- Email: [email protected]
Red blood cell microvesiculation during hypothermic storage
Abstracts / Cryobiology 63 (2011) 306–342 Forschungsgemeinschaft (DFG, German Research Foundation) for the Cluster of Excellence REBIRTH (From Regenerative Biology to Reconstructive Therapy). Conflict of interest: None declared. doi:10.1016/j.cryobiol.2011.09.021
Papers submitted for the Crystal Awards I
19. The ultra-rapid vitrification of human oocytes using low cryoprotectant concentrations. Heidi Elmoazzen 1, Diane L. Wright 2, Mehmet Toner 1, Thomas Toth 2, 1 Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School and Shriners Hospital for Children, Boston, USA, 2 Vincent Reproductive Medicine and IVF, Obstetrics and Gynecology Service, Massachusetts General Hospital, Boston, USA While oocyte cryopreservation is offered as an option to patients, current protocols are considered ‘‘experimental” by the American Society of Reproductive Medicine. The traditional applications of oocyte cryopreservation include: (1) the preservation of future fertility in women at risk for losing their reproductive functions due to cancer treatment and (2) the avoidance of the ethical and legal dilemmas surrounding the cryopreservation and long-term storage of human embryos. However, there has been an increasing social trend among women to delay initiating pregnancy due to social, professional or economic constraints and as a result, delaying family building using egg freezing is becoming more common. Also, there has been an increase in the use of donor eggs. Oocyte cryopreservation of donor eggs allows for more flexible timing and better precision of embryo transfer. There are currently two approaches to achieving cryopreservation of cells: the conventional slow freezing approach, which uses low cooling rates of less than 2 °C/min and vitrification which is a rapid cooling approach that uses cooling rates of over 1000 °C/minute. With either technique, cryoprotectants (CPAs) are used. With slow freezing, the CPAs are low viscosity solutions and the concentrations are quite low (1–2 molar) and are non-toxic to the cells. However, the challenge is that slow freezing is associated with injury due to ice formation. On the other hand, vitrification involves the solidification of a super-cooled liquid while maintaining the absence of ice, such that a glass is formed without any deleterious effects of ice formation. However, to achieve vitrification, rapid cooling rates and very high concentrations of cocktails of CPAs (4–8 molar) are required. These concentrations are toxic to most cells and as a result, multiple step are required to load and unload the CPAs and short exposure times to these high concentrations are need, all which make the process complicated. We have developed an ultra-rapid cooling method to cryopreserve human oocytes that requires a minimal amount of CPAs. We have combined the benefits of conventional slow freezing (i.e., reduced toxicity secondary to low CPA levels) with the benefits of vitrification (i.e., absence of intracellular ice crystal formation). Our novel method of ultra-rapid vitrification using micro-capillaries allows for cooling rates of 250,000 °C/min to be achieved and thus low CPA concentrations to be used. We vitrified over 100 human failed-to-fertilize oocytes; all oocytes were obtained from consenting patients undergoing IVF for infertility treatment. Oocytes were equilibrated in 0.75 M propylene glycol before being transferred to the vitrification solution of 0.5 M trehalose +1.5 M propylene glycol. The oocytes were then loaded into micro-capillaries, heat sealed and plunged into slush nitrogen. In the preliminary results with human fail-to-fertilize oocytes, 105/114 oocytes were recovered and 78% (82/105) of the oocytes survived and were completely intact after the cryopreservation and thawing procedure. The authors have no conflicts of interest. This research was supported +by the NIH. doi:10.1016/j.cryobiol.2011.09.022
20. Water transport processes during cooling of mammalian cells. Maryam Akhoondi * 1, Harriëtte Oldenhof 2, Willem F. Wolkers 1, 1 Institute of Multiphase Processes, Leibniz Universität Hannover, Hannover, Germany, 2 Clinic for Horses – Unit for Reproductive Medicine, University of Veterinary Medicine Hannover, Hannover, Germany Cell survival after cryopreservation is dependent on the biophysical response of the cell during freezing. During slow freezing, cells suffer from dehydration damage, whereas fast freezing results in intracellular ice formation, which is even more damaging. In order to be able to predict cryobiological outcomes with different cooling regimes, the cell specific membrane hydraulic permeability and corresponding activation energy for water transport need to be experimentally determined. One of the intriguing questions in the field of cryobiology is how ice formation affects the transport of water through cellular membranes. Subzero membrane permeability measurements; however, are experimentally difficult and infrequently reported. We have used Fourier transform infrared spectroscopy (FTIR) to study membrane phase behavior and ice formation during freezing of 3T3 cells and porcine mesenchymal stem cells, showing transport of membrane bound and free water separately in real
311
time. The membrane hydraulic permeability, Lp, at subzero temperatures, which was derived from the FTIR studies, was compared with the permeability to water at suprazero temperatures. Suprazero Lp values were derived from measurements of the cell volume response in hypertonic media using a Coulter counter particle sizer. An Arrhenius plot of the membrane hydraulic permeability values showed clear differences in Arrhenius behavior above and below the water-to-ice phase transition. The activation energy for water transport, ELp, increases almost three fold upon extracellular ice formation. In addition Lpg, the Lp at 0 °C, extrapolated from suprazero measurements is approximately five fold greater compared to the Lpg extrapolated from subzero measurements. This implies that subzero membrane permeability values cannot be extrapolated from suprazero measurements, which confirms the necessity to determine Lp in the presence of ice. Cryoprotective agents including Me2SO and glycerol were found to control the rate of cellular and membrane dehydration and decrease the activation energy for water transport to similar values as those at physiological temperatures. This work is supported by funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) for the Cluster of Excellence REBIRTH (From Regenerative Biology to Reconstructive Therapy). Conflict of interest: There are no conflicts of interest. doi:10.1016/j.cryobiol.2011.09.023
21. Red blood cell microvesiculation during hypothermic storage. Ruqayyah J. Almizraq * 1,2, Jayme D.R. Tchir 2, Jelena L. Holovati 1, Jason P. Acker 1,2, 1 Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, AB, Canada T6G 2R8, 2 Research and Development, Canadian Blood Services, Edmonton, AB, Canada T6G 2R8 Red blood cell microparticles (RMPs) are phospholipid vesicles less than 1 lm in size produced as a response to cellular stimulation or eryptosis. They exist in patients’ blood with greater number than healthy people. Therefore, RMPs have been clinically used as a means of diagnosis in several kinds of diseases. Furthermore, in vivo red cell microvesiculation play an important role in the removal of aged or damaged cells in circulation via phagocytosis. Recently, it has been indicated that the red blood cell (RBC) microvesiculation increases during the hypothermic storage (HS) and cryopreservation, which may adversely affect transfusion patient outcomes. Moreover, phosphatidylserine (PS) exposure and changes in CD47 expression have been suggested to contribute to the RBC membrane injury. The aim of this study was to measure the hypothermic-storage induced changes in CD47 and PS on RBCs as well as quantitatively and qualitatively evaluate RMPs using the flow cytometry. Leukoreduced pRBC units (n = 10) were collected in saline–adenine–glucose–mannitol (SAGM) and hypothermically stored (1–6 °C) for up to 49 days. RMPs, PS exposure, and CD47 expression on RBC and on RMP populations were evaluated weekly during the hypothermic storage period on a FACSCalibur instrument. Glycophorin A was used as a marker for RBCs and RMPs, while APC- annexin V and PE- anti mouse CD47 antibody were used to label and detect PS and CD47, respectively. In addition, 1.0 lm sized beads were used to define the size range of RBC microparticles. N-Ethylmaleimide treatment to induce PS exposure on the RBC was used as the positive control. The results show that the number of RMPs increased with length of storage and reached a significant level by day 42. We observed a significant decrease in the number of RMPs expressing PS and CD47 after 28 d of hypothermic storage with a statistically significant increase in the mean fluorescence intensity of PS and CD47 on RMPs by the end of storage (days 42 and 49). In contrast the number of RBCs expressing PS or CD47 did not change during hypothermic storage, but the overall MFI of both PS and CD47 on the stored RBC was shown to decrease. In conclusion, RBS membrane changes during HS occur after day 28, as evidenced by the significant increase in RBC microvesiculation, PS externalization and loss of CD47. As current blood banking standards permit ex vivo hypothermic storage of RBCs for up to 42 days before transfusion, more studies need to be done to determine clinical significance of increased RBC microvesiculation and PS expression in a blood product. Novel biopreservation approaches are needed to reduce the RBC membrane changes contributing to the hypothermic storage lesion and transfusion-associated adverse patient outcomes. This work was supported by the Canadian Institute for Health Research and Canadian Blood Services. Conflict of interest: Authors declare no conflict of interest. doi:10.1016/j.cryobiol.2011.09.024
22. Development of in vitro culture method for zebrafish ovarian tissue fragment. S. Anil *, T. Zampolla, T. Zhang, Bedfordshire, Luton, Bedfordshire LU2 8DL, UK Cryopreservation of ovarian tissues is a viable alternative to cryopreservation of oocytes and embryos in many species, but has not been successful in fish. In recent years, studies on mammals have demonstrated that ovarian tissue cryopreservation enables the storage of large number of oocytes within the primordial follicles. The main aim of the present study was to develop an optimum in vitro culture condition
Papers submitted for the Crystal Awards I
19. The ultra-rapid vitrification of human oocytes using low cryoprotectant concentrations. Heidi Elmoazzen 1, Diane L. Wright 2, Mehmet Toner 1, Thomas Toth 2, 1 Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School and Shriners Hospital for Children, Boston, USA, 2 Vincent Reproductive Medicine and IVF, Obstetrics and Gynecology Service, Massachusetts General Hospital, Boston, USA While oocyte cryopreservation is offered as an option to patients, current protocols are considered ‘‘experimental” by the American Society of Reproductive Medicine. The traditional applications of oocyte cryopreservation include: (1) the preservation of future fertility in women at risk for losing their reproductive functions due to cancer treatment and (2) the avoidance of the ethical and legal dilemmas surrounding the cryopreservation and long-term storage of human embryos. However, there has been an increasing social trend among women to delay initiating pregnancy due to social, professional or economic constraints and as a result, delaying family building using egg freezing is becoming more common. Also, there has been an increase in the use of donor eggs. Oocyte cryopreservation of donor eggs allows for more flexible timing and better precision of embryo transfer. There are currently two approaches to achieving cryopreservation of cells: the conventional slow freezing approach, which uses low cooling rates of less than 2 °C/min and vitrification which is a rapid cooling approach that uses cooling rates of over 1000 °C/minute. With either technique, cryoprotectants (CPAs) are used. With slow freezing, the CPAs are low viscosity solutions and the concentrations are quite low (1–2 molar) and are non-toxic to the cells. However, the challenge is that slow freezing is associated with injury due to ice formation. On the other hand, vitrification involves the solidification of a super-cooled liquid while maintaining the absence of ice, such that a glass is formed without any deleterious effects of ice formation. However, to achieve vitrification, rapid cooling rates and very high concentrations of cocktails of CPAs (4–8 molar) are required. These concentrations are toxic to most cells and as a result, multiple step are required to load and unload the CPAs and short exposure times to these high concentrations are need, all which make the process complicated. We have developed an ultra-rapid cooling method to cryopreserve human oocytes that requires a minimal amount of CPAs. We have combined the benefits of conventional slow freezing (i.e., reduced toxicity secondary to low CPA levels) with the benefits of vitrification (i.e., absence of intracellular ice crystal formation). Our novel method of ultra-rapid vitrification using micro-capillaries allows for cooling rates of 250,000 °C/min to be achieved and thus low CPA concentrations to be used. We vitrified over 100 human failed-to-fertilize oocytes; all oocytes were obtained from consenting patients undergoing IVF for infertility treatment. Oocytes were equilibrated in 0.75 M propylene glycol before being transferred to the vitrification solution of 0.5 M trehalose +1.5 M propylene glycol. The oocytes were then loaded into micro-capillaries, heat sealed and plunged into slush nitrogen. In the preliminary results with human fail-to-fertilize oocytes, 105/114 oocytes were recovered and 78% (82/105) of the oocytes survived and were completely intact after the cryopreservation and thawing procedure. The authors have no conflicts of interest. This research was supported +by the NIH. doi:10.1016/j.cryobiol.2011.09.022
20. Water transport processes during cooling of mammalian cells. Maryam Akhoondi * 1, Harriëtte Oldenhof 2, Willem F. Wolkers 1, 1 Institute of Multiphase Processes, Leibniz Universität Hannover, Hannover, Germany, 2 Clinic for Horses – Unit for Reproductive Medicine, University of Veterinary Medicine Hannover, Hannover, Germany Cell survival after cryopreservation is dependent on the biophysical response of the cell during freezing. During slow freezing, cells suffer from dehydration damage, whereas fast freezing results in intracellular ice formation, which is even more damaging. In order to be able to predict cryobiological outcomes with different cooling regimes, the cell specific membrane hydraulic permeability and corresponding activation energy for water transport need to be experimentally determined. One of the intriguing questions in the field of cryobiology is how ice formation affects the transport of water through cellular membranes. Subzero membrane permeability measurements; however, are experimentally difficult and infrequently reported. We have used Fourier transform infrared spectroscopy (FTIR) to study membrane phase behavior and ice formation during freezing of 3T3 cells and porcine mesenchymal stem cells, showing transport of membrane bound and free water separately in real
311
time. The membrane hydraulic permeability, Lp, at subzero temperatures, which was derived from the FTIR studies, was compared with the permeability to water at suprazero temperatures. Suprazero Lp values were derived from measurements of the cell volume response in hypertonic media using a Coulter counter particle sizer. An Arrhenius plot of the membrane hydraulic permeability values showed clear differences in Arrhenius behavior above and below the water-to-ice phase transition. The activation energy for water transport, ELp, increases almost three fold upon extracellular ice formation. In addition Lpg, the Lp at 0 °C, extrapolated from suprazero measurements is approximately five fold greater compared to the Lpg extrapolated from subzero measurements. This implies that subzero membrane permeability values cannot be extrapolated from suprazero measurements, which confirms the necessity to determine Lp in the presence of ice. Cryoprotective agents including Me2SO and glycerol were found to control the rate of cellular and membrane dehydration and decrease the activation energy for water transport to similar values as those at physiological temperatures. This work is supported by funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) for the Cluster of Excellence REBIRTH (From Regenerative Biology to Reconstructive Therapy). Conflict of interest: There are no conflicts of interest. doi:10.1016/j.cryobiol.2011.09.023
21. Red blood cell microvesiculation during hypothermic storage. Ruqayyah J. Almizraq * 1,2, Jayme D.R. Tchir 2, Jelena L. Holovati 1, Jason P. Acker 1,2, 1 Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, AB, Canada T6G 2R8, 2 Research and Development, Canadian Blood Services, Edmonton, AB, Canada T6G 2R8 Red blood cell microparticles (RMPs) are phospholipid vesicles less than 1 lm in size produced as a response to cellular stimulation or eryptosis. They exist in patients’ blood with greater number than healthy people. Therefore, RMPs have been clinically used as a means of diagnosis in several kinds of diseases. Furthermore, in vivo red cell microvesiculation play an important role in the removal of aged or damaged cells in circulation via phagocytosis. Recently, it has been indicated that the red blood cell (RBC) microvesiculation increases during the hypothermic storage (HS) and cryopreservation, which may adversely affect transfusion patient outcomes. Moreover, phosphatidylserine (PS) exposure and changes in CD47 expression have been suggested to contribute to the RBC membrane injury. The aim of this study was to measure the hypothermic-storage induced changes in CD47 and PS on RBCs as well as quantitatively and qualitatively evaluate RMPs using the flow cytometry. Leukoreduced pRBC units (n = 10) were collected in saline–adenine–glucose–mannitol (SAGM) and hypothermically stored (1–6 °C) for up to 49 days. RMPs, PS exposure, and CD47 expression on RBC and on RMP populations were evaluated weekly during the hypothermic storage period on a FACSCalibur instrument. Glycophorin A was used as a marker for RBCs and RMPs, while APC- annexin V and PE- anti mouse CD47 antibody were used to label and detect PS and CD47, respectively. In addition, 1.0 lm sized beads were used to define the size range of RBC microparticles. N-Ethylmaleimide treatment to induce PS exposure on the RBC was used as the positive control. The results show that the number of RMPs increased with length of storage and reached a significant level by day 42. We observed a significant decrease in the number of RMPs expressing PS and CD47 after 28 d of hypothermic storage with a statistically significant increase in the mean fluorescence intensity of PS and CD47 on RMPs by the end of storage (days 42 and 49). In contrast the number of RBCs expressing PS or CD47 did not change during hypothermic storage, but the overall MFI of both PS and CD47 on the stored RBC was shown to decrease. In conclusion, RBS membrane changes during HS occur after day 28, as evidenced by the significant increase in RBC microvesiculation, PS externalization and loss of CD47. As current blood banking standards permit ex vivo hypothermic storage of RBCs for up to 42 days before transfusion, more studies need to be done to determine clinical significance of increased RBC microvesiculation and PS expression in a blood product. Novel biopreservation approaches are needed to reduce the RBC membrane changes contributing to the hypothermic storage lesion and transfusion-associated adverse patient outcomes. This work was supported by the Canadian Institute for Health Research and Canadian Blood Services. Conflict of interest: Authors declare no conflict of interest. doi:10.1016/j.cryobiol.2011.09.024
22. Development of in vitro culture method for zebrafish ovarian tissue fragment. S. Anil *, T. Zampolla, T. Zhang, Bedfordshire, Luton, Bedfordshire LU2 8DL, UK Cryopreservation of ovarian tissues is a viable alternative to cryopreservation of oocytes and embryos in many species, but has not been successful in fish. In recent years, studies on mammals have demonstrated that ovarian tissue cryopreservation enables the storage of large number of oocytes within the primordial follicles. The main aim of the present study was to develop an optimum in vitro culture condition