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58. Effects of glycerol on recovery and antioxidant enzyme activities of lyophilized red blood cells
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Abstracts / Cryobiology 53 (2006) 367–446
funding: Grant-in-Aid for Scientific Research, 15500302, from the Japan Society for the Promotion of Science.) doi:10.1016/j.cryobiol.2006.10.057
57. Alternative strategies for the improvement of cryopreservation of equine spermatazoa. Delene Draper a, Jon E. Green a, Brian W. Grout a, George J. Morris b, a Centre for Equine and Animal Studies, Writtle College, Chelmsford CM1 3RR, United Kingdom; b Asymptote Ltd., St. Johns Innovation Centre, Cambridge CB4 0WS, United Kingdom Cryoprotected stallion semen has been cooled in 0.25 ml straws at rates from 0.5 to 330 °C/min, thawed in water at 37 °C and then evaluated for survival using viability (fluorescence/propidium iodide double-staining), motility and progressive motility as indicators, estimated using Computer Assisted Sperm Analysis. The data indicated no significant difference in survival in samples cooled between 4 and 150 °C/min. Freezefracture electron microscopy indicated that intracellular ice does not form in the frozen spermatozoa. Subsequent warming rate experiments indicated that the survival levels achieved in a bath at 37 °C, or 5 °C, are indistinguishable and optimal, with lower rates dramatically reducing the proportion of motile sperm. These data suggest that a considerable latitude in freeze–thaw procedures for equine sperm can be tolerated without significant, practical reduction in the motility or viability of the recovered material, and that concentrating on the quality of the sperm sample collected might be more fruitful in improving the technique. Studies might include further investigation of the physical collection and processing of samples prior to cryopreservation and impact of nutriceuticals, particularly fatty acids, on semen quality. With regard to processing, preliminary data has shown the positive impact that seminal plasma can have on sperm motility when it is added back to chilled, transported samples, including differential improvement that depends upon the particular plasma employed. Such benefit could not be transferred to thawed samples as the conventional, egg-based extender interacts physically with seminal plasma. (For this reason, spermatozoa are separated from plasma by centrifugation in preparation for freezing). Once the benefits of adding back seminal plasma are confirmed we expect to revisit the established cryoprotocols with the intention of finding a replacement for egg-based extender and utilising seminal plasma, frozen-stored for practical convenience. (Conflict of interest: None declared. Source of funding: None declared). doi:10.1016/j.cryobiol.2006.10.058
58. Effects of glycerol on recovery and antioxidant enzyme activities of lyophilized red blood cells. Hui He, Baolin Liu, Tse-Chao Hua, Institute of Cryomedicine and Food Refrigeration, University of Shanghai for Science and Technology, 200093 Shanghai, People’s Republic of China Successful storage of red blood cells (RBCs) by freeze-drying has important implications in blood transfusion and clinical medicine. Glycerol is a permeable protective agent. Pretreatment of red blood cells with glycerol can increase the concentration in the cytoplasm of the RBCs and then enhance the possibility of glass transition during the freeze-drying process. In order to
understand the protective mechanism of glycerol on lyophilized human red blood cells, the effect of glycerol on the activity of antioxidant enzymes and the recovery of lyophilized red blood cells was investigated in this paper. Glycerol solutions with concentrations of 0%, 20%, and 40% were used to treat the RBCs before the regular freeze–drying process. After pretreatment with the glycerol solution, red blood cells suspended in the protective agents were lyophilized in a freezedrier (Freezone 2.5, Labconco). The freeze–drying process included a primary lyophilization at 70 °C for 30 h and a secondary lyophilization at 10 °C for 10 h. The results demonstrated that glycerol had remarkable effects on cell viability. The maximum viability of red blood cells was 55.3 ± 4.26% and the recovery of hemoglobin was 53.49 ± 3.85% after pretreatment with the 40% glycerol solution, both of which were significantly higher than the control group (0% glycerol). The effect of storage time on the activity of antioxidant enzymes and recovery of lyophilized RBCs was studied using a spectrophotometer. The results showed that recovery of lyophilized RBC superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) activity all decreased significantly after 180 days storage at room temperature. The enzyme activity showed a similar decreasing pattern with the recovery of lyophilized RBCs during the preservation processes. These results provide fundamental information for the long-term preservation of RBCs by the freeze–drying technique. (Conflict of interest: None declared. Source of funding: NSFC (50376040, 50436030, 50576059), Shanghai Leading Academic Discipline Project (P0502), Shanghai Municipal Foundation for Returned Overseas Chinese Scholars and SRF for ROCS, SEM). doi:10.1016/j.cryobiol.2006.10.059
59. On possibility of low-dose ozone increasing the survival of cryopreserved erythrocytes. Iryna A. Musina a, Yelizaveta L. Volovelskaya b, a Department of biophysics, Institute for problems of cryobiology and cryomedicine, 61015 Kharkov, Ukraine; b Department of cell cryophysiology, Institute for problems of cryobiology and cryomedicine, 61015 Kharkov, Ukraine Erythrocytes are among those biological objects that are actively investigated by cryobiologists. However, the problem of increasing the survival of erythrocytes is very topical. Recently, ozone at low doses has been reported to promote the increase of the resistance of some biological objects to low temperatures. In the present work we studied the survival of cryopreserved erythrocytes. We froze the erythrocytes in cryoprotective media and then treated them with ozone at the stage of cryoprotectant removal. Erythrocytes were isolated from freshly donated blood by centrifugation at 800 g and then purified by three cycles of resuspension in and washing with 0.15 M NaCl solution with careful removal of the supernatant. We mixed the deposited erythrocytes with cryoprotective solutions (dimethylsulphoxide, glucose, sucrose, NaCl and dextran 10000) in the volumetric ratio1:1. Cryoprotective solutions with different dextran concentrations (5, 10, 15, 20% w/v), were used. After incubation in the cryoprotective solutions for 30 min at room temperature we froze the samples by immersing them in liquid nitrogen. Part of the frozen samples was stored for 12 days at 196 °C, and other part of the samples was stored for 12 days at +4 °C without preliminary freezing. The frozen samples were thawed in a water bath at
Abstracts / Cryobiology 53 (2006) 367–446
funding: Grant-in-Aid for Scientific Research, 15500302, from the Japan Society for the Promotion of Science.) doi:10.1016/j.cryobiol.2006.10.057
57. Alternative strategies for the improvement of cryopreservation of equine spermatazoa. Delene Draper a, Jon E. Green a, Brian W. Grout a, George J. Morris b, a Centre for Equine and Animal Studies, Writtle College, Chelmsford CM1 3RR, United Kingdom; b Asymptote Ltd., St. Johns Innovation Centre, Cambridge CB4 0WS, United Kingdom Cryoprotected stallion semen has been cooled in 0.25 ml straws at rates from 0.5 to 330 °C/min, thawed in water at 37 °C and then evaluated for survival using viability (fluorescence/propidium iodide double-staining), motility and progressive motility as indicators, estimated using Computer Assisted Sperm Analysis. The data indicated no significant difference in survival in samples cooled between 4 and 150 °C/min. Freezefracture electron microscopy indicated that intracellular ice does not form in the frozen spermatozoa. Subsequent warming rate experiments indicated that the survival levels achieved in a bath at 37 °C, or 5 °C, are indistinguishable and optimal, with lower rates dramatically reducing the proportion of motile sperm. These data suggest that a considerable latitude in freeze–thaw procedures for equine sperm can be tolerated without significant, practical reduction in the motility or viability of the recovered material, and that concentrating on the quality of the sperm sample collected might be more fruitful in improving the technique. Studies might include further investigation of the physical collection and processing of samples prior to cryopreservation and impact of nutriceuticals, particularly fatty acids, on semen quality. With regard to processing, preliminary data has shown the positive impact that seminal plasma can have on sperm motility when it is added back to chilled, transported samples, including differential improvement that depends upon the particular plasma employed. Such benefit could not be transferred to thawed samples as the conventional, egg-based extender interacts physically with seminal plasma. (For this reason, spermatozoa are separated from plasma by centrifugation in preparation for freezing). Once the benefits of adding back seminal plasma are confirmed we expect to revisit the established cryoprotocols with the intention of finding a replacement for egg-based extender and utilising seminal plasma, frozen-stored for practical convenience. (Conflict of interest: None declared. Source of funding: None declared). doi:10.1016/j.cryobiol.2006.10.058
58. Effects of glycerol on recovery and antioxidant enzyme activities of lyophilized red blood cells. Hui He, Baolin Liu, Tse-Chao Hua, Institute of Cryomedicine and Food Refrigeration, University of Shanghai for Science and Technology, 200093 Shanghai, People’s Republic of China Successful storage of red blood cells (RBCs) by freeze-drying has important implications in blood transfusion and clinical medicine. Glycerol is a permeable protective agent. Pretreatment of red blood cells with glycerol can increase the concentration in the cytoplasm of the RBCs and then enhance the possibility of glass transition during the freeze-drying process. In order to
understand the protective mechanism of glycerol on lyophilized human red blood cells, the effect of glycerol on the activity of antioxidant enzymes and the recovery of lyophilized red blood cells was investigated in this paper. Glycerol solutions with concentrations of 0%, 20%, and 40% were used to treat the RBCs before the regular freeze–drying process. After pretreatment with the glycerol solution, red blood cells suspended in the protective agents were lyophilized in a freezedrier (Freezone 2.5, Labconco). The freeze–drying process included a primary lyophilization at 70 °C for 30 h and a secondary lyophilization at 10 °C for 10 h. The results demonstrated that glycerol had remarkable effects on cell viability. The maximum viability of red blood cells was 55.3 ± 4.26% and the recovery of hemoglobin was 53.49 ± 3.85% after pretreatment with the 40% glycerol solution, both of which were significantly higher than the control group (0% glycerol). The effect of storage time on the activity of antioxidant enzymes and recovery of lyophilized RBCs was studied using a spectrophotometer. The results showed that recovery of lyophilized RBC superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) activity all decreased significantly after 180 days storage at room temperature. The enzyme activity showed a similar decreasing pattern with the recovery of lyophilized RBCs during the preservation processes. These results provide fundamental information for the long-term preservation of RBCs by the freeze–drying technique. (Conflict of interest: None declared. Source of funding: NSFC (50376040, 50436030, 50576059), Shanghai Leading Academic Discipline Project (P0502), Shanghai Municipal Foundation for Returned Overseas Chinese Scholars and SRF for ROCS, SEM). doi:10.1016/j.cryobiol.2006.10.059
59. On possibility of low-dose ozone increasing the survival of cryopreserved erythrocytes. Iryna A. Musina a, Yelizaveta L. Volovelskaya b, a Department of biophysics, Institute for problems of cryobiology and cryomedicine, 61015 Kharkov, Ukraine; b Department of cell cryophysiology, Institute for problems of cryobiology and cryomedicine, 61015 Kharkov, Ukraine Erythrocytes are among those biological objects that are actively investigated by cryobiologists. However, the problem of increasing the survival of erythrocytes is very topical. Recently, ozone at low doses has been reported to promote the increase of the resistance of some biological objects to low temperatures. In the present work we studied the survival of cryopreserved erythrocytes. We froze the erythrocytes in cryoprotective media and then treated them with ozone at the stage of cryoprotectant removal. Erythrocytes were isolated from freshly donated blood by centrifugation at 800 g and then purified by three cycles of resuspension in and washing with 0.15 M NaCl solution with careful removal of the supernatant. We mixed the deposited erythrocytes with cryoprotective solutions (dimethylsulphoxide, glucose, sucrose, NaCl and dextran 10000) in the volumetric ratio1:1. Cryoprotective solutions with different dextran concentrations (5, 10, 15, 20% w/v), were used. After incubation in the cryoprotective solutions for 30 min at room temperature we froze the samples by immersing them in liquid nitrogen. Part of the frozen samples was stored for 12 days at 196 °C, and other part of the samples was stored for 12 days at +4 °C without preliminary freezing. The frozen samples were thawed in a water bath at