- Email: [email protected]
41. Regulatory volume decrease in COS-7 cells at 22 °C and it’s influence on the determination of the osmotically inactive volume Vb
374
Abstracts / Cryobiology 61 (2010) 362–408
cytoplasmic structures present in the parasite, which is sequestered inside a red blood cell. In all the techniques used parasite membranes are visible though they vary in size from 6 nm thick to 14 nm thick for room temperature section. Aggregation artefacts are also apparent in the nuclear regions of parasites resulting in a large grained uneven texture, in freeze substitution, room temperature and Tokuyasu rather that the homogeneous fine grain seen in CEMOVIS sections. Conflict of interest: None declared. Source of funding: Internal NIBSC funding. doi:10.1016/j.cryobiol.2010.10.043
40. Rectification of the water permeability in COS-7 cells at 22 °C, 10 °C, and 0 °C. Diana B. Peckys * 1,2, Peter Mazur 1, 1 Dept. of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, TN 37996-0840, United States, 2 Center for Environmental Biotechnology, The University of Tennessee, Knoxville, TN 379961605, United States Knowledge of the permeability of cells to water is crucial for the development of effective cryopreservation protocols. Here, we report the hydraulic conductivity (Lp) of the COS-7 cell line to both exosmosis in hypertonic sucrose/Tyrodes and endosmosis in hypotonic Tyrodes at 22 °C, 10 °C, and 0 °C. The value of Lp during swelling was several-fold higher than that during shrinking – a phenomenon referred to as rectification. COS-7 are large (20 lm) cells derived from green African monkey kidney fibroblasts and are broadly used in cell biology. Lp’s were determined by the classical method in which cells are abruptly placed in solutions of nonpermeating solutes of known osmolality and their rates of shrinkage or swelling imaged under the microscope with time. Measurements at 22 °C were made using a Microcell slide. Measurements at 10 °C and 0 °C were made with our Linkam cryostage. Lp is a major parameter in the thermodynamic equation for water permeability. We estimated it by determining the value of Lp that provided the best fit between experimental and theoretical shrink swell curves. For the determination of water permeability (Lp), we performed swell/shrink experiments with two different anisotonic solutions (157 and 602 mOsm/kg) at three different temperatures (0 °C, 10 °C and 22 °C). The time-dependent volume changes of 358 single cells were measured and analyzed. The cell suspension was mixed 1:4 with the anisotonic solution, yielding the desired final osmolality. Images of the cells were recorded digitally at 10-s intervals for the first 5 min and followed at 1-min intervals over the ensuing 5 min. The lag time between initial mixing of the cell suspension and the acquisition of the first image was 20–35 s. Measured diameters were converted into volumes and the calculated volumes of the individual cells were averaged for each recorded point in time and transformed into swell/shrink curves. Average Lp’s for all six combinations of osmolality and temperature were calculated using the volume values of 12–15 time intervals from the dynamic part of each averaged swell/shrink curve and solving the integrated equation for Lp [1]. The Lp’s at 0 °C, 10 °C and 22 °C were 0.31 ± 0.02, 0.50 ± 0.03 and 0.42 ± 0.04, for exosmotic flow and were 1.00 ± 0.07, 2.20 ± 0.09 and 2.08 ± 0.13 lm/min/atm (mean ± SEM) for endosmotic flow. Thus, all endosmotic Lp’s were several fold higher than their exosmotic counterparts (p < 0.05), the ratios being 3.2, 4.4 and 4.9, for 0 °C, 10 °C and 22 °C. We also observed that Lp was affected by temperature, but the effects were smaller and more anomalous than expected. Rectification of water permeability has not been widely reported. Our results, however, show a large (3-fold or so) effect in COS-7 cells. The only other reported comparable effect we know of is in human granulocytes [2]. The mechanism of this rectification in COS-7 is unknown. Also unknown are its implications for cryopreservation. Note that events during cooling and freezing involve exosmosis only. Conflict of interest: None declared. Source of funding: Research supported by NIH Grant R01-RR018470. DP thanks Dr. Shinsuke Seki for training on the Linkam cryostage system.
References [1] D.A.T. Dick, Cell Water, Butterworths, Washington, 1966, p. 87 (Eq. 6.16).. [2] Toupin CJ et al.. Permeability of human granulocytes to water: rectification of osmotic flow. Cryobiology 1989;26(5):431–44. doi:10.1016/j.cryobiol.2010.10.044
41. Regulatory volume decrease in COS-7 cells at 22 °C and it’s influence on the determination of the osmotically inactive volume Vb. Diana Peckys, Peter Mazur *, Fundamental and Applied Cryobiology Group, Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, United States A basic premise in cryobiology and in the underlying osmotic relations of cells is that in solutions of nonpermeating solutes, only water traverses the plasma membrane and it does so at a rate and to an extent determined by passive physical flows.
One consequence of this is the Boyle van’t Hoff plot of cell volume vs. the reciprocal of the osmotic pressure or osmolality of the external medium. In many cells, such a plot yields a straight line and such cells are said to behave as ideal osmometers. An extrapolation of such a line to infinite osmolality yields the quantity Vb, the fractional volume of solids. In a number of cells, however, a cell volume excursion above or below the isotonic volume, engenders an active volume regulatory response that tends to drive the cell volume back towards isotonic. For swollen cells in hypotonic media, the ensuing shrinkage back towards isotonic size is referred to as Regulatory Volume Decrease (RVD). We have observed a strong RVD in COS-7 cells held in half-isotonic Tyrodes (157 mosm) for 30 min. During the first 5 min in that hypotonic medium, the cells, as expected, undergo a passive near doubling in volume, swelling from 3293 lm3 to 5941 lm3. But over the ensuing hour, the volume of the cells decreases to 3534 lm3 after 30 min and 3123 lm3 after 60 min. This RVD does not occur at 0 °C, supporting the view that it is an energetically driven process. Nor does volume regulation occur in COS-7 cells that have undergone passive osmotic shrinkage by being placed in double-strength hypertonic Tyrodes (602 mOsm). That is quite common. An important and unexpected finding was that the existence a large RVD can have a major effect on the slope of the BVH plot and accordingly can exert a major effect on the value of Vb. We exposed the COS-7 cells to 5 anisotonic test solutions ranging from 77 to 1010 mOsm/kg and determined the volumes of some 225–329 cells per test solution after 5 min or 30 min. The mean volumes, normalized to the isotonic volume, were then plotted against the reciprocal of the measured osmolality of the test solutions. The curve for the cells exposed to the solutions for 5 min and that for cells exposed to the same solutions for 30 min yielded two distinctly different Boyle–vanHoff plots The values of Vb were 0.33 and 0.62 respectively. This difference was mainly due to a flattening by a factor of two between the slope after 5 min (0.2057), and the one after 30 min (0.0981), resulting from divergent 5 and 30 min volume data of the farther from isotonicity deviating test solutions (especially, of the hypotonic ones). In contrast, normalized volumes of the cells in the 602 mOsm/ kg test solution were the same after 5 or 30 min, consistent with a lack of RVI in hypertonic solutions. Conflict of interest: None declared. Source of funding: None declared. doi:10.1016/j.cryobiol.2010.10.045
42. Race for the pace: Is the universal cryoprotocol a dream or reality?. Igor. I. Katkov, CELLTRONIX, San Diego, CA, USA With the development of Peter Mazur’s equations and work of other cryobiologists on slow (equilibrium) freezing in 1960s, it became clear that a particular cell would need its own optimal cryopreservation protocol, that would largely depend on the cell cryobiological and physiological parameters as well as on the type of cryoprotective agents (CPAs) used. With advance of fast freezing and vitrification (VF), introduced by Rall and Fahy in 1980s, the focus shifted toward searching for nontoxic cocktails and decreasing concentration of the vitrificants by increasing rates of cooling and warming. Yet, the protocols of this type of VF are largely customized to the cell type due to difference in the cell osmotic fragility and chemical tolerance to the vitrificants. The universal cryoprotocol, that would fit ALL types of cells, at least if they are in suspension on make a thin layer, would be the Holy Grail of Cryobiology. Here is my hypothesis for your consideration: 1. Every cell has it own critical rate of cooling and thawing, at which rate and at HIGHER rates the cell can be vitrified during cooling (B_cr_cool) and will not devitrify during warming (B_cr_warm) WITHOUT any external ‘‘cryoprotectants” (they must be called ‘‘vitrificants” in this case). OR, it might be just that non-lethal ice (i.e., cubical vs. hexagonal ‘‘killer ice”) is formed during cooling and its transformation (recrystallization) to hexagonal type is precluded during warming. In any case, at rates higher than those two B_cr’s, the cell will survive without any exogenous compounds. 2. Those rates are substantially LOWER than predicted by the most common theories (Rall and Fahy, Boutron’s work, Cravalho’s school: Toner, Karlsson, et al.). I will not go into the details of the thermodynamics of the glassy state but the main reasons are three: (A) cell internal vitrificants; (B) small compartmentalized intracellular milieu; and (C) no ‘‘true” VF is needed for survival. In any scenario, the cell SURVIVES if the pace of cooling and warming is higher than those two B-cr’s, and that is what matters! 3. The distribution and average values of those B-cr’s depend on the species of cells, particularly on the abundance of endogenous vitrificants, how ‘‘watery” those cells are, the level of compartmentalization, the size of the compartments, etc. It may well be that the same species (such embryos) might have VERY different B-cr’s at different stages of their development. 4. I predict that while those speeds are relatively high for the most of the cell species (in range 20,000–1,000,000 C/min), we have already achieved those critical rates in one well documented case, namely human sperm, thanks to the early work in 1930s and in this century by the Isachenkos.
Abstracts / Cryobiology 61 (2010) 362–408
cytoplasmic structures present in the parasite, which is sequestered inside a red blood cell. In all the techniques used parasite membranes are visible though they vary in size from 6 nm thick to 14 nm thick for room temperature section. Aggregation artefacts are also apparent in the nuclear regions of parasites resulting in a large grained uneven texture, in freeze substitution, room temperature and Tokuyasu rather that the homogeneous fine grain seen in CEMOVIS sections. Conflict of interest: None declared. Source of funding: Internal NIBSC funding. doi:10.1016/j.cryobiol.2010.10.043
40. Rectification of the water permeability in COS-7 cells at 22 °C, 10 °C, and 0 °C. Diana B. Peckys * 1,2, Peter Mazur 1, 1 Dept. of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, TN 37996-0840, United States, 2 Center for Environmental Biotechnology, The University of Tennessee, Knoxville, TN 379961605, United States Knowledge of the permeability of cells to water is crucial for the development of effective cryopreservation protocols. Here, we report the hydraulic conductivity (Lp) of the COS-7 cell line to both exosmosis in hypertonic sucrose/Tyrodes and endosmosis in hypotonic Tyrodes at 22 °C, 10 °C, and 0 °C. The value of Lp during swelling was several-fold higher than that during shrinking – a phenomenon referred to as rectification. COS-7 are large (20 lm) cells derived from green African monkey kidney fibroblasts and are broadly used in cell biology. Lp’s were determined by the classical method in which cells are abruptly placed in solutions of nonpermeating solutes of known osmolality and their rates of shrinkage or swelling imaged under the microscope with time. Measurements at 22 °C were made using a Microcell slide. Measurements at 10 °C and 0 °C were made with our Linkam cryostage. Lp is a major parameter in the thermodynamic equation for water permeability. We estimated it by determining the value of Lp that provided the best fit between experimental and theoretical shrink swell curves. For the determination of water permeability (Lp), we performed swell/shrink experiments with two different anisotonic solutions (157 and 602 mOsm/kg) at three different temperatures (0 °C, 10 °C and 22 °C). The time-dependent volume changes of 358 single cells were measured and analyzed. The cell suspension was mixed 1:4 with the anisotonic solution, yielding the desired final osmolality. Images of the cells were recorded digitally at 10-s intervals for the first 5 min and followed at 1-min intervals over the ensuing 5 min. The lag time between initial mixing of the cell suspension and the acquisition of the first image was 20–35 s. Measured diameters were converted into volumes and the calculated volumes of the individual cells were averaged for each recorded point in time and transformed into swell/shrink curves. Average Lp’s for all six combinations of osmolality and temperature were calculated using the volume values of 12–15 time intervals from the dynamic part of each averaged swell/shrink curve and solving the integrated equation for Lp [1]. The Lp’s at 0 °C, 10 °C and 22 °C were 0.31 ± 0.02, 0.50 ± 0.03 and 0.42 ± 0.04, for exosmotic flow and were 1.00 ± 0.07, 2.20 ± 0.09 and 2.08 ± 0.13 lm/min/atm (mean ± SEM) for endosmotic flow. Thus, all endosmotic Lp’s were several fold higher than their exosmotic counterparts (p < 0.05), the ratios being 3.2, 4.4 and 4.9, for 0 °C, 10 °C and 22 °C. We also observed that Lp was affected by temperature, but the effects were smaller and more anomalous than expected. Rectification of water permeability has not been widely reported. Our results, however, show a large (3-fold or so) effect in COS-7 cells. The only other reported comparable effect we know of is in human granulocytes [2]. The mechanism of this rectification in COS-7 is unknown. Also unknown are its implications for cryopreservation. Note that events during cooling and freezing involve exosmosis only. Conflict of interest: None declared. Source of funding: Research supported by NIH Grant R01-RR018470. DP thanks Dr. Shinsuke Seki for training on the Linkam cryostage system.
References [1] D.A.T. Dick, Cell Water, Butterworths, Washington, 1966, p. 87 (Eq. 6.16).. [2] Toupin CJ et al.. Permeability of human granulocytes to water: rectification of osmotic flow. Cryobiology 1989;26(5):431–44. doi:10.1016/j.cryobiol.2010.10.044
41. Regulatory volume decrease in COS-7 cells at 22 °C and it’s influence on the determination of the osmotically inactive volume Vb. Diana Peckys, Peter Mazur *, Fundamental and Applied Cryobiology Group, Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, United States A basic premise in cryobiology and in the underlying osmotic relations of cells is that in solutions of nonpermeating solutes, only water traverses the plasma membrane and it does so at a rate and to an extent determined by passive physical flows.
One consequence of this is the Boyle van’t Hoff plot of cell volume vs. the reciprocal of the osmotic pressure or osmolality of the external medium. In many cells, such a plot yields a straight line and such cells are said to behave as ideal osmometers. An extrapolation of such a line to infinite osmolality yields the quantity Vb, the fractional volume of solids. In a number of cells, however, a cell volume excursion above or below the isotonic volume, engenders an active volume regulatory response that tends to drive the cell volume back towards isotonic. For swollen cells in hypotonic media, the ensuing shrinkage back towards isotonic size is referred to as Regulatory Volume Decrease (RVD). We have observed a strong RVD in COS-7 cells held in half-isotonic Tyrodes (157 mosm) for 30 min. During the first 5 min in that hypotonic medium, the cells, as expected, undergo a passive near doubling in volume, swelling from 3293 lm3 to 5941 lm3. But over the ensuing hour, the volume of the cells decreases to 3534 lm3 after 30 min and 3123 lm3 after 60 min. This RVD does not occur at 0 °C, supporting the view that it is an energetically driven process. Nor does volume regulation occur in COS-7 cells that have undergone passive osmotic shrinkage by being placed in double-strength hypertonic Tyrodes (602 mOsm). That is quite common. An important and unexpected finding was that the existence a large RVD can have a major effect on the slope of the BVH plot and accordingly can exert a major effect on the value of Vb. We exposed the COS-7 cells to 5 anisotonic test solutions ranging from 77 to 1010 mOsm/kg and determined the volumes of some 225–329 cells per test solution after 5 min or 30 min. The mean volumes, normalized to the isotonic volume, were then plotted against the reciprocal of the measured osmolality of the test solutions. The curve for the cells exposed to the solutions for 5 min and that for cells exposed to the same solutions for 30 min yielded two distinctly different Boyle–vanHoff plots The values of Vb were 0.33 and 0.62 respectively. This difference was mainly due to a flattening by a factor of two between the slope after 5 min (0.2057), and the one after 30 min (0.0981), resulting from divergent 5 and 30 min volume data of the farther from isotonicity deviating test solutions (especially, of the hypotonic ones). In contrast, normalized volumes of the cells in the 602 mOsm/ kg test solution were the same after 5 or 30 min, consistent with a lack of RVI in hypertonic solutions. Conflict of interest: None declared. Source of funding: None declared. doi:10.1016/j.cryobiol.2010.10.045
42. Race for the pace: Is the universal cryoprotocol a dream or reality?. Igor. I. Katkov, CELLTRONIX, San Diego, CA, USA With the development of Peter Mazur’s equations and work of other cryobiologists on slow (equilibrium) freezing in 1960s, it became clear that a particular cell would need its own optimal cryopreservation protocol, that would largely depend on the cell cryobiological and physiological parameters as well as on the type of cryoprotective agents (CPAs) used. With advance of fast freezing and vitrification (VF), introduced by Rall and Fahy in 1980s, the focus shifted toward searching for nontoxic cocktails and decreasing concentration of the vitrificants by increasing rates of cooling and warming. Yet, the protocols of this type of VF are largely customized to the cell type due to difference in the cell osmotic fragility and chemical tolerance to the vitrificants. The universal cryoprotocol, that would fit ALL types of cells, at least if they are in suspension on make a thin layer, would be the Holy Grail of Cryobiology. Here is my hypothesis for your consideration: 1. Every cell has it own critical rate of cooling and thawing, at which rate and at HIGHER rates the cell can be vitrified during cooling (B_cr_cool) and will not devitrify during warming (B_cr_warm) WITHOUT any external ‘‘cryoprotectants” (they must be called ‘‘vitrificants” in this case). OR, it might be just that non-lethal ice (i.e., cubical vs. hexagonal ‘‘killer ice”) is formed during cooling and its transformation (recrystallization) to hexagonal type is precluded during warming. In any case, at rates higher than those two B_cr’s, the cell will survive without any exogenous compounds. 2. Those rates are substantially LOWER than predicted by the most common theories (Rall and Fahy, Boutron’s work, Cravalho’s school: Toner, Karlsson, et al.). I will not go into the details of the thermodynamics of the glassy state but the main reasons are three: (A) cell internal vitrificants; (B) small compartmentalized intracellular milieu; and (C) no ‘‘true” VF is needed for survival. In any scenario, the cell SURVIVES if the pace of cooling and warming is higher than those two B-cr’s, and that is what matters! 3. The distribution and average values of those B-cr’s depend on the species of cells, particularly on the abundance of endogenous vitrificants, how ‘‘watery” those cells are, the level of compartmentalization, the size of the compartments, etc. It may well be that the same species (such embryos) might have VERY different B-cr’s at different stages of their development. 4. I predict that while those speeds are relatively high for the most of the cell species (in range 20,000–1,000,000 C/min), we have already achieved those critical rates in one well documented case, namely human sperm, thanks to the early work in 1930s and in this century by the Isachenkos.