Curve fitting approach for measurement of cellular osmotic properties by the electrical sensing zone method. I. Osmotically inactive volume

We have investigated the confounding effects of dynamic range limitations on measurement of the osmotically inactive volume using electrical sensing zone instruments (e.g., Coulter counters), and propose an improved approach to parameter estimation. The conventional approach for analysis of cell size distributions measured by such particle sizing instruments requires data truncation: the mean cell volume is computed after exclusion of data below a specified lower bound (typically chosen to remove artifacts due to small-volume noise) and above an upper bound (typically governed by instrument limitations). The osmotically inactive volume is then estimated from a Boyle–van’t Hoff plot of the averaged volume data obtained after exposure to various solution osmolalities. We demonstrate that systematic exclusion of data in the conventional approach introduces bias that results in erroneously high estimates of the osmotically inactive volume fraction. To minimize this source of error, we have devised a new algorithm based on fitting a bimodal distribution model to the non-truncated volume data. In experiments with mouse insulinoma (MIN6) cells, the osmotically inactive volume fraction was estimated to be 0.15 ± 0.01 using the new method, which was significantly smaller than the estimate of 0.37 ± 0.02 obtained using the conventional method (p < 0.05). In silico experiments indicated that the parameter estimate obtained by the new method was accurate within 5%, whereas the error associated with the conventional approach was approximately 150%. Parametric analysis was used to elucidate the sensitivity of errors to variations in instrument dynamic range and cell volume distribution width.     

THE OSMOTIC PROPERTIES OF LIVING CELLS (EGGS OF ARBACIA PUNCTULATA)

We have attempted to answer the question: How nearly ideal, as an osmometer, is the unfertilized Arbacia egg? The following conclusion have been reached: 1. Volumes can be measured accurately over a wide range of pressures since the cell is in general spherical and does not suffer deformation from its own weight or other factors. 2. The product of volume and pressure is approximately constant, if allowance be made for osmotically inactive cell contents. It is computed that from 7 to 14 per cent of cell volume is occupied by osmotically inactive material. 3. Evidence is presented that no appreciable escape of cell contents occurs while the cell is in hypotonic sea water; that, therefore, the semipermeability of the membrane is approximately perfect, so long as injury to the cell is avoided. 4. In comparison with osmotic pressure the influence of other forces, such as elasticity or surface tension, on cell volume must in these experiments be slight.     

Osmotic factors of dehardening in cornus Florida L

The killing temperature for cortical cells from the flowering dogwood changes abruptly from −25 C to −15 C during dehardening. Cell sap concentration, minimum critical cell volume, and osmotically inactive cell volume show a progressive change during dehardening, but only cell sap concentration is correlated directly with the killing temperature, showing the same step change. There is a limit to the extent to which hardy dogwood cells can be osmotically reduced in volume. Beyond this limiting volume, the extracellular osmotically can be increased without further volume reduction. Ultimately the cell succumbs, presumably to an osmotic pressure gradient. Nonhardy cells do not exhibit this resistance to shrinkage. The ability to resist volume reduction is probably a crucial factor in the freezing resistance of dogwood cortical cells.     

A non-ideal replacement for the Boyle van't Hoff equation

A non-ideal osmotic equilibrium equation is proposed as a replacement for the Boyle van’t Hoff equation to describe the equilibrium volume of a living cell as a function of external osmolality. Contrary to common understanding, the Boyle van’t Hoff equation is only thermodynamically correct for ideal, dilute solutions. However, the Boyle van’t Hoff equation is commonly used to determine the osmotically inactive fraction of the cell. This involves extrapolating to infinite osmolality, which violates the ideal, dilute solution constraint. It has been noted that the osmotically inactive fractions obtained from the Boyle van’t Hoff equation for human erythrocytes are markedly larger than measured values of the dry volume fraction of the cell. Using the new osmotic equilibrium equation to analyze experimental osmotic equilibrium data reduces the inferred osmotically inactive fraction of human erythrocytes by approximately 20%.     

Volume Control by Muscle Fibers of the Blue Crab : Volume readjustment in hypotonic salines

Single isolated muscle fibers from the walking legs of the blue crab, Callinectes sapidus act as Boyle-van't Hoff osmometers with an osmotically inactive volume of 33 %. Fibers in hypotonic salines undergo a spontaneous volume readjustment toward the initial volumes of the cells found in isotonic salines. The volume readjustment is initiated by the increase in cell volume in hypotonic salines and appears to be dependent on the duration of exposure of the fiber to external sodium, the sodium concentration, and the pH of the external medium. The volume-readjusted cells continue to behave as osmometers, but with an increased relative osmotically inactive volume and a decreased internal resistivity. The decreases in cell volumes appear to be, in large part, due to losses of osmotically active nonelectrolytes from the cells.     

Curve fitting approach for measurement of cellular osmotic properties by the electrical sensing zone method. I. Osmotically inactive volume

We have investigated the confounding effects of dynamic range limitations on measurement of the osmotically inactive volume using electrical sensing zone instruments (e.g., Coulter counters), and propose an improved approach to parameter estimation. The conventional approach for analysis of cell size distributions measured by such particle sizing instruments requires data truncation: the mean cell volume is computed after exclusion of data below a specified lower bound (typically chosen to remove artifacts due to small-volume noise) and above an upper bound (typically governed by instrument limitations). The osmotically inactive volume is then estimated from a Boyle–van’t Hoff plot of the averaged volume data obtained after exposure to various solution osmolalities. We demonstrate that systematic exclusion of data in the conventional approach introduces bias that results in erroneously high estimates of the osmotically inactive volume fraction. To minimize this source of error, we have devised a new algorithm based on fitting a bimodal distribution model to the non-truncated volume data. In experiments with mouse insulinoma (MIN6) cells, the osmotically inactive volume fraction was estimated to be 0.15 ± 0.01 using the new method, which was significantly smaller than the estimate of 0.37 ± 0.02 obtained using the conventional method (p < 0.05). In silico experiments indicated that the parameter estimate obtained by the new method was accurate within 5%, whereas the error associated with the conventional approach was approximately 150%. Parametric analysis was used to elucidate the sensitivity of errors to variations in instrument dynamic range and cell volume distribution width.     

The osmotic sensitivity of rat growth plate chondrocytes in situ; clarifying the mechanisms of hypertrophy

Bone elongation is predominantly driven by the volume expansion of growth plate chondrocytes. This mechanism was initially believed to be "hypertrophy", describing a proportional increase of cell water and organelles. However, morphometrical analysis subsequently assumed the increase to be "swelling", resulting in a disproportionate increase of cell water (osmotically active fraction). Histological approaches were performed on fixed tissue, and for the "swelling" assumption to be valid, the osmotic sensitivity of living cells before and during volume increase should differ. To test this, analysis of images acquired by 2-photon laser scanning microscopy (2PLSM) were used to determine the osmotic sensitivity, and osmotically active/inactive proportions of in situ chondrocytes from 15 living rat growth plates exposed to varying media osmolarities ( approximately 0-580 mOsm). The dimensions of cell volume swelling in hypotonic media were different to the preferential lengthening seen in vivo, confirming the complexity of directional cell volume increase. Boyle-van't Hoff analysis of cell volume over the range of media osmolarity indicated no significant difference (Student's t-test) in the osmotically inactive fraction, 39.5 +/- 2.9% and 47.0 +/- 4.3% (n = 13) for proliferative and hypertrophic zones, respectively, or the sensitivity of volume to changes in media osmolarity (proliferative 15.5 +/- 0.8 and hypertrophic zone 15.5 +/- 1.2%volume . Osm). The osmotic fractions did not change as chondrocytes progress from proliferative to hypertrophic regions of the growth plate. Our data suggest cell volume increase by hypertrophy may play a greater role in cell enlargement than swelling, and should be re-evaluated as a mechanism responsible for growth plate chondrocyte volume increase and hence bone elongation.     

Volume regulation during dehydration of desert beetles

In arid areas in East Africa, dietary water is available only during the rainy seasons. Since the rainy seasons are separated by dry seasons, which may last for many months and in extreme cases for more than a year, the beetles may lose more than 80% of their body water. The water loss takes place mainly at the expense of the extracellular fluid, i.e. as the haemolymph volume drops to zero, the cell volume is only moderately reduced. The protection of cell volume at the expense of the haemolymph requires that solutes are removed from the haemolymph. The solutes are either excreted from the body or sequestered within the body in an osmotically inactive state. In predatory beetles of the family Carabidae, where Na is the dominating extracellular solute, Na is excreted, but it can easily be replaced from the diet. In most herbivorous beetles, such as the Tenebrionidae, which feed on a low Na diet, and which have low extracellular Na levels, Na is usually, but not always, deposited within the body. Free amino acids are moved from haemolymph to cells, but some seem to be made osmotically inactive by polymerization to peptides. As beetles become rehydrated, the peptides are rapidly depolymerized and the amino acids released to the haemolymph. Another factor, which may be important in the stabilisation of cell volume, is the colloid osmotic contribution of intracellular proteins, which may have a steep increase in their osmotic activity with increasing concentration.     

STUDIES ON OSMOTIC EQUILIBRIUM AND ON THE KINETICS OF OSMOSIS IN LIVING CELLS BY A DIFFRACTION METHOD

1. Osmotic equilibrium and kinetics of osmosis of living cells (unfertilized eggs of Arbacia punctulata) have been studied by a diffraction method. This method consists of illuminating a suspension of cells by parallel monochromatic light and measuring, by means of telescope and scale, the angular dimensions of the resulting diffraction pattern from which the average volume of the cells may be computed. The method is far less laborious and possesses several advantages over direct measurement of individual cells. The average size of a large number of cells is obtained from a single measurement of the diffraction pattern and thus individual variability is averaged out. The observations can be made at intervals of a few seconds, permitting changes in volume to be followed satisfactorily. During the measurements the cells are in suspension and are constantly stirred. 2. Volumes of cells in equilibrium with solutions of different osmotic pressure have been determined. In agreement with our previous experiments, based upon direct microscope measurements, we have confirmed the applicability of the law of Boyle-van''t Hoff to these cells; that is to say, the product of volume and pressure has been found to be approximately constant if allowance be made for the volume of osmotically inactive material of the cell contents. The volume of osmotically inactive material was found to be, on the average, 12 per cent of the initial cell volume; in eggs from different animals this value ranged from 6 to 20 per cent. 3. Permeability to water of the Arbacia egg has been found to average, at 22°C., 0.106 cubic micra of water per square micron of cell surface, per minute, per atmosphere of difference in osmotic pressure. 4. Permeability to ethylene glycol has been found to average, at 24°C., 4.0 x 10^–15 mols, per square micron of cell surface, per minute, for a concentration difference of 1 mol per liter. This is in agreement with the values reported by Stewart and Jacobs.     

Electrolyte and non-electrolyte distribution in the ehrlich ascites tumor cells during the cell cycle

In a previous study, evidence was presented for changes in the state of water and osmotically active solutes during the cell cycle. Total water was constant at 82% (w/w), while the fraction of water that was osmotically active decreased from a maximum during S to a minimum at mitosis. Total Na⁺, K⁺, and C1^? in milliequivalents per liter of cell water remained constant. Therefore, electrolytes are sequestered in the osmotically inactive water. Evidence is now presented that Na⁺ exists primarily as one compartment, with a second, slower compartment appearing during S and disappearing during G2. Na⁺ is completely exchangeable during the entire cell cycle. The distribution of other penetrating solutes was also investigated. When placed in hyperosmotic ethylene glycol solutions, cells first shrink, then swell to their original volumes. ¹⁴C-ethylene glycol distributes in 89% of cell water throughout the cell cycle. However, ¹⁴C-urea distributes in anywhere from 86–100% of the cell water, depending on the stage in the cell cycle. Both solutes are at chemical equilibrium in water in which they are distributed, but they differ in their effects on cell volume. The final volume at which cells equilibrate in urea varies with the concentration of urea in the environment and with time into the cell cycle. Results suggest a loss of osmotically active particles or decreased osmotic activity of urea.     

Is the osmotically inactive sodium storage pool fixed or variable?

Recently, there is renewed interest in the role of osmotically inactive Na(+) storage during Na(+) retention. Although it is well accepted that a portion of the total exchangeable Na(+) reservoir is osmotically inactive, there is current controversy as to whether the osmotically inactive Na(+) storage pool is fixed or variable during Na(+) retention. In this article, we analyze the current scientific evidence to assess whether the osmotically inactive Na(+) storage pool can be dynamically regulated. Our analysis supports the assertion that the osmotically inactive Na(+) storage pool is fixed rather than variable.     

Osmotic tolerance limits and effects of cryoprotectants on the motility, plasma membrane integrity and acrosomal integrity of rat sperm

Osmotic stress is an important factor that can result in cell damage during cryopreservation. The objectives of this study were to determine: (1) isosmotic sperm cell volume; (2) osmotically inactive volume; (3) osmotic tolerance limits of rat sperm; and (4) the effects of addition and removal of glycerol (Gly), ethylene glycol (EG), propylene glycol (PG) or dimethyl sulfoxide (Me(2)SO) on rat sperm function. Sperm from Fischer 344 and Sprague-Dawley rats were used in this study. An electronic particle counter was used to measure the cell volume of rat sperm. Computer-assisted sperm motility analysis and flow-cytometric analysis were used to assess sperm motility, plasma membrane and acrosomal integrity. The isosmotic sperm cell volumes of the two strains were 37.0+/-0.1 and 36.2+/-0.2 microm(3), respectively. Rat sperm behaved as linear osmometers from 260 to 450 mOsm, and the osmotically inactive sperm volumes of the two strains were 79.8+/-1.5% and 81.4+/-2.2%, respectively. Rat sperm have very limited osmotic tolerances. The sperm motility and the sperm plasma membranes of both strains were sensitive to anisosmotic treatments, but the acrosomes of both strains were more sensitive to hyposmotic than hyperosmotic conditions. The one-step addition and removal of Me(2)SO showed the most deleterious effect on rat sperm motility, plasma membrane integrity, and acrosomal integrity among the four cryoprotectants. These data characterizing rat sperm osmotic behavior, osmotic and cryoprotectant tolerance will be used to design cryopreservation protocols for rat sperm.     

Osmotic tolerance and membrane permeability characteristics of rhesus monkey (Macaca mulatta) spermatozoa

Biophysical characteristics of the plasma membrane, such as osmotic sensitivity and water and cryoprotectant permeability are important determinants of the function of spermatozoa after cryopreservation. A series of experiments was conducted with rhesus macaque spermatozoa at 23 degrees C to determine their: (1) cell volume and osmotically inactive fraction of the cell volume; (2) permeability coefficients for water and the cryoprotectants dimethyl sulfoxide, glycerol, propylene glycol, and ethylene glycol; (3) tolerance to anisosmotic conditions; and (4) motility after a one step addition and removal of the four cryoprotectants. An electronic particle counter and computer aided semen analysis were used to determine the cell volume and permeability coefficients, and motility, respectively. Rhesus spermatozoa isosmotic cell volume was 27.7+/-3.0 microm3 (mean+/-SEM) with an osmotically inactive cell fraction of 51%. Hydraulic conductivity in the presence of dimethyl sulfoxide, glycerol, propylene glycol, and ethylene glycol was 1.09+/-0.30, 0.912+/-0.27, 1.53+/-0.53, and 1.94+/-0.47 microm/min/atm, respectively. Cryoprotectant permeability was 1.39+/-0.31, 2.21+/-0.32, 3.38+/-0.63, and 6.07+/-1.1 (x10(-3)cm/min), respectively. Rhesus sperm tolerated all hyposmotic exposures. However, greater than 70% motility loss was observed after exposure to solutions of 600 mOsm and higher. A one step addition and removal of all four cryoprotectants did not cause significant motility loss. These data suggest that rhesus sperm are tolerant to hyposmotic conditions, and ethylene glycol may be the most appropriate cryoprotectant for rhesus sperm cryopreservation, as it has the highest permeability coefficient of the tested cryoprotectants.     

Osmotic tolerance limits and effects of cryoprotectants on motility of bovine spermatozoa

This study was conducted to determine the osmotic properties of bull spermatozoa, including the effects of osmotic stress and cryoprotectant agent (CPA) addition and removal, on sperm motility. Semen from beef bulls was collected by electroejaculation and extended 1:3 in TL-Hepes containing 100 micro g/ml pyruvate and 6 mg/ml BSA. In solutions of 150-1200 mOsmolal (mOsm), bull spermatozoa behaved as linear osmometers (r(2) = 0.97) with an osmotically inactive cell volume of 61%. The isosmotic cell volume was 23.5 micro m(3). Motility was determined after exposure to anisosmotic solutions ranging from 35 to 2400 mOsm and after return to isosmotic conditions. Retention of at least 90% of isosmotic motility could be maintained only between 270-360 mOsm. Bull spermatozoa were calculated to retain 90% of their isosmotic motility at 92-103% of their isosmotic cell volume. Motility following a one-step addition and removal of 1 M glycerol, dimethyl sulfoxide, and ethylene glycol was reduced by 31%, 90%, and 6%, respectively, compared with CPA addition only. These data indicate that, during bull spermatozoa cryopreservation, osmotically driven cell volume excursions must be limited by exposure to a very narrow range that may be facilitated by the use of ethylene glycol as a CPA.     

OSMOTIC PROPERTIES OF THE EGG CELLS OF THE OYSTER (OSTREA VIRGINICA)

Investigations of the osmotic properties of oyster eggs by a diffraction method for measuring volumes have led to the following conclusions: 1. The product of cell volume and osmotic pressure is approximately constant, if allowance is made for osmotically inactive cell contents (law of Boyle-van''t Hoff). The space occupied by osmotically inactive averages 44 per cent of cell volume. 2. Volume changes over a wide range of pressures are reversible, indicating that the semipermeability of the cell during such changes remains intact. 3. The kinetics of endosmosis and of exosmosis are described by the equation, See PDF for Equation, where dV is rate of volume change; S, surface area of cell, (P-P_e), the difference in osmotic pressure between cell interior and medium, and K, the permeability of the cell to water. 4. Permeability to water during endosmosis is 0.6µ³ of water per minute, per square micron of cell surface, per atmosphere of pressure. The value of permeability for exosmosis is closely the same; in this respect the egg cell of the oyster appears to be a more perfect osmometer than the other marine cells which have been studied. Permeability to water computed by the equation given above is in good agreement with computations by the entirely different method devised by Jacobs. 5. Permeability to diethylene glycol averages 27.2, and to glycerol 20.7. These values express the number of mols x 10^–15 which enter per minute through each square micron of cell surface at a concentration difference of 1 mol per liter and a temperature of 22.5°C. 6. Values for permeability to water and to the solutes tested are considerably higher for the oyster egg than for other forms of marine eggs previously examined. 7. The oyster egg because of its high degree of permeability is a natural osmometer particularly suitable for the study of the less readily penetrating solutes.     

Cryopreservation and biophysical properties of articular cartilage chondrocytes

In order to successfully cryopreserve articular cartilage chondrocytes, it is important to characterize their osmotic response during the cryopreservation process, as the ice forms and the solutes concentrate. In this study, experimental work was undertaken to determine the osmotic parameters of articular cartilage chondrocytes. The osmotically inactive volume of articular cartilage chondrocytes was determined to be 44% of the isotonic volume. The membrane hydraulic conductivity parameters for water were determined by fitting a theoretical water transport model to the experimentally obtained volumetric shrinkage data; the membrane hydraulic conductivity parameter L(Pg) was found to be 0.0633 microm/min/atm, and the activation energy E, 8.23 kcal/mol. The simulated cooling process, using the osmotic parameters obtained in this study, suggests a cooling rate of 80 degrees C/min for the cryopreservation of the articular cartilage chondrocytes of hogs. The data obtained in this study could serve as a starting point for those interested in cryopreservation of chondrocytes from articular cartilage in other species in which there is clinical interest and there are no parameters for prediction of responses.     

Studies on membrane permeability of zebrafish (Danio rerio) oocytes in the presence of different cryoprotectants

Investigation into fish oocyte membrane permeability is essential for developing successful protocols for their cryopreservation. The aim of the present work was to study the permeability of the zebrafish (Danio rerio) oocyte membrane to water and cryoprotectants before cryopreservation protocol design. The study was conducted on stage III and stage V zebrafish oocytes. Volumetric changes of stage III oocytes in different concentrations of sucrose were measured after 20 min exposure at 22 degrees C and the osmotically inactive volume of the oocytes (Vb) was determined using the Boyle-van't Hoff relationship. Volumetric changes of oocytes during exposure to different cryoprotectant solutions were also measured. Oocytes were exposed to 2 M dimethyl sulphoxide (DMSO), propylene glycol (PG), and methanol for 40 min at 22 degrees C. Stage III oocytes were also exposed to 2 M DMSO at 0 degrees C. Oocyte images were captured on an Olympus BX51 cryomicroscope using Linkham software for image recording. Scion Image was used for image analysis and diameter measurement. The experimental data were fitted to a two-parameter model using Berkeley Madonna 8.0.1 software. Hydraulic conductivity (L(p)) and solute (cryoprotectant) permeability (Ps) were estimated using the model. The osmotically inactive volume of stage III zebrafish oocytes was found to be 69.5%. The mean values+/-SE of Lp were found to be 0.169+/-0.02 and 0.196+/-0.01 microm/min/atm in the presence of DMSO and PG, respectively, at 22 degrees C, assuming an internal isosmotic value for the oocyte of 272 mOsm. The Ps values were 0.000948+/-0.00015 and 0.000933+/-0.00005 cm/min for DMSO and PG, respectively. It was also shown that the membrane permeability of stage III oocytes decreased significantly with temperature. No significant changes in cell volume during methanol treatment were observed. Fish oocyte membrane permeability parameters are reported here for the first time. The Lp and Ps values obtained for stage III zebrafish oocytes are generally lower than those obtained from successfully cryopreserved mammalian oocytes and higher than those obtained with fish embryos and sea urchin eggs. It was not possible to estimate membrane permeability parameters for stage V oocytes using the methods employed in this study because stage V oocytes experienced the separation of outer oolemma membrane from inner vitelline during exposure to cryoprotectants.     

Osmotic water permeability measurements using confocal laser scanning microscopy

 We have developed a method for measurement of plasma membrane water permeability (P _f) in intact cells using laser scanning confocal microscopy. The method is based on confocal recording of the fluorescence intensity emitted by calcein-loaded adherent cells during osmotic shock. P _f is calculated as a function of the time constant in the fluorescence intensity change, the cell surface-to-volume ratio and the fractional content of the osmotically active cell volume. The method has been applied to the measurement of water permeability in MDCK cells. The cells behaved as linear osmometers in the interval from 100 to 350 mosM. About 57% of the total cell volume was found to be osmotically inactive. Water movement across the plasma membrane in intact MDCK cells was highly temperature dependent. HgCl₂ had no effect on water permeability, while amphotericin B and DMSO significantly increased P _f values. The water permeability in MDCK cells transfected with aquaporin 2 was an order of magnitude higher than in the intact MDCK cell line. The water permeability of the nuclear membrane in both cell lines was found to be unlimited. Thus the intranuclear fluid belongs to the osmotically active portion of the cell. We conclude that the use of confocal microscopy provides a sensitive and reproducible method for measurement of water permeability in different types of adherent cells and potentially for coverslip-attached tissue preparations. Received: 12 June 1999 / Revised version: 21 February 2000 / Accepted: 25 February 2000     

Freezing response and optimal cooling rates for cryopreserving sperm cells of striped bass, Morone saxatilis

This study explored the optimization of techniques for sperm cryopreservation of an economically important fish species, the striped bass Morone saxatilis. The volumetric shrinkage or the water transport response during freezing of sperm cells was obtained using a differential scanning calorimeter (DSC) technique. Water transport was obtained in the presence of extracellular ice at a cooling rate of 20 degrees C/min in two different media: (1) without cryoprotective agents (CPAs), and (2) with 5% (v/v) dimethyl sulfoxide (DMSO). The sperm cell was modeled as a cylinder of length of 22.8 microm and diameter 0.288 microm and was assumed to have an osmotically inactive cell volume (V(b)) of 0.6 V(0), where V(0) is the isotonic or initial cell volume. By fitting a model of water transport to the experimentally determined water transport data, the best fit membrane permeability parameters (reference membrane permeability to water, L(pg) or L(pg)[cpa] and the activation energy, E(Lp) or E(Lp)[cpa]) were determined and ranged from L(pg)=0.011-0.001 microm/min-atm, and E(Lp)=40.2-9.2 kcal/mol). The parameters obtained in this study suggested that the optimal rate of cooling for striped bass sperm cells in the presence and absence of DMSO range from 14 to 20 degrees C/min. These theoretically predicted rates of optimally freezing M. saxatilis sperm compared quite closely with independent and experimentally determined optimal rates of cooling striped bass sperm.     

Permeability of cultured megakaryocytopoietic cells of the rat to dimethyl sulfoxide

The permeability of the membrane of the rat megakaryocytopoietic cell to dimethyl sulfoxide was measured to assess its availability to the intracellular compartment. The method used was osmotic, and measured the initial loss of cell water followed by a reswelling to isotonic volume when cells were placed in culture media containing 0.6 M DMSO. Values for the hydraulic coefficient, Lp, the permeability of the membrane to DMSO, wRT , and the reflection coefficient were calculated from the equations of Kedem and Katchalsky . The average value at 25 degrees C for Lp was 0.46 micron min-1 atm1 ; wRT was 9.3 micron min-1, and the reflection coefficient was 0.65. At these cell volumes, 50% equilibration occurred in 5 sec. Cells equilibrated in 0.6 M DMSO increased their volume of osmotically inactive water. Coupled with this phenomenon of stabilization of water was a reduction in the hydraulic coefficient by 50%. These findings are discussed in the context of current hypotheses about cellular viability during freezing and thawing in the presence and absence of cryoprotectants.