Cryomicroscopic determination of the membrane osmotic properties of human monocytes at subfreezing temperatures

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BRIEF COMMUNICATIONS Cryomicroscopic Determination Properties of Human Monocytes CAMERON McCAA,*,’

of the Membrane Osmotic at Subfreezing Temperatures

K. R. DILLER,* S. J. AGGARWAL,* T. TAKAHASHI-i

AND

*Department of Mechanical Engineering, Bio-Heat Transfer Laboratory, Biomedical Engineering Program, The University of Texas at Austin, Austin, Texas 78712; and tAmerican Red Cross, Jerome H. Holland Laboratory for the Biomedical Sciences, Rockville, Maryland 20855 Monocytes were isolated from fresh whole human blood and resuspended in Hanks balanced salt solution; a portion of the cells was mixed with an equal volume of 2 M dimethyl sulfoxide (DMSO) to form a 1 M solution. Microliter volumes of cell suspension were placed directly onto a computercontrolled cryostage and cooled to a predetermined subzero temperature. Ice was nucleated in the extracellular medium and a continuous video record was made of the subsequent osmotically induced volume changes of individual cells owing to exposure to the concentrated extracellular solutes. Selected micrographs emphasizing the initial transient data were digitized for computer analysis with an interactive boundary tracing algorithm to determine metric parameters of specific cells, and apparent volume changes were measured as a function of elapsed time after nucleation. The KedemKatchalsky-coupled transport equations were fit to the data using a network thermodynamic model implemented on a microcomputer to determine values for the permeability properties L,, o, and (r. Experiments were performed over the temperature range from - 7” to - 10°C. Cells pre-equilibrated with DMSO had a lower L, and a higher activation energy, LW, than without additive, although the statistical significance of the difference could not be substantiated. It was found that the movement of DMSO across the plasma membrane in response to extracellular freezing was apparently so much smaller than the water flux that values for o and o could not be determined from the data base. 8 1991 Academic

Press, Inc.

The preservation of living cells and tissues at cryogenic temperatures has become quite common, although protocols may vary widely as a function of cell membrane properties as well as other biophysical and environmental factors. Reliable procedures for the frozen preservation of monocytes have been developed and published (IO, 18, 28, 30). Critical physical events during the cooling of monocyte cell suspensions, derived from correlations of cell mortality to the state of water in frozen preparations have been reported by Takahashi et al. (29). The current study was designed to determine the osmotic characteristics of the

monocyte plasma membrane in the presence of extracellular ice as a function of temperature. Numerous modeling studies, starting with and building on the work of Mazur in 1963 (14), have demonstrated that the osmotic behavior of a cell during cryopreservation is governed by the plasma membrane permeability to water and cryoprotective additive. However, experimental data for these properties have been very difficult to obtain, and they remain scarce. In this paper we report the results of some initial experiments to measure the value of the membrane water permeability for human monocytes as obtained by cryomicroscopic investigations over a limited range of subzero temperatures.

Received July 20, 1987; accepted October 11, 1990. i Present address: CryoLife, Inc., 2211 New Market Pkwy. Suite 142, Marietta, GA 30067.

MATERIALS

Monocyte

AND METHODS

cell suspensions were pre-

391 001l-2240/91 $3.00 Copyright 0 1991 by Academic Press, Inc. Au rights of reproduction in any form reserved.

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pared at the American Red Cross Blood Re- croscopic field of observation was selected search Laboratory in Maryland, packed on to include multiple cells, following which ice in insulated shipping containers, and extracellular ice was nucleated in the specsent by overnight air delivery to the Uni- imen by directing a fine jet of liquid N, to a versity of Texas at Austin. Osmotic re- peripheral area of the stage away from the sponse experiments were performed on a field of view. Throughout the subsequent cryomicroscope within 36 h of blood collec- isothermal holding period of the experiment tion. The isolation procedure for the mono- the response of the cells to the presence of cytes has been described by Takahashi elevated solute concentration in the extra(29). Purified monocytes were suspended in cellular solution was followed continuHanks balanced salt solution (HBSS) with- ously. out calcium and magnesium, but containing In general the limit of temperature reso5% (voYvol) fetal calf serum. lution on the cryostage during an experiSome of the cells were used for freezing mental protocol was approximately trials as received from the Red Cross, and -+OS”C. This thermal uncertainty translates the remainder were modified by the addi- directly into a band of uncertainty in the tion of a cryoprotective agent (CPA), di- composition of the extracellular medium methyl sulfoxide (DMSO). A 2 M DMSO which was used to elicit an osmotic resolution was added in small increments sponse from the test cells. It is well known while mixing at 4°C over a period of 30 min from aqueous phase diagrams that the sento achieve a final 1: 1 mixture yielding a cell sitivity of the solution composition to temperature is greatest near the equilibrium suspension in a I M CPA concentration. Fluorescein diacetate (FDA) and trypan phase change state (EPCS), and it deblue assays were performed on cell samples creases progressively as the temperature is upon receipt in Texas. Results were consis- reduced. Therefore, in order to maintain tent and indicated viability of no less than the most stable chemical environment dur95%. The cells were tested both in the un- ing the osmotic tests, it was desirable to modified HBSS and in the 1 M DMSO so- operate at a temperature somewhat relution. moved from the EPCS; in practice this translated to -7°C for the 1 M DMSO soExperimental Procedure lutions in the present series of trials. A secA typical experiment was initiated by ond limiting factor for the operating state of first defining a desired freezing protocol on the system was spontaneous intracellular a programmable temperature controller ice formation, the probability of which in(26). Approximately 1 ~1 of cell suspension creased with reduced temperature. This was placed directly onto a convection cryo- phenomenon made it problematic to constage (15). The temperature was manually duct trials at temperatures significantly belowered to 0°C by slowly increasing the low - 10°C. In particular, cells suspended flow rate of refrigerated nitrogen gas in only HBSS usually experienced intracelthrough the stage, at which time the tem- lular ice formation within 5 s or less subseperature controller was activated. A video quent to extracellular nucleation, making it recorder and an electronic timer were acti- difficult to collect osmotic response data vated, and the stage temperature was then over an extended period of time. Thus, only further decreased at a rate of 6Wmin to the a relatively small window of temperatures preprogrammed nucleation value, where it between -7” and - 10°C was available for was then held constant for 300 s while the obtaining data with a practical degree of osmotic stress trial was conducted. The mi- consistency during the present trials.

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Cell Volume Analysis Although more than 100 trials were executed, less than 15% were suitable for observing the osmotic behavior of the monocytes. In nearly half of the trials intracellular ice formed before any significant dehydration occurred. Other problems that precluded volumetric analysis included cellular deformation so that extrapolation of the geometry to an ideal spherical shape was not justifiable and obscuring of cell boundaries by the growing extracellular ice mass or by air bubbles coming out of the solution. However, a small fraction of the trials yielded micrographs which could be used for analysis of cell volume changes. A continuous video record, incorporating a temperature and time display, was made for each experimental trial. From these video recordings selected individual frames at specific time intervals of 1 s or greater were digitized on an IBM PC AT computer. With the digitized image “frozen” on a video screen an interactive boundary tracing and analysis program (1) was used to outline the perimeter of the cell via an operator-guided mouse. In general, with this imaging and analysis procedure, cell volumes may be determined to within the range of 23 to 5%. When the tracing sequence was complete for all frames of a particular experiment, a report file was generated to provide both individual cell and aggregate (global) metric data. The volumes of the individual cells in each frame were normalized to the values at the time of extracellular ice formation in the field of view. Individual cell reports indicate metric values for single cells from frame to frame; the global reports present the averaged data for all cells in a frame. Apparent cell volumes were determined by a simple spherical extrapolation of the enclosed cross-sectional area data to three dimensions. Cells which were observed to contain intracellular ice, manifested by

“flashing” to a dark, opaque image, or which appeared to form intracellular gas bubbles were not analyzed subsequent to the event. Determination of Membrane Permeability Parameters The volume/time data were used as input values for a network thermodynamic computer program (8) to calculate values of the parameters for coupled membrane transport as defined by Kedem and Katchalsky (11). The model was fit to data values averaged over like trial protocols rather than attempting to determine an effective permeability for every cell measured; this approach was adopted in order to minimize the effects of variations and uncertainty in the individual volume measurements. A chi-square statistical comparison criterion was used to determine the optimum agreement between the model results and the experimental data. Input to the software consisted of the experimental time/volume data, along with the initial concentrations of salt and DMSO, the metric properties of monocyte cells, and the ice nucleation temperature. The program varied the parameters L,, u, and o to minimize the chi-square value until an optimal match between the theoretical and experimental dehydration curves was detected. The output consists of the determined values of L,, o, and u as well as a simulation of the volumetric response to the trial protocol based on the derived transport coefficients. RESULTS

Initial analysis of the data consisted of determining the volume of each cell during the duration of the protocol for which it was observed. Since all frames from the video record were evaluated at uniform 1 s intervals, it was straightfoward to calculate the average and standard deviation values for the normalized volume for each protocol executed. These values are presented in

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Table 1, and Fig. 1 illustrates the overall osmotic behavior expressed in terms of the average volume and limiting range values of the standard deviation. These data were used to calculate permeability values for the monocytes. The transient volume data were evaluated in the NT program according to the procedure outlined above to obtain a single set of permeability coefficients for each specific experimental condition. The calculated values of the membrane water permeability, Lp, at each state are given in Table 2. It was assumed in the theoretical model (16) that the permeability values followed an Arrhenius behavior at subfreezing temperatures .

where T is absolute temperature, Lpg is specified at a reference temperature T,, = 273.15”K, AE is the activation energy, and C&is the universal gas constant. For data interpreted in terms of this model, when the value of ln(LP) is evaluated as a function of (l/T - l/T,,), the slope of an imposed linear function is equal to - AE/%, and ln(L,,) is the Y-intercept. The activation energy (AL?) was calculated from the slope of this linear function tit to the data. For cells frozen in HBSS, the calculated activation energy was AE = 61 [kJ/mol], and in 1 M DMSO

protocol 1.0 0.9

0.3 0

s 1

E 2

-

-ST, I-IBSS

-c

-lO”C, - 7T, -ST, -lOT,

.

' 3

HBSS 1M DMSO 1M DMSO 1MDMSO

E 4

' 5

time (SIX)

1. Average transient cell volume data for monocytes frozen at the indicated temperatures and solution compositions. Maximum and minimum standard deviation values are shown at each temperature, along with intermediate values where they may be discriminated. See Table 1 for a listing of all data. FIG.

solution the activation energy was AE = 145 [kJ/molJ. DISCUSSION

Intracellular Ice Formation Intracellular ice formation was assumed to have occurred when there was a visual “flashing” of a cell from a light to a relatively dark shading. In some experiments, small, light-colored dots formed within the cells, presumably owing to the presence of either gas bubbles or intracellular ice (19,

TABLE 1 Normalized Volume Measurements for All Data Sets Analyzed, Showing Average Values and Standard Deviations HBSS

1 M DMSO

Time (s)

-8°C (10)

- 10°C (12)

- 7°C (7)

0 1 2 3 4 5

1.0 0.82 2 0.08 0.64 2 0.17

1.0 0.81 k 0.06 0.62 k 0.12

1.0 0.79 + 0.07 0.61 * 0.09 0.46 2 0.05 0.42 k 0.05 0.41 2 0.04

Note. Values in parentheses denote number of cells measured.

- 8°C (3) 0.80 0.69 0.60 0.53 0.45

1.0 2 0.10 2 0.07 2 0.03 2 0.04 2 0.03

- 10°C (6) 1.0 0.81 2 0.16 0.70 k 0.16 0.62 f 0.14 0.56 2 0.16 0.52 2 0.14

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TABLE

2

Calculated Membrane Water Permeability Properties for the Indicated Experimental Protocols HBSS

No. cells measured Lp (pm/atm . min) AE (kJ/mol)

1 M DMSO

-8°C

- 10°C

-7°C

- 8°C

- 10°C

10 0.352

12 0.292

7 0.406

3 0.265 145

6 0.186

61

25, 27). Consequently, cells exhibiting these phenomena were excluded from further analysis. Intracellular ice formed within the first 3 to 5 s after extracellular nucleation for virtually all experiments performed without a cryoprotective additive. Conversely, virtually no cells frozen in the presence of DMSO exhibited intracellular ice formation at any time. A major difference between the cells with and without a CPA is that the prefreezing mixing with DMSO causes the loss of considerable intracellular water prior to exposure to an environment containing ice. Consequently, there is less intracellular water available for subsequent freezing. Other relevant factors relating to nucleation kinetics are beyond the scope of this discussion. Rate of Cellular Dehydration The rate of water loss from a cell during freezing is a function of both the hydraulic permeability Lp and the driving chemical potential Ap+,,.The extracellular electrolyte concentration becomes greater as the nucleation temperature is reduced, which in turn causes a larger driving potential for dehydration. On the other hand, the value of Lp decreases exponentially with temperature reduction, thus raising the impedance to water loss. These two phenomena present a competition between retarding and enhancing the transmembrane water flux. Figure 1 illustrates dehydration rates for cells frozen in 1 M DMSO and in HBSS. The data indicate that the reduction in Lp dominates over the increase in driving po-

tential within the given temperature range in that the rate of volume loss is reduced with lower temperatures. Effects of Dimethyl &&oxide on Hydraulic Permeability The calculated values of Lp in Table 2 are lower by an average of 36% for cells suspended in a DMSO solution in comparison with those suspended in HBSS. This trend is in agreement with the data of prior investigators. Hempling and White have investigated the effects of DMSO on the permeability of rat megakaryocytopoietic cells (9). Experiments were performed at room temperature in a diffusion chamber, and the data were analyzed by the Kedem and Katchalsky equations for water and solute transport. Their results indicated that there was a reduction in the hydraulic permeability owing to the interaction between the coupled flows of water and additive. For experiments in which the cells were equilibrated in a 0.6 M DMSO solution, the values of L, were an average of 55% less than those for experiments in which no additive was present. The data of Rule et al. (22) for V-79 Chinese hamster lung fibroblasts also show a 50% or greater reduction in the hydraulic permeability in the presence of a 0.6 it4 DMSO solution. No value of the reflection coefftcient was available as an indication of the degree of coupling between the mobile species. Papanek (20) also measured a significant reduction in the water permeability of human red cells in the range 25°C to - 10°C when ethylene glycol was added as a cryoprotective agent.

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There appear to be no data in the literature that deal specifically with the possible alteration of Lp by the presence of a CPA for cells exposed to ice in the extracellular environment. In general it has not been determined whether the presence of extracellular ice has an effect on the actual or apparent membrane permeability properties of cells. It is possible that the lower permeability measured in the presence of DMSO is associated with the protective effects of the CPA against freezing injury. With no cryoprotectant in the suspension, the cell membrane may experience biophysical alterations owing to extracellular ice formation. The relatively high values of L, measured in experiments without an additive could then be attributed to injury during the freezing process, resulting in a leaky membrane. Clearly, the present data are not a proof of such a hypothesis. However, we have measured this effect in similar experiments on human granulocytes (32), using glycerol as the CPA, in which cells that sustain injury during freezing have a higher water permeability. Comparison with Prior Reported Lp and AE Values Permeability values for cells at subzero temperatures in the presence of extracellular ice are very sparse. Levin (13) and Schwartz and Diller (24) have measured the water permeability of yeast from transient volume data obtained during freezing. In both instances values were reported approximately an order of magnitude smaller than those of the present experiment within the same temperature range, although there was considerable variation among the data. Papanek determined the hydraulic permeability of human erythrocytes at subzero temperatures by the stopped flow method in which there is no ice in the extracellular medium (20). As anticipated, the erythrocyte permeability is much larger than the measured values for monocytes. Schwartz and Diller have measured the water perme-

ability of human granulocytes by the identical cryomicroscope technique employed in the present study (23) and obtained values within the same order of magnitude as for the monocytes and which were consistent with other measurements performed at above-zero temperatures (3). The activation energies determined in the present experiments, 61 and 145 (kJ/mol), are quite high in comparison with values typically reported for the temperature coefficient of permeability. However, similarly high activation energies have also been measured for mouse ovum (54 kJ/mol) (12), human keratinocytes (45 kJ/mol) (2), human granulocytes (218 kJ/mol) (23), and yeast (19 to 101 and 45 to 143 kJ/mol, as a function of cooling rate) (13, 24) frozen in the absence of a CPA, and granulocytes frozen with glycerol (67 kJ/mol) (32). Admittedly, the small size of the present data set renders meaningful statistical analysis impossible; thus it is not possible to assign any quantitative values to the uncertainty in the calculated values of the permeability and activation energy. However, it is likely that +50% is not an unreasonably large range to consider. In particular, it is likely that the values are biased to the large side. The temperature coefficients for permeability of cells in the presence of extracellular ice appear to be significantly higher than those observed for similar cells at suprazero temperatures. Several possible hypotheses could be considered to explain this effect. The desire to explore the suggestion that the transport properties L, and hE at low temperature may very well not be simple extrapolations from ambient temperatures (13) is a primary rationale for experimental studies such as the present one. The present experiments do not provide any indication of mechanisms that might be involved in the apparent increased activation energy at subzero temperatures. However, possible phenomena could include phase changes in the membrane material within this temperature range or an in-

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crease in the diffusional impedance of the immediately adjacent environmental medium or boundary layer, perhaps in conjunction with the formation and growth of the extracellular ice phase. Further dedicated experiments of directed design will be required to test the validity of this effect to identify its phenomenalogical basis. Experimental Determination of o and u The transient volume data from the freezing experiments with a CPA were applied to determine values for w and u at subzero temperatures. All three permeability parameters were optimally fit to the experimental data sets as described above. These calculations provided consistent values for L,, but in contrast o varied by orders of magnitude among the individual experimental trials with no apparent phenomenalogical correlation. The values of o were always much smaller than those of LP. In addition, the values of u were (in virtually all cases) equal to 1.0, which was set as an upper available limit in the software program, indicating no apparent coupling between the water and additive fluxes. Interpretation of this data is, however, far from simple and straight forward. To provide a rational basis for interpreting the analysis of these data, we have performed simulations of isothermal freezing experiments with the network thermodynamics computer model using temperatures and ratios of LJo typical of measurements at above zero temperatures. These simulations indicate that during isothermal freezing experiments, because the flux of CPA is so small in comparison to the water flux, volume data are insufficiently sensitive to accurately determine values of w and u (8). This conclusion is in agreement with prior analysis of coupled water and additive transport during cell freezing at constant cooling velocities (5-7). When cells pre-equilibrated with a penetrating CPA are frozen, volume changes are dominated by the flux of water. The contri-

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bution of CPA flux to alterations in the total volume cannot be measured by our present techniques. Therefore, determination of o and u values will have to be performed by other types of experiments in which it is possible to produce a significant volume change by movement of CPA. These experiments in general involve addition and/or removal of CPA in a liquid state system by direct manipulation of the chemical composition of the cell environment. The stopped flow mixing technique implemented either macroscopically (20) or microscopically (3) can effect the necessary fluxes, as can the membrane diffusion chamber technique of McGrath (17) and the perfusion cryomicroscope of Walcerz and Diller (31). In all of these techniques the cells may be subjected to osmotic stress in the absence of extracellular ice, but trials can be executed over at most only a limited range of subzero temperatures. The perfusion cryomicroscope (31) allows independent control of both the chemical and thermal environments of the specimen over a broad and continuous range of temperatures both above and below 0°C. One of the above systems could be used to cause the transmembrane movement of additive to contribute significantly to alterations in cell volume and thus provide the sensitivity requisite to measure the associated transport coefficients. However, the absence of an extracellular ice phase may provide considerably different net impedance to the membrane than could be created by freezing. The data presented in this paper must be considered in light of an inherent limitation to the experimental technique. The cell volume, which is by definition a threedimensional quantity, is estimated from the measured two-dimensional cell crosssectional area. These data are extrapolated to estimate the volume via the assumption that the cell geometry is spherical, with the implication that the cellular dehydration is isometric in three dimensions. Human monocyte cells are approximately spherical

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in an isotonic environment; however, as the cells dehydrate, deviations from a uniform geometry are visually detectable in even a two-dimensional image. Resolution of this problem will require true three-dimensional analysis of cell volumes. An additional factor relevant to the interpretation of the data presented in this paper is the small sample size from which the permeability values are derived. This limitation was not a planned feature of the experimental protocol; however, the difficulty of obtaining quality photomicrographic records from which adequate measurements could be obtained describing alterations in cell size through the duration of an experimental trial precluded the use of much of the data that was collected. The cryomicroscopic examination of monocytes did not prove an easy procedure to execute or to analyze. Consequently, the conclusions from the investigation cannot be presented on as statistically justifiable a basis as is desirable. Nonetheless, the phenomena seen in the data are in line with measurements obtained in other cell types, and they contribute to our emerging understanding of the osmotic behavior of living cells when subjected to the stress of freezing. ACKNOWLEDGMENTS

This research was sponsored by a grant from the Texas Advanced Technology Research Program. We acknowledge the American Red Cross Blood Research Laboratory for providing a consistent supply of monocyte cells. Our thanks also to Dr. Stanley P. Leibo for technical dialogue and guidance provided during the investigations. REFERENCES

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3. Bradley, D. A., and Diller, K. R. Measurement of the water permeability of single human granulocytes on a microscopic stopped-flow mixing system. Trans. ASME J. Biomech. Eng. 106, 384-393 (1984). 4. Diller, K. R., Beaman, J. J., McCaa, C., Montoya, J. P., and Takahashi, T. Analysis of the osmotic response of human monocytes to freezing with a cryoprotective additive. Model. Simul. 18, 561-566 (1987). 5. Diller, K. R., and Lynch, M. E. An irreversible thermodynamic analysis of cell freezing in the presence of membrane permeable additives. I. Numerical model and transient cell volume data. Cryo Lett. 4, 295-308 (1983). 6. Diller, K. R., and Lynch, M. E. An irreversible thermodynamic analysis of cell freezing in the presence of membrane permeable additives. II. Transient electrolyte and additive fluxes. Cryo Left. 5, 117-130 (1984). 7. Diller, K. R., and Lynch, M. E. An irreversible thermodynamic analysis of cell freezing in the presence of membrane permeable additives. III. Transient electrolyte and additive concentrations. Cry0 Lett. 5, 131-144 (1984). 8. Diller, K. R., Beaman, J. J., Montoya, J. P., and Breedfield, P. C. Network thermodynamic modeling with bond graphs for membrane transport during cell freezing. Trans. ASME J. Heat Transfer 110, 938-945 (1988). 9. Hempling, H. G., and White, S. Permeability of cultured megakaryocytopoietic cells of the rat to dimethyl sulfoxide. Cryobiology 21, 133-143 (1984). 10. Hunt, S. M., Lionetti, F. J., Valeri, C. R., and Callahan, A. B. Cryogenic preservation of monocytes from human blood and platelet pheresis cellular residues. Blood 57, 592-598 (1981). 11. Kedem, O., and Katchalsky, A. Thermodynamic analysis of the permeability of biological membranes to electrolytes. Biochim. Biophys. Acta 27, 229-246 (1958). 12. Leibo, S. P. Water permeability and its activation energy of fertilized and unfertilized mouse ova. J. Membr. Biol. 53, 179-188 (1980). 13. Levitt, R. L. Water permeability of yeast cells at sub-zero temperature. J. Membr. Biol. 46, 91124 (1979). 14. Mazur, P. Kinetics of water loss from cells at subzero temperatures and the likelihood of intracellular freezing. J. Gen. Physiol. 47, 347-369 (1963). 15. McCaa, C., and Diller, K. R. A new convection cryostage design based on simplified fabrication procedures. Cryo Lett. 8, 168-175 (1987). 16. McCaa, C. Cryomicroscopic determination of the

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