- Email: [email protected]
44. New developments in lyophilized platelets for arrest of bleeding in transfusion medicine
324
Abstracts / Cryobiology 57 (2008) 315–340
Successful cryopreservation demands there be little or no intracellular ice. One procedure for achieving this is classical slow equilibrium freezing, and it has been successful in many cases. However, for some important cell types, including some mammalian oocytes, it has not. For the latter, there are increasing attempts to cryopreserve them by vitrification. However, even if intracellular ice formation (IIF) is prevented during cooling, it can still occur during the warming of a vitrified sample. Here, we examine two aspects of this occurrence in mouse oocytes. One takes place in oocytes that are partly dehydrated by an initial hold for 12 minutes at 25 °C. They are then cooled rapidly to 70 °C and warmed slowly or they are warmed rapidly to intermediate temperatures and held. These oocytes undergo no IIF during cooling but blacken from IIF during warming. The blackening rate increases about 5-fold for each 5-degree rise in temperature. Upon thawing, they are dead. The second aspect involved oocytes that had been vitrified by suspension in a concentrated solution of cryoprotectants, cooled rapidly to 196 °C and warmed at rates ranging from 140 to 3,300 °C/min. Survivals after warming at 140 and 250 °C/min were low (<30%). Survivals after warming at P2,200 °C/min were high (80%). When warmed slowly, they are killed apparently by the recrystallization of previously formed small internal ice crystals. The similarities and differences in the consequences of the two types of freezing will be discussed. (Conflicts of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2008.10.044
a part of the treatment. Graft vs. host disease (GvHD) made patients less likely to relapse. Cells were integral to the curative process, so engineering the cells using compounds that changed the internal molecular structure could make them more powerful fighters of disease. A translational medicine example uses binding RNA fragments, aptamers in clinical application. It was hypothesized that if engineered RNA molecules could be used to stimulate immunity to specific tumors or to bind to specific tumor antigens, become incorporated into the cells, and use a silencing RNA pathway to kill the cells, then anti-tumor therapy could be done with less noxious agents. The costimulatory aptamer 4-1BB was isolated using the SELEX method [J.O. McNamara et al., J. Clin. Invest. 118 (2008) 376–385]. High affinity RNA ligands were processed through interactive rounds of co-incubation of the target protein and a partially randomized RNA sequence. For the PSMA siRNA Plk1 aptamer double-stranded DNA templates were generated to produce the desired RNA aptamers [J.O. McNamara et al., Nat. Biotechnol. 24 (2006) 1005–1015]. The compounds were tested by flow cytometry and against the appropriate cells. The receptor activating molecule targeting the costimulatory receptor, 4-1BB is active in cancer immunotherapy. The agonist costimulates CD8+ cells specifically and is specific. PSMA-binding aptamer delivered siRNA intracellularly inhibiting PLK1, a tumor growth gene for prostate cancer. Human xenografts of prostate cancer in nude mice showed apoptosis at 10 days. Aptamers use RNA molecules instead of antibodies to target cell protein sequences. The first compound enhances immune cell activity against tumors. The second binds to a membranes receptor to gain entry for the silencing RNA. Both approaches could serve as a platform for treating a number of conditions. (Conflicts of interest: None declared. Source of funding: None declared.)
Contributed papers 6. Cell and gene therapies doi:10.1016/j.cryobiol.2008.10.046 44. New developments in lyophilized platelets for arrest of bleeding in transfusion medicine. Arthur P. Bode a, Thomas H. Fischer b, Anne S. Hale c, a Department of Pathology and Laboratory Medicine, East Carolina University, Greenville, NC, USA, b Department of Pathology, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA, c MidWest Animal Blood Services, Stockbridge, MI, USA Platelets are small anuclear cells introduced into the blood stream by membrane demarcation and fragmentation of the cytoplasmic body of marrow megakaryocytes. Their main function is to adhere to sites of vascular injury and to arrest hemorrhage by forming a cellular plug and accelerating clot generation to secure it in place. The platelet is generally consumed in this process, or in its other function of maintaining intravascular integrity of capillary vessels by plugging separated gap junctions, or senesces from circulation within 10 days. Patients with severe platelet dysfunction bleed frequently and severely; several million transfusions of platelet concentrates are conducted each year to try to limit or prevent such bleeding. We have developed a means to stabilize blood platelets in freeze-dried form for long-term storage to increase their availability and reduce the hazards and wastage associated with current methods of concentrate production and transfusion. Our process involves steps which reduce the viral and bacterial bioburden of donor platelet preparations, and extend the shelf-life from 5 days in room temperature storage to years in refrigeration. In vitro data and animal model testing suggest that the reconstituted lyophilized platelets will be safe and efficacious in several hemorrhagic scenarios. The remaining steps in commercialization and pharmaceutical production will focus on decisions concerning batch size and quality control measures, as well as field of first clinical exposure. To that end, the first clinical trials will actually be conducted in the veterinary setting with freeze-dried canine platelets administered to canine patients presenting with active bleeding associated with low platelet count or suspected platelet dysfunction. Preclinical and anecdotal usage of test units has already indicated that the hemostatic effectiveness and its persistence in recipients with initially high bleeding scores is not predicted by count of particles in circulation, so the standard blood banking transfusion monitor of corrected count increment in circulation will be of little value. Other in vitro testing is being explored to identify either a function or physical signature of the lyophilized platelets which would either certify their utility and safety, or rigorously exclude them from clinical use due to a proven profile of increased risk and reduced efficacy. Minimal effective dose and maximal tolerated dose will be defined in the clinical studies; batch size and economy of scale in production will be examined at the bench. Results in the veterinary trials should go far in setting up clinical trials with the human-source product. The practice of transfusion support for hemorrhagic patients will benefit greatly from the availability of a safe and efficacious platelet-based hemostatic agent that can be stored for a year or more and is ready for use as simply as adding water and stirring. (Conflicts of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2008.10.045
45. Molecular-based translational medicine. N. Rebecca Haley, Bruce Sullenger, Duke University, Durham, NC, USA The original concept of bone marrow transplantation was to rescue the patient after fatal or near-fatal doses of chemo/radiotherapy. Allogeneic infused cells were
46. Microfluidic processing of cells for preservation. Allison Hubel, Clara Mata, Katie K.F. Glass, Ellen K. Longmire, University of Minnesota, Minneapolis, MN, USA Cryopreservation involves the use of specialized solution that must be introduced and removed and conventional methods for doing that have typically involved centrifugation and resuspension of the cells. This process is labor intensive and the quality of the outcome is operator dependent. One alternative is to use microfluidics to introduce and remove cryopreservation solutions. This technology has the potential to reduce the labor and time involved in cell preservation and improve outcome by semi-automating the process. To that end, studies have been performed to remove Me2SO from a frozen and thawed solution using a microfluidic device. A cell stream containing 10% v/v Me2SO entered the device with a wash stream that did not contain Me2SO. Both streams exited the device and the outlet concentration of Me2SO was determined for both streams. In addition, the total number of cells entering and leaving the device was quantified. Me2SO removal and cell recovery (viable cells leaving the device divided by viable cells entering the device) for a single stage device was determined and validated for flow rate fractions ranging between 0.10 and 0.37. The extraction of Me2SO was within 4% of that predicted by a theoretical model. Cell recoveries for a single stage device were greater than 93%. Additional studies were performed with a multistage device capable of extracting Me2SO to a level appropriate for infusion into patients. For a flow rate fraction of 0.23, a three-stage device removed 95% of the Me2SO with a cell recovery greater than 85%. Microfluidic devices can be used to reduce labor involved with cell preservation and improve outcome. (Conflicts of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2008.10.047
47. Cryobiology in translational reproductive medicine: The oncofertility consortium. John K. Critser, University of Missouri, USA Significant advances in cancer treatment during the past few decades have significantly improved the survival of individuals diagnosed with this devastating disease. The 5-year relative survival rate for all cancers diagnosed between 1995 and 2001 is 65%. While the incidence of childhood cancers is relatively low (9500 new cases of cancer are expected to be diagnosed in 2006 for children between the ages of 0–14), the cure rate is often quite high: 80% overall (American Cancer Society, Cancer Facts and Figures 2006. American Cancer Society: Atlanta). These numbers suggest that a significant proportion of the adult population in the future will be survivors of cancer. In fact, it has been estimated that, by 2010, 1 in 250 people in the adult population will be cancer survivors. The remarkable success of improved cancer therapy has generated a significant societal need: namely, the development of effective means to preserve the fertility of individuals undergoing such therapies. While fertility preservation for males using sperm cryopreservation has been available for several decades, attempts to preserve mature oocytes from women have, in general, been disappointing. In fact, all forms of female fertility preservation are still considered experimental (ASRM Practice Committee, Ovarian tissue and oocyte cryopreservation. Fertil. Steril., 82 (2004) 993–998). For most female cancer patients the only option
Abstracts / Cryobiology 57 (2008) 315–340
Successful cryopreservation demands there be little or no intracellular ice. One procedure for achieving this is classical slow equilibrium freezing, and it has been successful in many cases. However, for some important cell types, including some mammalian oocytes, it has not. For the latter, there are increasing attempts to cryopreserve them by vitrification. However, even if intracellular ice formation (IIF) is prevented during cooling, it can still occur during the warming of a vitrified sample. Here, we examine two aspects of this occurrence in mouse oocytes. One takes place in oocytes that are partly dehydrated by an initial hold for 12 minutes at 25 °C. They are then cooled rapidly to 70 °C and warmed slowly or they are warmed rapidly to intermediate temperatures and held. These oocytes undergo no IIF during cooling but blacken from IIF during warming. The blackening rate increases about 5-fold for each 5-degree rise in temperature. Upon thawing, they are dead. The second aspect involved oocytes that had been vitrified by suspension in a concentrated solution of cryoprotectants, cooled rapidly to 196 °C and warmed at rates ranging from 140 to 3,300 °C/min. Survivals after warming at 140 and 250 °C/min were low (<30%). Survivals after warming at P2,200 °C/min were high (80%). When warmed slowly, they are killed apparently by the recrystallization of previously formed small internal ice crystals. The similarities and differences in the consequences of the two types of freezing will be discussed. (Conflicts of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2008.10.044
a part of the treatment. Graft vs. host disease (GvHD) made patients less likely to relapse. Cells were integral to the curative process, so engineering the cells using compounds that changed the internal molecular structure could make them more powerful fighters of disease. A translational medicine example uses binding RNA fragments, aptamers in clinical application. It was hypothesized that if engineered RNA molecules could be used to stimulate immunity to specific tumors or to bind to specific tumor antigens, become incorporated into the cells, and use a silencing RNA pathway to kill the cells, then anti-tumor therapy could be done with less noxious agents. The costimulatory aptamer 4-1BB was isolated using the SELEX method [J.O. McNamara et al., J. Clin. Invest. 118 (2008) 376–385]. High affinity RNA ligands were processed through interactive rounds of co-incubation of the target protein and a partially randomized RNA sequence. For the PSMA siRNA Plk1 aptamer double-stranded DNA templates were generated to produce the desired RNA aptamers [J.O. McNamara et al., Nat. Biotechnol. 24 (2006) 1005–1015]. The compounds were tested by flow cytometry and against the appropriate cells. The receptor activating molecule targeting the costimulatory receptor, 4-1BB is active in cancer immunotherapy. The agonist costimulates CD8+ cells specifically and is specific. PSMA-binding aptamer delivered siRNA intracellularly inhibiting PLK1, a tumor growth gene for prostate cancer. Human xenografts of prostate cancer in nude mice showed apoptosis at 10 days. Aptamers use RNA molecules instead of antibodies to target cell protein sequences. The first compound enhances immune cell activity against tumors. The second binds to a membranes receptor to gain entry for the silencing RNA. Both approaches could serve as a platform for treating a number of conditions. (Conflicts of interest: None declared. Source of funding: None declared.)
Contributed papers 6. Cell and gene therapies doi:10.1016/j.cryobiol.2008.10.046 44. New developments in lyophilized platelets for arrest of bleeding in transfusion medicine. Arthur P. Bode a, Thomas H. Fischer b, Anne S. Hale c, a Department of Pathology and Laboratory Medicine, East Carolina University, Greenville, NC, USA, b Department of Pathology, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA, c MidWest Animal Blood Services, Stockbridge, MI, USA Platelets are small anuclear cells introduced into the blood stream by membrane demarcation and fragmentation of the cytoplasmic body of marrow megakaryocytes. Their main function is to adhere to sites of vascular injury and to arrest hemorrhage by forming a cellular plug and accelerating clot generation to secure it in place. The platelet is generally consumed in this process, or in its other function of maintaining intravascular integrity of capillary vessels by plugging separated gap junctions, or senesces from circulation within 10 days. Patients with severe platelet dysfunction bleed frequently and severely; several million transfusions of platelet concentrates are conducted each year to try to limit or prevent such bleeding. We have developed a means to stabilize blood platelets in freeze-dried form for long-term storage to increase their availability and reduce the hazards and wastage associated with current methods of concentrate production and transfusion. Our process involves steps which reduce the viral and bacterial bioburden of donor platelet preparations, and extend the shelf-life from 5 days in room temperature storage to years in refrigeration. In vitro data and animal model testing suggest that the reconstituted lyophilized platelets will be safe and efficacious in several hemorrhagic scenarios. The remaining steps in commercialization and pharmaceutical production will focus on decisions concerning batch size and quality control measures, as well as field of first clinical exposure. To that end, the first clinical trials will actually be conducted in the veterinary setting with freeze-dried canine platelets administered to canine patients presenting with active bleeding associated with low platelet count or suspected platelet dysfunction. Preclinical and anecdotal usage of test units has already indicated that the hemostatic effectiveness and its persistence in recipients with initially high bleeding scores is not predicted by count of particles in circulation, so the standard blood banking transfusion monitor of corrected count increment in circulation will be of little value. Other in vitro testing is being explored to identify either a function or physical signature of the lyophilized platelets which would either certify their utility and safety, or rigorously exclude them from clinical use due to a proven profile of increased risk and reduced efficacy. Minimal effective dose and maximal tolerated dose will be defined in the clinical studies; batch size and economy of scale in production will be examined at the bench. Results in the veterinary trials should go far in setting up clinical trials with the human-source product. The practice of transfusion support for hemorrhagic patients will benefit greatly from the availability of a safe and efficacious platelet-based hemostatic agent that can be stored for a year or more and is ready for use as simply as adding water and stirring. (Conflicts of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2008.10.045
45. Molecular-based translational medicine. N. Rebecca Haley, Bruce Sullenger, Duke University, Durham, NC, USA The original concept of bone marrow transplantation was to rescue the patient after fatal or near-fatal doses of chemo/radiotherapy. Allogeneic infused cells were
46. Microfluidic processing of cells for preservation. Allison Hubel, Clara Mata, Katie K.F. Glass, Ellen K. Longmire, University of Minnesota, Minneapolis, MN, USA Cryopreservation involves the use of specialized solution that must be introduced and removed and conventional methods for doing that have typically involved centrifugation and resuspension of the cells. This process is labor intensive and the quality of the outcome is operator dependent. One alternative is to use microfluidics to introduce and remove cryopreservation solutions. This technology has the potential to reduce the labor and time involved in cell preservation and improve outcome by semi-automating the process. To that end, studies have been performed to remove Me2SO from a frozen and thawed solution using a microfluidic device. A cell stream containing 10% v/v Me2SO entered the device with a wash stream that did not contain Me2SO. Both streams exited the device and the outlet concentration of Me2SO was determined for both streams. In addition, the total number of cells entering and leaving the device was quantified. Me2SO removal and cell recovery (viable cells leaving the device divided by viable cells entering the device) for a single stage device was determined and validated for flow rate fractions ranging between 0.10 and 0.37. The extraction of Me2SO was within 4% of that predicted by a theoretical model. Cell recoveries for a single stage device were greater than 93%. Additional studies were performed with a multistage device capable of extracting Me2SO to a level appropriate for infusion into patients. For a flow rate fraction of 0.23, a three-stage device removed 95% of the Me2SO with a cell recovery greater than 85%. Microfluidic devices can be used to reduce labor involved with cell preservation and improve outcome. (Conflicts of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2008.10.047
47. Cryobiology in translational reproductive medicine: The oncofertility consortium. John K. Critser, University of Missouri, USA Significant advances in cancer treatment during the past few decades have significantly improved the survival of individuals diagnosed with this devastating disease. The 5-year relative survival rate for all cancers diagnosed between 1995 and 2001 is 65%. While the incidence of childhood cancers is relatively low (9500 new cases of cancer are expected to be diagnosed in 2006 for children between the ages of 0–14), the cure rate is often quite high: 80% overall (American Cancer Society, Cancer Facts and Figures 2006. American Cancer Society: Atlanta). These numbers suggest that a significant proportion of the adult population in the future will be survivors of cancer. In fact, it has been estimated that, by 2010, 1 in 250 people in the adult population will be cancer survivors. The remarkable success of improved cancer therapy has generated a significant societal need: namely, the development of effective means to preserve the fertility of individuals undergoing such therapies. While fertility preservation for males using sperm cryopreservation has been available for several decades, attempts to preserve mature oocytes from women have, in general, been disappointing. In fact, all forms of female fertility preservation are still considered experimental (ASRM Practice Committee, Ovarian tissue and oocyte cryopreservation. Fertil. Steril., 82 (2004) 993–998). For most female cancer patients the only option