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Toxicity of cryoprotective agents. I. Effects on fetal myocardial cells
ANNUAL
MEETING
6. Influence of Molecular Weight on the Cryoprotective Properties of Polyvinylpyrrolidone (PVP) and Dextran Towards Bacteria and Erythrocytes. M. J. ASHWOOD-SMITH AND CAROL WARBY* (Department of Biology, University of Victoria, Victoria, B. C., Canada). Published molecular weight values for commercially available PVP samples are probably misleading. Our studies with Plasdone C(K30) and 14C-labeled K36PVP indicate a range of molecular weights between 200,000 and 1,000 daltons, values much wider than the published figures. Both samples contained approximately 12% of their total weight as molecules smaller than 12,000 daltons. These new figures were based on gel exclusion chromatography of PVP samples on Sephadex G-100 and G-200 and on dialysis. Molecular weight studies on I’C-labeled K36PVP using density gradient centrifugation on sucrose (5-20%) for 18 hr at 2.5 X 10’ g indicate much lower weights (approximately 14,000 daltons). These results are expected as PVP molecules are nondense and linear. As centrifugation and viscosity measurements have been the basis of most studies on PVP molecular weight, the accepted molecular weight values for PVP are understandably incorrect. However, the gel filtration results are unequivocal. Plasdone C, if dialyzed before use. gave much better protection to Pseudomonas F 8 against freeze-thaw damage than undialyzed. Dextran samples of characterized molecular weight showed no improvement of already good protective properties to Pseudomonas after dialysis. Both bacteria and washed rabbit red cells are protected better by increasing concentrations of either PVP or dextran (5-10%). Increasing molecular weight (10,000 to 500,000 daltons) in the case of dextran (5%) does little to increase protection from freesethaw damage in either bacteria or red cells which remains, under the experimental conditions, essentially steady at about 40%. There is an indication, however, of a slight decrease in effectiveness of dextran samples in the molecular weight range of 20,000-50.000 daltons against freeze-thaw damage in Pseudomonas F 8. (Supported by Canadian Medical Research Council Grant MA4055.) 7. Cryoprotective Potentials of Hydroxyethyl Starch for Nucleated Mammalian Cells. MAXIM D. PERSIDSKY AND MILDRED H. ELLETT* (Institute of Chemical Biology, University of San Francisco, San Francisco, California 94117). Reported effectiveness of hydroxyethyl starch (HES) as a cryoprotective agent in preservation of blood and platelets prompted us to investigate
ABSTRACTS
‘ cc <3 II
its potentials for cryopreservation of nucleated mammalian cells. This high-molecular-weight extracellular additive is nontoxic and completely biodegradable and therefore is acceptable for clinical use. Rat bone marrow cells were used to test HES. For the purpose of comparison PVP and DMSO were also employed as cryopreservatives. Cell viability was assessed by their ability t.o incorporate “C-glycerine, as measured by the radioactive count with liquid scintillation counter. Results indicate that HES affords cryoprotection to the bone marrow cells which is at least as high as PVP but is somewhat lower than DMSO. Details of this investigation will be discussed. (Supported by ONR Contract N00014-68~C-0367.) 8. Toxicity of Cryoprotective Agents. I. Effects on Fetal Myocardial Cells. D. M. ROBINSON AND J. F. S~PSON* (American Red Cross Blood Research Laboratory, 9312 Old Georgetown Road, Bethesda, Maryland 20014). We have prepared primary cultures of contractile myocardial cells from embryonic rats in which individual cells beat spontaneously and regularly for several days. We have exposed these cells to several concentrations of the following potential cryoprotective agents (CPA) : glycerol, ethylene glycol, dimethyl sulfoxide, thiourea, ammonium salts, and the salts of mono-, tri-, and tetramethylamine. Measurements have been made of changes in the rate of contraction and subjective estimates made of changes in contractile force following exposure to the CPA. We have established concentration levels at which beating stops and the upper concentration limits beyond which beating is never resumed on removal of the CPA. On the basis of these observations, glycerol and ethylene glycol are nontoxic at concentrations up to 5 M. All other CPA tested show toxic manifestations at lower concentrations. We shall discuss the nature of these effects as they relate to the choice of a CPA for whole heart preservation at, low temperatures. 9. Toxicity of Cryoprotective Agents. II. Effects on Cardiac Microsomal Adenosine Triphosphatase. D. M. ROBINSON AND P. K. SCHORK* (American Red Cross Blood Research Laboratory, 9312 Old Georgetown Road, Bethesda, Maryland 20014). One way cells avoid freezing injury is by the development of a reversible leak in the plasma membrane, with the inward movement of concentrated external solute, in order to escape the
MEETING
6. Influence of Molecular Weight on the Cryoprotective Properties of Polyvinylpyrrolidone (PVP) and Dextran Towards Bacteria and Erythrocytes. M. J. ASHWOOD-SMITH AND CAROL WARBY* (Department of Biology, University of Victoria, Victoria, B. C., Canada). Published molecular weight values for commercially available PVP samples are probably misleading. Our studies with Plasdone C(K30) and 14C-labeled K36PVP indicate a range of molecular weights between 200,000 and 1,000 daltons, values much wider than the published figures. Both samples contained approximately 12% of their total weight as molecules smaller than 12,000 daltons. These new figures were based on gel exclusion chromatography of PVP samples on Sephadex G-100 and G-200 and on dialysis. Molecular weight studies on I’C-labeled K36PVP using density gradient centrifugation on sucrose (5-20%) for 18 hr at 2.5 X 10’ g indicate much lower weights (approximately 14,000 daltons). These results are expected as PVP molecules are nondense and linear. As centrifugation and viscosity measurements have been the basis of most studies on PVP molecular weight, the accepted molecular weight values for PVP are understandably incorrect. However, the gel filtration results are unequivocal. Plasdone C, if dialyzed before use. gave much better protection to Pseudomonas F 8 against freeze-thaw damage than undialyzed. Dextran samples of characterized molecular weight showed no improvement of already good protective properties to Pseudomonas after dialysis. Both bacteria and washed rabbit red cells are protected better by increasing concentrations of either PVP or dextran (5-10%). Increasing molecular weight (10,000 to 500,000 daltons) in the case of dextran (5%) does little to increase protection from freesethaw damage in either bacteria or red cells which remains, under the experimental conditions, essentially steady at about 40%. There is an indication, however, of a slight decrease in effectiveness of dextran samples in the molecular weight range of 20,000-50.000 daltons against freeze-thaw damage in Pseudomonas F 8. (Supported by Canadian Medical Research Council Grant MA4055.) 7. Cryoprotective Potentials of Hydroxyethyl Starch for Nucleated Mammalian Cells. MAXIM D. PERSIDSKY AND MILDRED H. ELLETT* (Institute of Chemical Biology, University of San Francisco, San Francisco, California 94117). Reported effectiveness of hydroxyethyl starch (HES) as a cryoprotective agent in preservation of blood and platelets prompted us to investigate
ABSTRACTS
‘ cc <3 II
its potentials for cryopreservation of nucleated mammalian cells. This high-molecular-weight extracellular additive is nontoxic and completely biodegradable and therefore is acceptable for clinical use. Rat bone marrow cells were used to test HES. For the purpose of comparison PVP and DMSO were also employed as cryopreservatives. Cell viability was assessed by their ability t.o incorporate “C-glycerine, as measured by the radioactive count with liquid scintillation counter. Results indicate that HES affords cryoprotection to the bone marrow cells which is at least as high as PVP but is somewhat lower than DMSO. Details of this investigation will be discussed. (Supported by ONR Contract N00014-68~C-0367.) 8. Toxicity of Cryoprotective Agents. I. Effects on Fetal Myocardial Cells. D. M. ROBINSON AND J. F. S~PSON* (American Red Cross Blood Research Laboratory, 9312 Old Georgetown Road, Bethesda, Maryland 20014). We have prepared primary cultures of contractile myocardial cells from embryonic rats in which individual cells beat spontaneously and regularly for several days. We have exposed these cells to several concentrations of the following potential cryoprotective agents (CPA) : glycerol, ethylene glycol, dimethyl sulfoxide, thiourea, ammonium salts, and the salts of mono-, tri-, and tetramethylamine. Measurements have been made of changes in the rate of contraction and subjective estimates made of changes in contractile force following exposure to the CPA. We have established concentration levels at which beating stops and the upper concentration limits beyond which beating is never resumed on removal of the CPA. On the basis of these observations, glycerol and ethylene glycol are nontoxic at concentrations up to 5 M. All other CPA tested show toxic manifestations at lower concentrations. We shall discuss the nature of these effects as they relate to the choice of a CPA for whole heart preservation at, low temperatures. 9. Toxicity of Cryoprotective Agents. II. Effects on Cardiac Microsomal Adenosine Triphosphatase. D. M. ROBINSON AND P. K. SCHORK* (American Red Cross Blood Research Laboratory, 9312 Old Georgetown Road, Bethesda, Maryland 20014). One way cells avoid freezing injury is by the development of a reversible leak in the plasma membrane, with the inward movement of concentrated external solute, in order to escape the