- Email: [email protected]
56. Cryopreserved Indian gerbil (Tatera indica) spermatozoa maintain their fertility
Abstracts / Cryobiology 53 (2006) 367–446 study was performed on VS4 and on VS4-impregnated ovarian tissue fragments to evaluate the critical cooling rates to ensure against ice-crystal formation in the tissue and in the VS4. These cooling criteria were compared to the cooling rates applied to the ovaries. Results showed that the perfusion conditions applied failed to provide sufficiently homogeneous VS4 impregnation of the cortex to ensure its systematic vitrification under the implemented operating conditions. On the contrary, the ovarian lumbar pedicle vessels were especially well impregnated and vitrified more easily. NMR studies of VS4 impregnation in ovarian tissue are considered to optimize the perfusion protocol to enable vitrification of the whole ovary. A preliminary study undertaken on vitrified-thawed intact sheep ovaries showed that transplantation is technically feasible, using artery and vein anastomosis to the contralateral ovarian pedicle, with a good prospect regarding immediate restoration of vascular supply and ovarian hormonal function. (Conflict of interest: None declared. Source of funding: This work was funded by institutional sources of the CNRS and INSERM.) doi:10.1016/j.cryobiol.2006.10.055
55. Successful cryopreservation of embryoid bodies. Ali Eroglu, Sean Liour, Shruti Sharma, Jacek Rzucidlo, Institute of Molecular Medicine and Genetics, Medical College of Georgia, 30912 Augusta, GA, USA Embryonic stem (ES) cells are pluripotent cells that can differentiate into all kind of cell in the body and be expanded indefinitely. Therefore, ES cells offer unique opportunities for medical therapies for debilitating diseases. However, further comprehensive research is needed to understand signaling pathways controlling the differentiation of ES cells into different cell lineages and accordingly to develop directed differentiation protocols. One of the limiting steps for differentiation studies is proper culture and expansion of ES cells, which is labor-intensive, expensive, and requires a great deal of expertise. This limiting step can be overcome by the successful banking and distribution of embryoid bodies (EBs) that are aggregates of ES cells and typically the starting point of differentiation protocols. The objective of this study was to investigate the feasibility of EB banking by studying the survival and functionality of frozen-thawed EBs. To this end, EBs were formed by culturing mouse 129 ES cells in the absence of leukemia inhibitory factor in hanging drops or suspension using non-adherent dishes, and then subjecting them to different cryopreservation protocols. In the first series of experiments, we tested the morphological survival of EBs frozen using various concentrations (10, 5, and 2.5%) of dimethylsulfoxide and ethylene glycol alone or in combination with extracellular trehalose (0.1 to 0.2 M). EBs between 100 and 300 lm in diameter were selected, loaded with cryoprotectants, cooled to 80 °C at different cooling rates (1, 5, and 10 °C/ min) using a controlled-rate freezer, and then plunged into liquid nitrogen. Frozen EBs were thawed in air, and cryosurvival of individual EB cells was assessed by a standard red/green staining using Syto 13 and Ethidium Homodimer. Experiments were repeated three times. All tested conditions yielded high survival rates ranging from 80 to 95%. In the second series of experiments, we analyzed growth and functionality of cryopreserved EBs. After thawing, EBs were cultured for more than two weeks, and their growth and differentiation into various cell types was
391
evaluated. In all experimental groups, over 80% of the cryopreserved EBs grew further and differentiated into various cell types such as fibroblasts, myocardiocytes, neurons and red blood cells. Taken together, our results suggest that EBs are tolerant of cryopreservation-associated stresses and retain their pluripotency after freezing and thawing using different protocols. Thus, our data support the feasibility of EB banking, which would greatly facilitate advancement of ES cell-based therapies. (Conflict of interest: None declared. Source of funding: This work was supported by intramural funding of the Medical College of Georgia to A.E.) doi:10.1016/j.cryobiol.2006.10.056
56. Cryopreserved Indian gerbil (Tatera indica) spermatozoa maintain their fertility. Chihiro Koshimoto a, Daisuke Watanabe b, Akio Shinohara a, Tetsuo Morita b, a Division of Bioresources, Frontier Science Research Center, University of Miyazaki, 889-1692 Kiyotake, Miyazaki, Japan; b Faculty of Agriculture, University of Miyazaki, 889-2192 Miyazaki, Japan The Indian gerbil (Tatera indica) is one of the largest species in the subfamily Gerbillinae and has a remarkable forestomach and cecum which some bacteria and protozoa related to fermentation inhabit. We have been developing this rodent as a new experimental animal for ruminants. This is the first attempt to freeze Indian gerbil spermatozoa. We examined the fertility of frozen/thawed sperm by the using the hamster test in order to apply cryopreservation technology to the maintenance of Tatera colonies in the future. First we examined the hyperosmotic tolerance of Indian gerbil spermatozoa by exposing them to 290–620 mOsmolal conditions for 5 min at room temperature prior to freezing. The percentage of total motile sperm was not significantly affected by 5 min hyperosmotic exposure, but the percentage of progressive motile spermatozoa after osmotic stress exposure began to drop at 400 mOsm and a significant decrease was observed at 620 mOsm (P < 0.01). This osmolality tolerance curve corresponds to that of mouse sperm exposed to similar osmotic conditions. According to the result, the osmolality of freezing solutions for Indian gerbil spermatozoa, in which 6–22% raffinose is present, was fixed at 430 mOsmolal which is the same for mouse sperm freezing solutions made by appropriate dilution of basic PBS. The sperm suspension was frozen by placing the sample straw in the vapor of liquid nitrogen for 5 min, which produced a cooling rate between 0 and 70 °C of 130 °C/min, followed by immersion in liquid nitrogen. Although the sperm survived all the raffinose conditions, the highest survival was observed when the sperm were frozen in the presence of 14 or 18% raffinose, in which the normalized motility was >40%. The fertility of cryopreserved Indian gerbil sperm was examined by using the zona-free hamster test as a substitute for in vitro fertilization or artificial insemination. Frozen thawed sperm adhered to most of the zona-free hamster oocyte surface (99 out of 113; 88%) and some of them were penetrated and exhibited swollen sperm head or male pronuclei, which we defined as fertilized. Although the fertilization rate of cryopreserved sperm with zona-free hamster eggs was significantly lower than that for fresh sperm (6% vs. 30%; p < 0.01), frozen thawed Indian gerbil spermatozoa have been demonstrated to retain their fertility. (Conflict of interest: None declared. Source of
392
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
55. Successful cryopreservation of embryoid bodies. Ali Eroglu, Sean Liour, Shruti Sharma, Jacek Rzucidlo, Institute of Molecular Medicine and Genetics, Medical College of Georgia, 30912 Augusta, GA, USA Embryonic stem (ES) cells are pluripotent cells that can differentiate into all kind of cell in the body and be expanded indefinitely. Therefore, ES cells offer unique opportunities for medical therapies for debilitating diseases. However, further comprehensive research is needed to understand signaling pathways controlling the differentiation of ES cells into different cell lineages and accordingly to develop directed differentiation protocols. One of the limiting steps for differentiation studies is proper culture and expansion of ES cells, which is labor-intensive, expensive, and requires a great deal of expertise. This limiting step can be overcome by the successful banking and distribution of embryoid bodies (EBs) that are aggregates of ES cells and typically the starting point of differentiation protocols. The objective of this study was to investigate the feasibility of EB banking by studying the survival and functionality of frozen-thawed EBs. To this end, EBs were formed by culturing mouse 129 ES cells in the absence of leukemia inhibitory factor in hanging drops or suspension using non-adherent dishes, and then subjecting them to different cryopreservation protocols. In the first series of experiments, we tested the morphological survival of EBs frozen using various concentrations (10, 5, and 2.5%) of dimethylsulfoxide and ethylene glycol alone or in combination with extracellular trehalose (0.1 to 0.2 M). EBs between 100 and 300 lm in diameter were selected, loaded with cryoprotectants, cooled to 80 °C at different cooling rates (1, 5, and 10 °C/ min) using a controlled-rate freezer, and then plunged into liquid nitrogen. Frozen EBs were thawed in air, and cryosurvival of individual EB cells was assessed by a standard red/green staining using Syto 13 and Ethidium Homodimer. Experiments were repeated three times. All tested conditions yielded high survival rates ranging from 80 to 95%. In the second series of experiments, we analyzed growth and functionality of cryopreserved EBs. After thawing, EBs were cultured for more than two weeks, and their growth and differentiation into various cell types was
391
evaluated. In all experimental groups, over 80% of the cryopreserved EBs grew further and differentiated into various cell types such as fibroblasts, myocardiocytes, neurons and red blood cells. Taken together, our results suggest that EBs are tolerant of cryopreservation-associated stresses and retain their pluripotency after freezing and thawing using different protocols. Thus, our data support the feasibility of EB banking, which would greatly facilitate advancement of ES cell-based therapies. (Conflict of interest: None declared. Source of funding: This work was supported by intramural funding of the Medical College of Georgia to A.E.) doi:10.1016/j.cryobiol.2006.10.056
56. Cryopreserved Indian gerbil (Tatera indica) spermatozoa maintain their fertility. Chihiro Koshimoto a, Daisuke Watanabe b, Akio Shinohara a, Tetsuo Morita b, a Division of Bioresources, Frontier Science Research Center, University of Miyazaki, 889-1692 Kiyotake, Miyazaki, Japan; b Faculty of Agriculture, University of Miyazaki, 889-2192 Miyazaki, Japan The Indian gerbil (Tatera indica) is one of the largest species in the subfamily Gerbillinae and has a remarkable forestomach and cecum which some bacteria and protozoa related to fermentation inhabit. We have been developing this rodent as a new experimental animal for ruminants. This is the first attempt to freeze Indian gerbil spermatozoa. We examined the fertility of frozen/thawed sperm by the using the hamster test in order to apply cryopreservation technology to the maintenance of Tatera colonies in the future. First we examined the hyperosmotic tolerance of Indian gerbil spermatozoa by exposing them to 290–620 mOsmolal conditions for 5 min at room temperature prior to freezing. The percentage of total motile sperm was not significantly affected by 5 min hyperosmotic exposure, but the percentage of progressive motile spermatozoa after osmotic stress exposure began to drop at 400 mOsm and a significant decrease was observed at 620 mOsm (P < 0.01). This osmolality tolerance curve corresponds to that of mouse sperm exposed to similar osmotic conditions. According to the result, the osmolality of freezing solutions for Indian gerbil spermatozoa, in which 6–22% raffinose is present, was fixed at 430 mOsmolal which is the same for mouse sperm freezing solutions made by appropriate dilution of basic PBS. The sperm suspension was frozen by placing the sample straw in the vapor of liquid nitrogen for 5 min, which produced a cooling rate between 0 and 70 °C of 130 °C/min, followed by immersion in liquid nitrogen. Although the sperm survived all the raffinose conditions, the highest survival was observed when the sperm were frozen in the presence of 14 or 18% raffinose, in which the normalized motility was >40%. The fertility of cryopreserved Indian gerbil sperm was examined by using the zona-free hamster test as a substitute for in vitro fertilization or artificial insemination. Frozen thawed sperm adhered to most of the zona-free hamster oocyte surface (99 out of 113; 88%) and some of them were penetrated and exhibited swollen sperm head or male pronuclei, which we defined as fertilized. Although the fertilization rate of cryopreserved sperm with zona-free hamster eggs was significantly lower than that for fresh sperm (6% vs. 30%; p < 0.01), frozen thawed Indian gerbil spermatozoa have been demonstrated to retain their fertility. (Conflict of interest: None declared. Source of
392
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