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The effect of ion concentration during exposure to suboptimal temperatures on survival of mammalian cells
ABSTRACTS,
582
12TH ANNUAL
We have previously shown (Fourteenth Annual Meeting AACB, p, 229a ( 1974) ) by scanning electron microscopy that dramatic changes occur at the surface of Chinese hamster (CHO) cells following their exposure to media made hyperosmola with NaCI. We have now explored the surface of cells exposed to hyperosmolal media containing various concentrations of dimethyl sulfoxide (DMSO), Identical observations were also made on monolayers treated with Pronase or neuraminidase. The results indicate that the surface projections of CHO cells, e.g., blebs, microvilli and ruffles, are entirely preserved when DMSO is incorporated into hyperosmolal media at 5 and 10 g%. Lesions similar to those induced by hyperosmolal exposure could be created by treatment with pronase. Under our experimental conditions, neuraminidase did not induce any detectable alterations of the surface projections, indicating that removal only of sialic acid moieties from surface glycoprotein is not sufficient to produce changes in cell surface morphology. 85. Hyperosmotb
Xnjury in Chinese Hamster Cells. III. Electronically Eiecorded Cell Size Changes after Exposure to Hyperosmokn with 07 without Cryoprotectants. Media
J. F. SIMPSON,’ ANI> S. MIRONESCU.* (American NationaI Bed Cross, Blood Research Laboratory, 9312 OId Georgetown Road, Bethesda, Maryland 20014). Introduced by H. T. Meryman. In order to explore the reIationship between cell injury and cell volume during exposure to hyperosmolality, celI volume was monitored in the Coulter Channelyzer at various times after exposing freshly trypsinized suspensions of Chinese hamster (CHO) cells to hyperosmolal media. On transfer to the hyperosmolal medium there is an instantaneous voIume reduction proportional to the osmolality of the medium. This is followed by a gradua1 cell swelling indicative of solute influx. The rate of swelling is proportional to the osmolahty of the medium. At 6000 IIIOSM, after the initial voIume reduction, cell swelling is very rapid and in seven minutes there is little difference between experimenta and isotonic control samples. With prolonged exposure, an increasing number of cells exceed isotonic voIume. The survival of ceils following hyperosmolal exposure appears to be related to the extent of cell swelling during the exposure. Cells that have not swelled survive while those that have do not, The biphasic survival curve reported for unprotected cells at 3000 mOSM is paralleled by volume changes, These cells first swell, then reduce in volume implying active ion pumping,
MEETING
then swell once more. The presence of dimcthyl sulfoxide (DMSO) in ,the hyperosmotic medium delays cell swelling. The effect of DMSO on volume changes of CHO cells can be interpreted as a membrane-stabilizing effect which helps to maintain the normal low permeability of the membrane. 86. The E%ect of Ian Concenimtion during Exposure to Suboptimal Temperatures on Survival of Mammalian Cells. G. P. RAAPHORST,* J, FRIM, L. E. MCGANN, AND J. KRUW. (Physics Department, University of Waterloo, Waterloo, Ontario, Canada). Since various saIts and pH can alter the phase transition temperatures and the state of fluidity of lipids normally found in biological membranes, the effect of salts and pH on survival of cells exposed to freeze-thaw damage or to thermal shock was investigated. Chinese hamster cells (Vi’9 ), suspended in various unbuffered salt solutions for 20 min at 22°C were either frozen to -196°C or exposed to 0°C for 10 min. Cells held at 22°C for 20 min in these solutions showed no significant changes in survival in the range of osmolalities studied (0.1-2.0 OSM). However, cells exposed to either 0 or -196°C had a distinct maximum in survival as a function of osmolality. This maximum occured at the same osmoIaIity for cells exposed to either 0 or -196°C in any one salt solution, However, the osmolalities of the survival maxima were different for NaCl, KCI, LiCl and CaCll solutions used. Cell v&me measurements revealed that the cells at the optimum osmolality in the various solutions were not of equal size. Furthermore, the absolute survival of the cells after exposure to -196°C was significantly higher in the monovalent salt solutions than in the CaCI, solution. The pH at the time of freezing also greatly affected the subsequent survival. The results will be discussed in terms of the effects of the salts and the pH on membrane lipids and on intracellular water structure. 87. A Model
System for Freeze-Thaw Damage: Mammalian Plateau Phase Cells. J, FRIM
AND J. KRUW. (Physics Department University of Waterloo, Waterloo, Ontario, Canada ) .
Most cells in organized tissue have intimate contact with other cells and are in a nonproliferating phase (C,) of the cell cycle. Both of these factors can influence the survival to freeze-thaw damage, However, the assessment of this damage in organized tissue is relativeIy difficult and unreIiabIe. Plateau phase celIs in tissue
582
12TH ANNUAL
We have previously shown (Fourteenth Annual Meeting AACB, p, 229a ( 1974) ) by scanning electron microscopy that dramatic changes occur at the surface of Chinese hamster (CHO) cells following their exposure to media made hyperosmola with NaCI. We have now explored the surface of cells exposed to hyperosmolal media containing various concentrations of dimethyl sulfoxide (DMSO), Identical observations were also made on monolayers treated with Pronase or neuraminidase. The results indicate that the surface projections of CHO cells, e.g., blebs, microvilli and ruffles, are entirely preserved when DMSO is incorporated into hyperosmolal media at 5 and 10 g%. Lesions similar to those induced by hyperosmolal exposure could be created by treatment with pronase. Under our experimental conditions, neuraminidase did not induce any detectable alterations of the surface projections, indicating that removal only of sialic acid moieties from surface glycoprotein is not sufficient to produce changes in cell surface morphology. 85. Hyperosmotb
Xnjury in Chinese Hamster Cells. III. Electronically Eiecorded Cell Size Changes after Exposure to Hyperosmokn with 07 without Cryoprotectants. Media
J. F. SIMPSON,’ ANI> S. MIRONESCU.* (American NationaI Bed Cross, Blood Research Laboratory, 9312 OId Georgetown Road, Bethesda, Maryland 20014). Introduced by H. T. Meryman. In order to explore the reIationship between cell injury and cell volume during exposure to hyperosmolality, celI volume was monitored in the Coulter Channelyzer at various times after exposing freshly trypsinized suspensions of Chinese hamster (CHO) cells to hyperosmolal media. On transfer to the hyperosmolal medium there is an instantaneous voIume reduction proportional to the osmolality of the medium. This is followed by a gradua1 cell swelling indicative of solute influx. The rate of swelling is proportional to the osmolahty of the medium. At 6000 IIIOSM, after the initial voIume reduction, cell swelling is very rapid and in seven minutes there is little difference between experimenta and isotonic control samples. With prolonged exposure, an increasing number of cells exceed isotonic voIume. The survival of ceils following hyperosmolal exposure appears to be related to the extent of cell swelling during the exposure. Cells that have not swelled survive while those that have do not, The biphasic survival curve reported for unprotected cells at 3000 mOSM is paralleled by volume changes, These cells first swell, then reduce in volume implying active ion pumping,
MEETING
then swell once more. The presence of dimcthyl sulfoxide (DMSO) in ,the hyperosmotic medium delays cell swelling. The effect of DMSO on volume changes of CHO cells can be interpreted as a membrane-stabilizing effect which helps to maintain the normal low permeability of the membrane. 86. The E%ect of Ian Concenimtion during Exposure to Suboptimal Temperatures on Survival of Mammalian Cells. G. P. RAAPHORST,* J, FRIM, L. E. MCGANN, AND J. KRUW. (Physics Department, University of Waterloo, Waterloo, Ontario, Canada). Since various saIts and pH can alter the phase transition temperatures and the state of fluidity of lipids normally found in biological membranes, the effect of salts and pH on survival of cells exposed to freeze-thaw damage or to thermal shock was investigated. Chinese hamster cells (Vi’9 ), suspended in various unbuffered salt solutions for 20 min at 22°C were either frozen to -196°C or exposed to 0°C for 10 min. Cells held at 22°C for 20 min in these solutions showed no significant changes in survival in the range of osmolalities studied (0.1-2.0 OSM). However, cells exposed to either 0 or -196°C had a distinct maximum in survival as a function of osmolality. This maximum occured at the same osmoIaIity for cells exposed to either 0 or -196°C in any one salt solution, However, the osmolalities of the survival maxima were different for NaCl, KCI, LiCl and CaCll solutions used. Cell v&me measurements revealed that the cells at the optimum osmolality in the various solutions were not of equal size. Furthermore, the absolute survival of the cells after exposure to -196°C was significantly higher in the monovalent salt solutions than in the CaCI, solution. The pH at the time of freezing also greatly affected the subsequent survival. The results will be discussed in terms of the effects of the salts and the pH on membrane lipids and on intracellular water structure. 87. A Model
System for Freeze-Thaw Damage: Mammalian Plateau Phase Cells. J, FRIM
AND J. KRUW. (Physics Department University of Waterloo, Waterloo, Ontario, Canada ) .
Most cells in organized tissue have intimate contact with other cells and are in a nonproliferating phase (C,) of the cell cycle. Both of these factors can influence the survival to freeze-thaw damage, However, the assessment of this damage in organized tissue is relativeIy difficult and unreIiabIe. Plateau phase celIs in tissue