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Platelets and propylene glycol I. Permeability parameters
552
ABSTRACTS,
23rd ANNUAL
ditions is 3.0%. Me,SO, glycerol, and hydroxyethyl starch (HES) were also tested for bleb formation and survival under these FT conditions. While the FT survival was high (60, 72, and 84%. respectively) with these three cryoprotective agents, bleb formation was greatly different (17, 34, and 0.4%, respectively) with HES almost completely protecting against bleb formation. A FT cycle in the presence of 0.07 M ethanol resulted in 97% blebs and 0.078% survival. Hence, while there is a general correlation between bleb formation and survival for slow cooling rates in the absence (and also presence) of serum, some of the individual cryoprotective agents show significant deviations from this general relationship. Furthermore, almost complete protection against bleb formation does not result in 100% survival. Hence, other modes of FT damage have to be protected against. One of many other possible modes of FT damage is protein denaturation. Proteins could denature during slow cooling for a number of reasons. High salt concentrations. changes in pH, and even low temperatures could all independently cause protein denaturation or unfolding. When cells are labeled with a membranepermeable fluorescent label [N-( I-pyrene)maleimide] that attaches to sulfhydryl groups, one can estimate if proteins have denatured after a FT cycle by examining either the emission or excitation spectra. The results, using both spectra, show that Me,SO and, to some extent, glycerol, partially protect against “slow cooling” FT-induced protein denaturation, while HES sensitizes. In these experiments, all the cell proteins were labeled and none of the proteins were lost since all of the FT medium was used for the measurement. Experiments with whole cells were also done where the areas under the endothermic protein peaks in the differential scanning calorimeter scans were measured before and after a FT cycle. The difference in area is a measure of any denaturation taking place during the FT cycle. The results again showed that Me,SO provided the best protection against FT-induced protein denaturation, with HES providing the worst. Hence, experiments with this cell line show that cryoprotective agents protect by at least two mechanisms. Me,SO and glycerol protect against both total cell protein denaturation and bleb formation, although not to the same extent. while HES only protects against the latter. While this may imply that protein denaturation is not important, 15% of the HES-protected cells without blebs still die. However, measurements of the activir)~ of the plasmalemma-bound Na+-K’ATPase indicate that HES and Me,SO protect equally well against FT-induced damage to the pump. Hence. HES may only protect the small fraction of proteins on the outer surface of the plasmalemma. Therefore, denaturation experiments with whole cells are in progress with several protein-binding fluorescent labels that are not able to cross the intact plasmalemma. Measurements will indicate how well the cryoprotectants pro-
MEETING
tect against protein denaturdtion on the extracellular side of the plasmalemma. 22. Freeze Tolercrncc, rtnd ICC Formution in Wood Frogs (Rma Sylvatica). JACK R. LAYNE, JR.* AND RICHARD E. LEE, JR.+ (*Ohio University Belmont, 45425 National Road, St. Clairsville, Ohio 43950, and tMiami University, 1601 Peck Boulevard, Hamilton, Ohio 45011. Freeze tolerance and ice formation were examined in a southern Ohio population of Ram sylvaticcr following their emergence from hibernation. Frogs were maintained at 5°C for a minimum of 2 weeks prior to testing. Survival of frogs frozen at - 2.0 to - 3.O”Cfor 48 hr was 95% (19/20); whereas freezing at -5.0 to -6.O”C for 48 hr was lethal to all frogs (016). Plasma glucose levels for frogs frozen at - 2.5 to - 3.O”C fat 48 hr were fourfold higher than values for 5°C acclimated frogs. Calorimetry determinations based solely on equivalent masses of ice indicated that frogs frozen 24 hr or longer had 56% 2 4% (i I SD) of their body water as ice. Moreover, inclusion of the specific heat of the body dry mass increased the body ice content estimates to 65% 2 3%. Ice formation reached a plateau 24 hr after nucleation and one-half the equilibrium ice content was obtained 6 hr after nucleation. Previous work on freeze tolerance in R. sylwfic,cr has focused only on animals from northern latitudes and tested prior to emergence from hibernation. Prolonged freezing may occur following the emergence of R. sx/wticu in the late winter and early spring, and it thus may be adaptive for these frogs to retain freeze tolerance during this time of the year. Secondly, the time course of ice formation for R. sylvatic~ccas first reported here may have important implications for the mechanism of freeze tolerance of this species since cryoprotectant mobilization does not occur prior to nucleation. (The Academic Excellence Fund, Miami University, provided support for this research.) 23.
The Effect of Cryoprofectunts on Ice-Induced Phase Separations in Liposome Memhrane.~. TH.FORSTER.E GRIMM,AND E AURKH (Iwan N. Stranski-Institut fiir Physikalische und Theoretische Chemie, Technische Universitat Berlin, West Berlin, West Germany).
(Paper not presented.) 24. Platelets and Propylene Glycol I. Permeuhility Parameters. F. ARNAUD AND D. E. PEGG (MRC Medical Cryobiology Group, University Department of Surgery, Cambridge, CB2 2AH. United Kingdom). Propylene glycol (PC), has a high glass-forming tendency, which suggests that it may afford better protection of ceils during freezing than existing cryopro-
ABSTRACTS,
23rd ANNUAL
TABLE-Abstract
Hydraulic conductivity L, (cm * seccl . atm-I) Solute permeability oRT (cm . set-I) Reflection coefficient lJ
MEETING
5.53
24
2°C
20°C
37°C
2 x IO-’
8 x IO-’
2.5 x IO-6
6 x IO-’
I.1 x IO-5
5 x IO-5
0.4
-
0.4
0.4
a dissecting microscope with attached video camera, time-date generator and video recorder. The video images were subsequently digitized and analyzed by a custom program which marks the periphery of the tissue, determines the cross-sectional area, and calculates the equivalent spherical volume. Analysis of each image is completed in less than 2 set on an IBM PC AT microcomputer with Oculus video digitizer board. The planimetry of the system was calibrated using the known dimensions of a hemocytometer, showing excellent linearity and reproducibility in determinations of area. In replicate measurements of the cross-sectional area using a calibrated reference, the standard deviation was less than 0.3% over a range from 0.0025 to 0.36 mm*. The system is extremely sensitive to changes in volume, and its accuracy and reproducibility exceeds the requirements for reliable documentation of osmotic responses in tissues. Measurements of the kinetics of osmotic volume changes 25. Measur-emrut of Osmotic Vol~~rnr Chrr~~ges it1 in islets exposed to test solutions of permeable and Tissues. BRETT BAUDIN. RAY V. RAJOTTE. impermeable solutes at 24°C clearly demonstrate the LOCKSLEY E. MCGANN, AND JEAN-MICHEL osmotic responses and illustrate the challenge of modTURC (Departments of Surgery and Pathology. eling water and solute movements in multicompartUniversity of Alberta, and Canadian Red Cross ment systems. (Supported by the Canadian Red Cross Blood Transfusion Service Edmonton, Alberta. Blood Transfusion Service. the Medical Research Council. and the Edmonton Civic Employees ChariCanada). table Assistance Fund.) The osmotic responses of cells are of primary importance to the recovery of cells after cryopreserva- 26. Ostnoiic Wottv Mm,etnent in Is&red Hmwn and tion, particularly during the addition and removal of LOCKSLEY E. Rut Islets of Langerhtrns. cryoprotectant, and during the freezing and thawing MCGANN, BRETT BAUDIN, RAY V. RAJOTTE. phases. The ability to visualize and document the osAND JEAN-MICHEL TURC (Departments of Pdmotic responses of model tissues, such as the Islets of thology and Surgery, University of Alberta, and Langerhans, represent an important step toward the Canadian Red Cross Blood Transfusion Serpreservation of whole organs. A functioning system vice. Edmonton, Alberta. Canada). has been developed to visually monitor the volume of tissue samples exposed to a sequence of osmotic conThe islets of Langerhans were used as a model ditions, optionally videotape the results, and digitally system to study osmotic water movements in tissues. analyze the images to give tissue volumes as a func- Video images recorded as islets were exposed to solution of time. A diffusion chamber, designed for ease of tions of different osmotic pressures at room temperaloading the tissue samples, was fabricated to separate ture, and subsequently analyzed to give a measure of tissue samples from the variable osmotic environment islet volume at I-min time intervals. by a sheet of dialysis tubing, similar to the design by Islets from the pancreas of rats and from humans McGrath. Experimental solutions flowing past the di- responded osmotically when exposed to hypertonic alysis sheet resulted in altered osmotic environments solutions of nonpermeating solutes, and when refor the sample tissue. The chamber was placed under turned to isotonic conditions. Osmotic response is de-
tectants (CPA). Hence, PG has been proposed as a CPA for platelets. Permeability of platelets to PG has been studied to optimize protocols of addition and removal, the aim being the maintenance of platelet volume within specified limits such that osmotic injury will not occur. Low concentrations of PG were added to platelet-rich plasma (PRP), and the change in optical density was used to monitor changes in volume. Time courses recorded at 2, 20. and 37°C were matched with theoretical time courses derived from the equations of Kedem and Katschal sky. See table for permeability parameters deduced. Thus. PG penetrates the cells rapidly and can therefore be added to or removed from platelets more conveniently and with less osmotic risk than glycerol. This implies that it may be possible to achieve higher CPA concentrations than have hitherto been used in platelet cryopreservation.
ABSTRACTS,
23rd ANNUAL
ditions is 3.0%. Me,SO, glycerol, and hydroxyethyl starch (HES) were also tested for bleb formation and survival under these FT conditions. While the FT survival was high (60, 72, and 84%. respectively) with these three cryoprotective agents, bleb formation was greatly different (17, 34, and 0.4%, respectively) with HES almost completely protecting against bleb formation. A FT cycle in the presence of 0.07 M ethanol resulted in 97% blebs and 0.078% survival. Hence, while there is a general correlation between bleb formation and survival for slow cooling rates in the absence (and also presence) of serum, some of the individual cryoprotective agents show significant deviations from this general relationship. Furthermore, almost complete protection against bleb formation does not result in 100% survival. Hence, other modes of FT damage have to be protected against. One of many other possible modes of FT damage is protein denaturation. Proteins could denature during slow cooling for a number of reasons. High salt concentrations. changes in pH, and even low temperatures could all independently cause protein denaturation or unfolding. When cells are labeled with a membranepermeable fluorescent label [N-( I-pyrene)maleimide] that attaches to sulfhydryl groups, one can estimate if proteins have denatured after a FT cycle by examining either the emission or excitation spectra. The results, using both spectra, show that Me,SO and, to some extent, glycerol, partially protect against “slow cooling” FT-induced protein denaturation, while HES sensitizes. In these experiments, all the cell proteins were labeled and none of the proteins were lost since all of the FT medium was used for the measurement. Experiments with whole cells were also done where the areas under the endothermic protein peaks in the differential scanning calorimeter scans were measured before and after a FT cycle. The difference in area is a measure of any denaturation taking place during the FT cycle. The results again showed that Me,SO provided the best protection against FT-induced protein denaturation, with HES providing the worst. Hence, experiments with this cell line show that cryoprotective agents protect by at least two mechanisms. Me,SO and glycerol protect against both total cell protein denaturation and bleb formation, although not to the same extent. while HES only protects against the latter. While this may imply that protein denaturation is not important, 15% of the HES-protected cells without blebs still die. However, measurements of the activir)~ of the plasmalemma-bound Na+-K’ATPase indicate that HES and Me,SO protect equally well against FT-induced damage to the pump. Hence. HES may only protect the small fraction of proteins on the outer surface of the plasmalemma. Therefore, denaturation experiments with whole cells are in progress with several protein-binding fluorescent labels that are not able to cross the intact plasmalemma. Measurements will indicate how well the cryoprotectants pro-
MEETING
tect against protein denaturdtion on the extracellular side of the plasmalemma. 22. Freeze Tolercrncc, rtnd ICC Formution in Wood Frogs (Rma Sylvatica). JACK R. LAYNE, JR.* AND RICHARD E. LEE, JR.+ (*Ohio University Belmont, 45425 National Road, St. Clairsville, Ohio 43950, and tMiami University, 1601 Peck Boulevard, Hamilton, Ohio 45011. Freeze tolerance and ice formation were examined in a southern Ohio population of Ram sylvaticcr following their emergence from hibernation. Frogs were maintained at 5°C for a minimum of 2 weeks prior to testing. Survival of frogs frozen at - 2.0 to - 3.O”Cfor 48 hr was 95% (19/20); whereas freezing at -5.0 to -6.O”C for 48 hr was lethal to all frogs (016). Plasma glucose levels for frogs frozen at - 2.5 to - 3.O”C fat 48 hr were fourfold higher than values for 5°C acclimated frogs. Calorimetry determinations based solely on equivalent masses of ice indicated that frogs frozen 24 hr or longer had 56% 2 4% (i I SD) of their body water as ice. Moreover, inclusion of the specific heat of the body dry mass increased the body ice content estimates to 65% 2 3%. Ice formation reached a plateau 24 hr after nucleation and one-half the equilibrium ice content was obtained 6 hr after nucleation. Previous work on freeze tolerance in R. sylwfic,cr has focused only on animals from northern latitudes and tested prior to emergence from hibernation. Prolonged freezing may occur following the emergence of R. sx/wticu in the late winter and early spring, and it thus may be adaptive for these frogs to retain freeze tolerance during this time of the year. Secondly, the time course of ice formation for R. sylvatic~ccas first reported here may have important implications for the mechanism of freeze tolerance of this species since cryoprotectant mobilization does not occur prior to nucleation. (The Academic Excellence Fund, Miami University, provided support for this research.) 23.
The Effect of Cryoprofectunts on Ice-Induced Phase Separations in Liposome Memhrane.~. TH.FORSTER.E GRIMM,AND E AURKH (Iwan N. Stranski-Institut fiir Physikalische und Theoretische Chemie, Technische Universitat Berlin, West Berlin, West Germany).
(Paper not presented.) 24. Platelets and Propylene Glycol I. Permeuhility Parameters. F. ARNAUD AND D. E. PEGG (MRC Medical Cryobiology Group, University Department of Surgery, Cambridge, CB2 2AH. United Kingdom). Propylene glycol (PC), has a high glass-forming tendency, which suggests that it may afford better protection of ceils during freezing than existing cryopro-
ABSTRACTS,
23rd ANNUAL
TABLE-Abstract
Hydraulic conductivity L, (cm * seccl . atm-I) Solute permeability oRT (cm . set-I) Reflection coefficient lJ
MEETING
5.53
24
2°C
20°C
37°C
2 x IO-’
8 x IO-’
2.5 x IO-6
6 x IO-’
I.1 x IO-5
5 x IO-5
0.4
-
0.4
0.4
a dissecting microscope with attached video camera, time-date generator and video recorder. The video images were subsequently digitized and analyzed by a custom program which marks the periphery of the tissue, determines the cross-sectional area, and calculates the equivalent spherical volume. Analysis of each image is completed in less than 2 set on an IBM PC AT microcomputer with Oculus video digitizer board. The planimetry of the system was calibrated using the known dimensions of a hemocytometer, showing excellent linearity and reproducibility in determinations of area. In replicate measurements of the cross-sectional area using a calibrated reference, the standard deviation was less than 0.3% over a range from 0.0025 to 0.36 mm*. The system is extremely sensitive to changes in volume, and its accuracy and reproducibility exceeds the requirements for reliable documentation of osmotic responses in tissues. Measurements of the kinetics of osmotic volume changes 25. Measur-emrut of Osmotic Vol~~rnr Chrr~~ges it1 in islets exposed to test solutions of permeable and Tissues. BRETT BAUDIN. RAY V. RAJOTTE. impermeable solutes at 24°C clearly demonstrate the LOCKSLEY E. MCGANN, AND JEAN-MICHEL osmotic responses and illustrate the challenge of modTURC (Departments of Surgery and Pathology. eling water and solute movements in multicompartUniversity of Alberta, and Canadian Red Cross ment systems. (Supported by the Canadian Red Cross Blood Transfusion Service Edmonton, Alberta. Blood Transfusion Service. the Medical Research Council. and the Edmonton Civic Employees ChariCanada). table Assistance Fund.) The osmotic responses of cells are of primary importance to the recovery of cells after cryopreserva- 26. Ostnoiic Wottv Mm,etnent in Is&red Hmwn and tion, particularly during the addition and removal of LOCKSLEY E. Rut Islets of Langerhtrns. cryoprotectant, and during the freezing and thawing MCGANN, BRETT BAUDIN, RAY V. RAJOTTE. phases. The ability to visualize and document the osAND JEAN-MICHEL TURC (Departments of Pdmotic responses of model tissues, such as the Islets of thology and Surgery, University of Alberta, and Langerhans, represent an important step toward the Canadian Red Cross Blood Transfusion Serpreservation of whole organs. A functioning system vice. Edmonton, Alberta. Canada). has been developed to visually monitor the volume of tissue samples exposed to a sequence of osmotic conThe islets of Langerhans were used as a model ditions, optionally videotape the results, and digitally system to study osmotic water movements in tissues. analyze the images to give tissue volumes as a func- Video images recorded as islets were exposed to solution of time. A diffusion chamber, designed for ease of tions of different osmotic pressures at room temperaloading the tissue samples, was fabricated to separate ture, and subsequently analyzed to give a measure of tissue samples from the variable osmotic environment islet volume at I-min time intervals. by a sheet of dialysis tubing, similar to the design by Islets from the pancreas of rats and from humans McGrath. Experimental solutions flowing past the di- responded osmotically when exposed to hypertonic alysis sheet resulted in altered osmotic environments solutions of nonpermeating solutes, and when refor the sample tissue. The chamber was placed under turned to isotonic conditions. Osmotic response is de-
tectants (CPA). Hence, PG has been proposed as a CPA for platelets. Permeability of platelets to PG has been studied to optimize protocols of addition and removal, the aim being the maintenance of platelet volume within specified limits such that osmotic injury will not occur. Low concentrations of PG were added to platelet-rich plasma (PRP), and the change in optical density was used to monitor changes in volume. Time courses recorded at 2, 20. and 37°C were matched with theoretical time courses derived from the equations of Kedem and Katschal sky. See table for permeability parameters deduced. Thus. PG penetrates the cells rapidly and can therefore be added to or removed from platelets more conveniently and with less osmotic risk than glycerol. This implies that it may be possible to achieve higher CPA concentrations than have hitherto been used in platelet cryopreservation.