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Ultrastructure and aggregation of platelets incubated in glycerol
5%
ABSTRACTS, 17th ANNUAL
glycerolization, the glycerol concentration can be reduced in the same manner. The glycerol gradient experienced by the cells can be controlled by adjusting the rate of introduction and the concentration of the glycerol solution. The appropriate gradient, in turn, can be calculated knowing the range of osmotic tolerance of the cells and the rates of influx and efflux of glycerol. Human granulocytes suspended in Hanks’ balanced salt solution have been circulated through the device. Thirty minutes of circulation in the absence of glycerol results in a 30% reduction in cell count but only a slight reduction in the chemotactic function of the remaining cells. Following glycerolization and deglycerolization, the cell recovery is more variable but good recovery of both count and function has been achieved following glycerolization to concentrations exceeding 3000 mOsm. 10. Effects of Glycerol on Structure and Functions of Platelets. TOKIO NEI. (Faculty of Pharmaceutical Sciences, Higashi Nippon Gakuen University, Ishikari-Tobetsu, Hokkaido, 061-02 Japan). Platelets which play a great role in blood coagulation systems possess complicated structures and functions. Although the freeze-preservation of platelets using cryoprotectants such as glycerol and Me,SO has been tried by many investigators, problems still remain to be solved. In the present study, effects of glycerol on morphology and physiology of platelets have been investigated with special reference to Me,SO and glutaraldehyde, a fixative. In results obtained, membrane permeation of glycerol depended upon its concentration. High concentrations of glycerol caused cell swelling and ultrastructural alteration of intact and glutaraldehyde-fixed cell membranes. Aggregation of platelets was inhibited with increased addition of glycerol and glutaraldehyde. There was a marked difference in mode of actions of glycerol and Me,SO to platelets. 11. Clinical
Uses of Platelets Frozen in 5% Glycerol Solution. S. SUMIDA (Department of Car-
diovascular Surgery and Medical Low Temperature Unit, Jonai 2-2 Fukuoka, 810, Japan). SF-10 solution containing 10% w/v glycerol, 1% w/v glucose, 1% w/v mannitol, and 0.68% w/v NaCl in water was used as a cryoprotective solution for platelets, Platelet concentrate (PC) was mixed well with the same volume of SF-10 in 3 min at room temperature. Usually, a unit of PC has 40 ml when produced from 500 ml of ACD whole blood. PC was introduced into a PVC bag containing 30 ml of ACD solution and mixed well. Totally 110 ml (40 ml PC, 30 ml ACD, and 40 ml SF-IO) of glycerolized PC was spun at 4000 rpm and the supernatant discarded. The glycerolized PC (final concentration 40 ml) is trans-
MEETING
ferred into a FBS freezing bag and cooled at 1 or 5”C/ min to -80°C and then immersed into liquid nitrogen. Thawing was done at 40°C in a water bath. One hundred milliliters of 0.9% saline was introduced into the bag to dilute thawed glycerolized PC. Diluted thawed glycerolized PC was transferred into an empty bag for centrifugation at 4000 rpm at +21”C. The supematant was discarded and the sediment platelets were resuspended in 40 ml of 0.9% saline. The thawed deglycerolized PC was transfused into the patients by use of a 200~pm filter. No side effect was seen. The in viva recovery of thawed platelets was 60% or more in comparison with 70% or more of the fresh platelets. The life span of thawed platelets was a little shorter than that of fresh platelets, and longer than that of platelets preserved for 24 hr at 5 or 22°C. The prolonged bleeding times by ingestion of aspirin (0.6- 1.O g) measured by Ivy and Duke methods were remarkably shortened by the administration of thawed PC. All of these in viva experiments were undertaken by autologous transfusion into the original donors. Clinically, aplastic anemia, idiophatic thrombocytopenic purpura, acute myelogenous leukemia, and shock were the diseases for which thawed PC were infused. Most cases showed dramatic improvement in platelet counts and bleeding tendency. 12. Ultrastructure
and Aggregation of Platelets Incubated in Glycerol. W. J. ARMITAGE AND C. J.
HUNT (MRC Medical Cryobiology Group, University Department of Surgery, Cambridge, United Kingdom). It has been reported previously that incubation of platelets in 1 mol/liter glycerol at 37°C initially depresses the active uptake of serotonin, but after 30 min in glycerol, this plasma membrane function returns to control levels (Armitage, Cryobiology 16, 601, (1979)). It was found, however, that 1 mohliter glycerol had the opposite effect on the aggregation response to ADP; with increasing incubation time in glycerol at 37°C the aggregation response decreased. Thirty minutes in 1 mol/liter glycerol reduced the aggregation response (measured photometrically) by a third. Presumably, as glycerol moved into the platelets, it started to interfere with one or more of the intracellular events involved in aggregation. Despite these functional changes, the osmotic effects of rapid addition of glycerol did not appear to cause any irreversible morphological changes. After 30 min in 1 mol/liter glycerol, the ultrastructure was similar to freshly isolated platelets. The circumferential microtubules were intact and the discoid form maintained, though some platelets had extended pseudopods. There was no indication that the platelets had undergone any internal transformation such as the centripetal movement of organelles. The discoid form is an energydependent state, therefore, the inhibition due to
ABSTRACTS,
17th ANNUAL
MEETING
597
subjected to preprogrammed linear cooling and warming rates and minimum holding temperatures. The cooling rates ranged from 2 to 10 Wmin, the warming rates were all at 120 K/min, the holding tem13. Cryopreservation of Cytotoxic T-Cell Clones and perature was -2l”C, and extracellular supercooling HARALD VON their Targets. S. P. LEIBO, ranged from -2K to -8.5K. The freeze/thaw process BOEHMER, WERNER HAAS, AND HANS HENGARTNER (Biology Division, Oak Ridge National was recorded continuously on videotape, and the apLaboratory, Oak Ridge, Tennessee, and Base1 parent cell volumes were measured from sequential photomicrographs. The volumetric variation was Institute for Immunology, Basel, Switzerland). typified by a progressive volume decrease concomiMany types of lymphocytes may be successfully tant with a decrease in temperature. The minimum preserved by what is referred to as a “standard” normalized cell volumes achieved ranged from 25 to freezing method. This consists of suspending the cells in -7-10% dimethyl sulfoxide (Me,SO), cooling at 50%. During thawing the cell volumes increased more rapidly than would be predicted by equilibrium condi-l”C/min, storing at - 196”C, thawing rapidly, and tions for an ideal osmometer. This behavior correthen diluting the cells in a single step out of the sponds to that observed earlier for yeast cells Me,SO. There are some cell types, however, for which this standard procedure yields few if any sur- (Cryoletters 1, 129-134 (1980)) and HeLa cells (J. Biomech. Engr. 102, 91-97 (1980)). Upon the comvivors. For example, only -1% of clones of H2pletion of thawing the cells swelled to volumes much restricted cytotoxic T lymphocytes (CTL) survive larger than their original value and burst. when frozen in that way. To develop a preservation procedure for these CTL, the freezing sensitivity of Indicator 15. Droplet Freezing of Antibody-Coupled three T-cell clones has now been studied: B6.1 specific Sheep Red Cells. D. E. PEGG, M. P. DIAPER, S. for H-Y antigen with H-2D”; C.SP.2 specific for the YEO, AND R. R. A. COOMBS (MRC Medical hapten [3+-sulfophenyldiazo)-4-hydroxylphenyl aceCryobiology Group, Department of Surgery and tic acid] and H-2K”; CB.FL 1.1 specific for the hapten Department of Immunology, Cambridge Unifluorescein and H-2K”. By examining the sensitivity of versity, United Kingdom). these CTL to dilution out of Me,SO, and to various Attempts to preserve sheep red cells that were trypcooling and warming rates, a procedure for their pressin treated and coupled with various y-globulins using ervation has been developed. The “optimum” procethe technique developed for native human red cells, A. dure consists of suspending the cells in 2 M Me,SO, cooling them at -l”C/min to -70°C or below, storing W. Rowe, and F. H. Allen (Transfusion 5, 379 (1965)) them at - 196”C, thawing them at -5 to ZXimin, and and A. W. Rowe, H. Borel, and F. H. Allen (VOX Sanguinis 22, 188 (1972)) gave very poor recovery diluting the thawed cells stepwise at room temperature or above. Conditions different from these yield many rates. Preliminary experiments to determine the relative importance of sucrose, glucose, sodium chloride, fewer survivors. Although only -15% of CTL ultiand a colloid (HES) in determining survival of mately survive when frozen under optimum conditions, those cells that do survive exhibit the same spe- trypsin-treated sheep red cells after droplet freezing, showed that the addition of sucrose or HES or both to cific and nonspecific cytotoxicity as unfrozen cells, and they grow normally in culture. In contrast to the saline greatly improved recovery whereas the additional inclusion of glucose was deterimental. It was extreme sensitivity of CTL, lymphoblasts, coupled with appropriate haptens to serve as target cells, can found that when the concentrations of sucrose and salt be easily frozen by the “two-step” procedure of Far- were varied, peak recovery occurred at a distinct concentration of each, and it was possible to determine an rantet al. (Nature (London) 249,452, (1974.)) No CTL optimal concentration of 5% sucrose and 0.45% survive when frozen by the two-step procedure. sodium chloride. Increasing the colloid concentration from 0 to 10% produced a corresponding small im14. Volumetric Response of Human Lymphocytes to provement in recovery, and increasing the temperaFreezing and Thawing. GARY .I. SCHWARTZ AND KENNETH R. DILLER (Biomedical Engi- ture of the thawing solution from 20 to 60°C had a neering Center, Department of Mechanical En- similar additive effect. Using a freezing solution containing 5% sucrose, 0.45% sodium chloride, and 10% gineering The University of Texas, Austin, HES or Dextran 40 it was possible to thaw directly into Texas). phosphate-buffered saline with 80-90% recovery. The volumetric response of individual human lymCryopreserved reagent cells are now being used in phocytes to freezing and thawing has been measured both titration and rosetting tests with completely experimentally on a cryomicroscope. Lymphocytes satisfactory results. separated from fresh whole blood by density gradient centrifugation were suspended in a basal medium in 16. Cryopreservation of Nonspecijic T Suppressor the absence of any cryophylactic agent. The cells were Cells. T. J. MILSON, JR., D. GREIFF, AND R. H. glycerol was probably not associated with a deleterious effect on energy metabolism.
ABSTRACTS, 17th ANNUAL
glycerolization, the glycerol concentration can be reduced in the same manner. The glycerol gradient experienced by the cells can be controlled by adjusting the rate of introduction and the concentration of the glycerol solution. The appropriate gradient, in turn, can be calculated knowing the range of osmotic tolerance of the cells and the rates of influx and efflux of glycerol. Human granulocytes suspended in Hanks’ balanced salt solution have been circulated through the device. Thirty minutes of circulation in the absence of glycerol results in a 30% reduction in cell count but only a slight reduction in the chemotactic function of the remaining cells. Following glycerolization and deglycerolization, the cell recovery is more variable but good recovery of both count and function has been achieved following glycerolization to concentrations exceeding 3000 mOsm. 10. Effects of Glycerol on Structure and Functions of Platelets. TOKIO NEI. (Faculty of Pharmaceutical Sciences, Higashi Nippon Gakuen University, Ishikari-Tobetsu, Hokkaido, 061-02 Japan). Platelets which play a great role in blood coagulation systems possess complicated structures and functions. Although the freeze-preservation of platelets using cryoprotectants such as glycerol and Me,SO has been tried by many investigators, problems still remain to be solved. In the present study, effects of glycerol on morphology and physiology of platelets have been investigated with special reference to Me,SO and glutaraldehyde, a fixative. In results obtained, membrane permeation of glycerol depended upon its concentration. High concentrations of glycerol caused cell swelling and ultrastructural alteration of intact and glutaraldehyde-fixed cell membranes. Aggregation of platelets was inhibited with increased addition of glycerol and glutaraldehyde. There was a marked difference in mode of actions of glycerol and Me,SO to platelets. 11. Clinical
Uses of Platelets Frozen in 5% Glycerol Solution. S. SUMIDA (Department of Car-
diovascular Surgery and Medical Low Temperature Unit, Jonai 2-2 Fukuoka, 810, Japan). SF-10 solution containing 10% w/v glycerol, 1% w/v glucose, 1% w/v mannitol, and 0.68% w/v NaCl in water was used as a cryoprotective solution for platelets, Platelet concentrate (PC) was mixed well with the same volume of SF-10 in 3 min at room temperature. Usually, a unit of PC has 40 ml when produced from 500 ml of ACD whole blood. PC was introduced into a PVC bag containing 30 ml of ACD solution and mixed well. Totally 110 ml (40 ml PC, 30 ml ACD, and 40 ml SF-IO) of glycerolized PC was spun at 4000 rpm and the supernatant discarded. The glycerolized PC (final concentration 40 ml) is trans-
MEETING
ferred into a FBS freezing bag and cooled at 1 or 5”C/ min to -80°C and then immersed into liquid nitrogen. Thawing was done at 40°C in a water bath. One hundred milliliters of 0.9% saline was introduced into the bag to dilute thawed glycerolized PC. Diluted thawed glycerolized PC was transferred into an empty bag for centrifugation at 4000 rpm at +21”C. The supematant was discarded and the sediment platelets were resuspended in 40 ml of 0.9% saline. The thawed deglycerolized PC was transfused into the patients by use of a 200~pm filter. No side effect was seen. The in viva recovery of thawed platelets was 60% or more in comparison with 70% or more of the fresh platelets. The life span of thawed platelets was a little shorter than that of fresh platelets, and longer than that of platelets preserved for 24 hr at 5 or 22°C. The prolonged bleeding times by ingestion of aspirin (0.6- 1.O g) measured by Ivy and Duke methods were remarkably shortened by the administration of thawed PC. All of these in viva experiments were undertaken by autologous transfusion into the original donors. Clinically, aplastic anemia, idiophatic thrombocytopenic purpura, acute myelogenous leukemia, and shock were the diseases for which thawed PC were infused. Most cases showed dramatic improvement in platelet counts and bleeding tendency. 12. Ultrastructure
and Aggregation of Platelets Incubated in Glycerol. W. J. ARMITAGE AND C. J.
HUNT (MRC Medical Cryobiology Group, University Department of Surgery, Cambridge, United Kingdom). It has been reported previously that incubation of platelets in 1 mol/liter glycerol at 37°C initially depresses the active uptake of serotonin, but after 30 min in glycerol, this plasma membrane function returns to control levels (Armitage, Cryobiology 16, 601, (1979)). It was found, however, that 1 mohliter glycerol had the opposite effect on the aggregation response to ADP; with increasing incubation time in glycerol at 37°C the aggregation response decreased. Thirty minutes in 1 mol/liter glycerol reduced the aggregation response (measured photometrically) by a third. Presumably, as glycerol moved into the platelets, it started to interfere with one or more of the intracellular events involved in aggregation. Despite these functional changes, the osmotic effects of rapid addition of glycerol did not appear to cause any irreversible morphological changes. After 30 min in 1 mol/liter glycerol, the ultrastructure was similar to freshly isolated platelets. The circumferential microtubules were intact and the discoid form maintained, though some platelets had extended pseudopods. There was no indication that the platelets had undergone any internal transformation such as the centripetal movement of organelles. The discoid form is an energydependent state, therefore, the inhibition due to
ABSTRACTS,
17th ANNUAL
MEETING
597
subjected to preprogrammed linear cooling and warming rates and minimum holding temperatures. The cooling rates ranged from 2 to 10 Wmin, the warming rates were all at 120 K/min, the holding tem13. Cryopreservation of Cytotoxic T-Cell Clones and perature was -2l”C, and extracellular supercooling HARALD VON their Targets. S. P. LEIBO, ranged from -2K to -8.5K. The freeze/thaw process BOEHMER, WERNER HAAS, AND HANS HENGARTNER (Biology Division, Oak Ridge National was recorded continuously on videotape, and the apLaboratory, Oak Ridge, Tennessee, and Base1 parent cell volumes were measured from sequential photomicrographs. The volumetric variation was Institute for Immunology, Basel, Switzerland). typified by a progressive volume decrease concomiMany types of lymphocytes may be successfully tant with a decrease in temperature. The minimum preserved by what is referred to as a “standard” normalized cell volumes achieved ranged from 25 to freezing method. This consists of suspending the cells in -7-10% dimethyl sulfoxide (Me,SO), cooling at 50%. During thawing the cell volumes increased more rapidly than would be predicted by equilibrium condi-l”C/min, storing at - 196”C, thawing rapidly, and tions for an ideal osmometer. This behavior correthen diluting the cells in a single step out of the sponds to that observed earlier for yeast cells Me,SO. There are some cell types, however, for which this standard procedure yields few if any sur- (Cryoletters 1, 129-134 (1980)) and HeLa cells (J. Biomech. Engr. 102, 91-97 (1980)). Upon the comvivors. For example, only -1% of clones of H2pletion of thawing the cells swelled to volumes much restricted cytotoxic T lymphocytes (CTL) survive larger than their original value and burst. when frozen in that way. To develop a preservation procedure for these CTL, the freezing sensitivity of Indicator 15. Droplet Freezing of Antibody-Coupled three T-cell clones has now been studied: B6.1 specific Sheep Red Cells. D. E. PEGG, M. P. DIAPER, S. for H-Y antigen with H-2D”; C.SP.2 specific for the YEO, AND R. R. A. COOMBS (MRC Medical hapten [3+-sulfophenyldiazo)-4-hydroxylphenyl aceCryobiology Group, Department of Surgery and tic acid] and H-2K”; CB.FL 1.1 specific for the hapten Department of Immunology, Cambridge Unifluorescein and H-2K”. By examining the sensitivity of versity, United Kingdom). these CTL to dilution out of Me,SO, and to various Attempts to preserve sheep red cells that were trypcooling and warming rates, a procedure for their pressin treated and coupled with various y-globulins using ervation has been developed. The “optimum” procethe technique developed for native human red cells, A. dure consists of suspending the cells in 2 M Me,SO, cooling them at -l”C/min to -70°C or below, storing W. Rowe, and F. H. Allen (Transfusion 5, 379 (1965)) them at - 196”C, thawing them at -5 to ZXimin, and and A. W. Rowe, H. Borel, and F. H. Allen (VOX Sanguinis 22, 188 (1972)) gave very poor recovery diluting the thawed cells stepwise at room temperature or above. Conditions different from these yield many rates. Preliminary experiments to determine the relative importance of sucrose, glucose, sodium chloride, fewer survivors. Although only -15% of CTL ultiand a colloid (HES) in determining survival of mately survive when frozen under optimum conditions, those cells that do survive exhibit the same spe- trypsin-treated sheep red cells after droplet freezing, showed that the addition of sucrose or HES or both to cific and nonspecific cytotoxicity as unfrozen cells, and they grow normally in culture. In contrast to the saline greatly improved recovery whereas the additional inclusion of glucose was deterimental. It was extreme sensitivity of CTL, lymphoblasts, coupled with appropriate haptens to serve as target cells, can found that when the concentrations of sucrose and salt be easily frozen by the “two-step” procedure of Far- were varied, peak recovery occurred at a distinct concentration of each, and it was possible to determine an rantet al. (Nature (London) 249,452, (1974.)) No CTL optimal concentration of 5% sucrose and 0.45% survive when frozen by the two-step procedure. sodium chloride. Increasing the colloid concentration from 0 to 10% produced a corresponding small im14. Volumetric Response of Human Lymphocytes to provement in recovery, and increasing the temperaFreezing and Thawing. GARY .I. SCHWARTZ AND KENNETH R. DILLER (Biomedical Engi- ture of the thawing solution from 20 to 60°C had a neering Center, Department of Mechanical En- similar additive effect. Using a freezing solution containing 5% sucrose, 0.45% sodium chloride, and 10% gineering The University of Texas, Austin, HES or Dextran 40 it was possible to thaw directly into Texas). phosphate-buffered saline with 80-90% recovery. The volumetric response of individual human lymCryopreserved reagent cells are now being used in phocytes to freezing and thawing has been measured both titration and rosetting tests with completely experimentally on a cryomicroscope. Lymphocytes satisfactory results. separated from fresh whole blood by density gradient centrifugation were suspended in a basal medium in 16. Cryopreservation of Nonspecijic T Suppressor the absence of any cryophylactic agent. The cells were Cells. T. J. MILSON, JR., D. GREIFF, AND R. H. glycerol was probably not associated with a deleterious effect on energy metabolism.