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The effect of DMSO on the water permeability and optimal cooling rate of tissue culture cells
ABSTRACTS. vice, University berta, Canada).
of Alberta,
Edmonton,
17th ANNUAL Al-
The decisions to be made before cryopreservation of a given cell or tissue are the nature and concentration of additive, the rates and temperature of addition and dilution of additive, and the rates of cooling and warming. These variables are inherently dependent on the permeability characteristics of the cells to water and to the additive. There is a necessity for easily applicable methods to estimate permeability characteristics to allow calculation of the addition and dilution protocols and the selection of cooling and warming rates. In these studies, volume measurements on human lymphocytes as a function of time following an osmotic change were used to determine permeability coefficients. The methods used in calculation did not assume any solution to the differential equations describing the movement of water and solute, therefore avoided limiting assumptions. These permeability coefficients were compared to values obtained using a mathematical expression of the differential equations. This equation, developed by Johnson and Wilson, involves simplifying assumptions, but the values of the coefficients were comparable to those obtained by the more rigorous method. The Johnson and Wilson method was used because a single experiment measuring volume as a function of time after the addition of or dilution from a solute allows reasonably accurate estimates of the permeability of the cells to water and to the solutes as well as the Staverman reflection coefficient. This simple method based on a straightforward experiment provides estimates of the parameters required to calculate optimal addition and dilution protocols, and to select cooling and warming rates. 59. The Effect of DMSO on the Water Permeability and Optimal Cooling Rate of Tissue Culture Cells. P. LAW, G. S. RULE, J. FRIM, J. R. LEPOCK, AND J. KRUUV (Physics Department, University of Waterloo, Waterloo, Ontario, Canada). The water permeability coefficient, k, of exponential (EP) and plateau phase (PP) V79 Chinese hamster cells in tissue culture was measured in the temperature range of 10 to 30°C. The presence of 0.5 M dimethyl sulfoxide (DMSO) decreased the k value of EP cells by a factor of 2, but had no significant effect on PP cells. Additionally, EP cells had significantly higher k values than PP cells in this temperature range. These results correlate with cooling rate versus survival studies of the same cell line in this laboratory. A decrease in k value in the presence of DMSO in EP cells implies that these cells need more time to shrink in response to the hypertonic solution produced by the freezing process. Indeed, the optimum cooling rate for survival of EP phase cells was about a factor of 2 slower in the presence compared to the absence of 0.5 M DMSO. In the
MEETING
613
case of PP cells, the presence of 0.5 M DMSO did not significantly change the k value or the optimum cooling rate. The water permeability measurements revealed a major discontinuity at about 20°C which coincides with a membrane lipid phase transition in this cell line. Hence, when calculating a temperature coefficient for the k value for possible use in cryobiology, only experimental points below this phase transition should be used. Even then, extrapolation to a frozen system may not be valid. 60. Diffusion of Glycerol into Monolayer and Suspension Cultures of Chinese Hamster Ovary Ceils: A Two-Compartment Analysis. D. DOOLEY (Cryobiology Laboratory, ARC Blood Services Labs, 9312 Old Georgetown Road, Bethesda, Maryland) In a previous study of Chinese hamster ovary (CHO) cells, we found the half-time for the first-order movement of [3H]glycerol across the cell membrane was constant over a broad range of glycerol concentrations. This paper summarizes a more detailed analysis of the mechanism and kinetics of glycerol permeation in CHO cells. Influx was measured after a 30-set exposure of cells to medium containing [3H]glycerol. To quantitate efflux, cells containing [3H]glycerol were transferred to glycerol-free medium, and the rate of outflow was measured after 30 sec. The velocities of entry and exit were directly proportional to the concentration of glycerol. At any given concentration, the rates of entry and exit were virtually identical. The direct proportionality between flux and glycerol concentration extended from 0.1 FM to 200 mM, the highest concentration examined. Within this range, therefore, it did not appear to be possible to saturate the permeation mechanism. Glycerol influx was not affected by membrane alkylation, depletion of ATP, or treatment with Persantine. The cells were not capable of glycerol countertransport. It is concluded that glycerol crosses the membrane by nonmediated diffusion. The kinetics of glycerol eftlux were biphasic for cells growing in monolayer or in suspension. Influx curves were also biphasic but distinctly different from those of efflux. By summing two first-order functions, we could obtain curves closely approximating the observed kinetics, suggesting that glycerol might be diffusing into and out of two intracellular compartments. However, using the calculations -of Kotyk and Janacek, who analyzed the diffusion of solute into compartments arranged in series or in parallel, our experimental data were found not to agree with the predictions of a two-compartment system. The mechanism behind the biphasic influx and efflux curves remains unknown. 61. The Response of Multilamellar Liposomes to Freezing and Thawing. G. J. MORRIS AND S. S. MCGRATH (Culture Centre of Algae and Pro-
of Alberta,
Edmonton,
17th ANNUAL Al-
The decisions to be made before cryopreservation of a given cell or tissue are the nature and concentration of additive, the rates and temperature of addition and dilution of additive, and the rates of cooling and warming. These variables are inherently dependent on the permeability characteristics of the cells to water and to the additive. There is a necessity for easily applicable methods to estimate permeability characteristics to allow calculation of the addition and dilution protocols and the selection of cooling and warming rates. In these studies, volume measurements on human lymphocytes as a function of time following an osmotic change were used to determine permeability coefficients. The methods used in calculation did not assume any solution to the differential equations describing the movement of water and solute, therefore avoided limiting assumptions. These permeability coefficients were compared to values obtained using a mathematical expression of the differential equations. This equation, developed by Johnson and Wilson, involves simplifying assumptions, but the values of the coefficients were comparable to those obtained by the more rigorous method. The Johnson and Wilson method was used because a single experiment measuring volume as a function of time after the addition of or dilution from a solute allows reasonably accurate estimates of the permeability of the cells to water and to the solutes as well as the Staverman reflection coefficient. This simple method based on a straightforward experiment provides estimates of the parameters required to calculate optimal addition and dilution protocols, and to select cooling and warming rates. 59. The Effect of DMSO on the Water Permeability and Optimal Cooling Rate of Tissue Culture Cells. P. LAW, G. S. RULE, J. FRIM, J. R. LEPOCK, AND J. KRUUV (Physics Department, University of Waterloo, Waterloo, Ontario, Canada). The water permeability coefficient, k, of exponential (EP) and plateau phase (PP) V79 Chinese hamster cells in tissue culture was measured in the temperature range of 10 to 30°C. The presence of 0.5 M dimethyl sulfoxide (DMSO) decreased the k value of EP cells by a factor of 2, but had no significant effect on PP cells. Additionally, EP cells had significantly higher k values than PP cells in this temperature range. These results correlate with cooling rate versus survival studies of the same cell line in this laboratory. A decrease in k value in the presence of DMSO in EP cells implies that these cells need more time to shrink in response to the hypertonic solution produced by the freezing process. Indeed, the optimum cooling rate for survival of EP phase cells was about a factor of 2 slower in the presence compared to the absence of 0.5 M DMSO. In the
MEETING
613
case of PP cells, the presence of 0.5 M DMSO did not significantly change the k value or the optimum cooling rate. The water permeability measurements revealed a major discontinuity at about 20°C which coincides with a membrane lipid phase transition in this cell line. Hence, when calculating a temperature coefficient for the k value for possible use in cryobiology, only experimental points below this phase transition should be used. Even then, extrapolation to a frozen system may not be valid. 60. Diffusion of Glycerol into Monolayer and Suspension Cultures of Chinese Hamster Ovary Ceils: A Two-Compartment Analysis. D. DOOLEY (Cryobiology Laboratory, ARC Blood Services Labs, 9312 Old Georgetown Road, Bethesda, Maryland) In a previous study of Chinese hamster ovary (CHO) cells, we found the half-time for the first-order movement of [3H]glycerol across the cell membrane was constant over a broad range of glycerol concentrations. This paper summarizes a more detailed analysis of the mechanism and kinetics of glycerol permeation in CHO cells. Influx was measured after a 30-set exposure of cells to medium containing [3H]glycerol. To quantitate efflux, cells containing [3H]glycerol were transferred to glycerol-free medium, and the rate of outflow was measured after 30 sec. The velocities of entry and exit were directly proportional to the concentration of glycerol. At any given concentration, the rates of entry and exit were virtually identical. The direct proportionality between flux and glycerol concentration extended from 0.1 FM to 200 mM, the highest concentration examined. Within this range, therefore, it did not appear to be possible to saturate the permeation mechanism. Glycerol influx was not affected by membrane alkylation, depletion of ATP, or treatment with Persantine. The cells were not capable of glycerol countertransport. It is concluded that glycerol crosses the membrane by nonmediated diffusion. The kinetics of glycerol eftlux were biphasic for cells growing in monolayer or in suspension. Influx curves were also biphasic but distinctly different from those of efflux. By summing two first-order functions, we could obtain curves closely approximating the observed kinetics, suggesting that glycerol might be diffusing into and out of two intracellular compartments. However, using the calculations -of Kotyk and Janacek, who analyzed the diffusion of solute into compartments arranged in series or in parallel, our experimental data were found not to agree with the predictions of a two-compartment system. The mechanism behind the biphasic influx and efflux curves remains unknown. 61. The Response of Multilamellar Liposomes to Freezing and Thawing. G. J. MORRIS AND S. S. MCGRATH (Culture Centre of Algae and Pro-