at a later stage, by inflammatory processes. We have been studying the mechanisms of these initial processes and we have now developed a new preservation solution, Custodiol-N, aiming to inhibit these early processes. Based on the previous mechanistic studies (hypoxic injury largely triggered by a glycine- and alanine-inhibitable influx of sodium, hypothermic injury by a cold-induced increase in cellular chelatable iron and preservation solution toxicity largely caused by the buffers histidine or phosphate), the new solution has a low sodium concentration, is buffered with the non-toxic histidine derivative N-acetylhistidine and contains glycine, alanine, the well-known iron chelator deferoxamine and the new, membrane-permeable iron chelator LK 614. When tested in cellular models of hypoxic injury (rat hepatocytes; gassing with N2) or hypothermic injury (hepatocytes and liver endothelial cells at 4 °C) or assessed for toxicity (both cell types), this new solution significantly decreased hypoxic injury, significantly decreased cold-induced injury and displayed far lower toxicity than UW or HTK solution. Therefore, the solution was then used in a mouse heart transplantation model (24 h cold ischemia at 4 °C; heterotopic transplantation into the abdomen). Serum creatine kinase activity 24 hours after transplantation was 25,998 ± 8471 U/l for HTK-stored grafts and 3093 ± 597 U/l for Custodiol-N-preserved grafts. At day 14 postop, 0/8 grafts preserved with HTK solution but 7/8 of grafts preserved with the new solution were still beating. In summary, the new preservation solution provided substantial inhibition of all three early components of preservation injury in cellular models and showed very

80. Development of a new vascular preservation solution. Timo Wille aStephanie Garbe b, Birgit Zatschler b, Herbert de Groot a, Klaus Matschke c, Andreas Deussen b, Ursula Rauen a, a Institut für Physiologische Chemie, Universitätsklinikum, Essen, Germany, b Institut für Physiologie, Medizinische Fakultät Carl Gustav Carus, Dresden, Germany, c Klinik für Kardiochirurgie, Herzzentrum Dresden, Dresden, Germany Our previous results suggested that the iron chelator deferoxamine and a high chloride concentration have protective effects on the endothelium of vessels during cold storage and rewarming. Based on these results we here developed and tested a new solution for the hypothermic storage of blood vessels. Segments of porcine aorta, rat mesenteric artery (RMA) and human internal mammary artery (HIMA) were stored for 1–21 days in diverse solutions at 4 °C. The percentage of dead endothelial cells, endothelial mitochondrial membrane potential (MMP) and thrombocyte adhesion were tested in porcine aortae, endothelium-dependent relaxation in segments of RMA and HIMA (MULVANY). An amino acid-containing base solution was optimized with regard to buffer (N-acetylhistidine), pH (7.0), main cation (potassium) and main anion (chloride). Combination of deferoxamine with a new, more membrane-permeable iron chelator, LK 614, further improved preservation so that even after 3 weeks of cold storage only 10 ± 1% of porcine aortic endothelial cells had died. In this optimized solution endothelial cell death was significantly lower than in HTK (83 ± 3%), UW (28 ± 4%), Perfadex (32 ± 6%) or physiological saline solution (PSS, 34 ± 4%). MMP, assessed after 14 days of cold storage, was also significantly better preserved. Moreover, when compared to HTK solution, thrombocyte adhesion was significantly reduced after cold storage in the optimized solution. The endotheliumdependent vasodilation of RMA, assessed after 4 days of cold storage, was significantly better in the new solution (69 ± 7% of non-stored control) than in HTK solution (48 ± 9%) or PSS (0%). HIMAs, typical arterial bypass grafts, still showed full function after 4 days and partial function even after 12 days of cold storage in the new solution whereas HIMAs stored in PSS had lost relaxatory reactivity completely after 15 h storage. The new vascular preservation solution improves endothelial cell survival and function after cold storage and should now be evaluated in clinical settings. (Conflicts

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