Methods for determination sf cell volurne in tissue culturea H. K. KIMELRERG,~ E. R. O'CONNOR, A N D P. SANKAR Division
of
A'k.urosurg and Depcarbnzenb of P I ~ u n n ~ ~ c ~and l o g yToxicology, Albany Medical Coblege, ACbciray, NY 12.208, U.S.A. AND
C. KEESE Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by Iowa State University on 12/17/14 For personal use only.
Rensseluea Po/ytecknic lt;lstibutc., Troy, ArY 12180, U.S. A.
Rcccivcd Noven-nber '7. 1991
KIME,LBP,RG, H.K . , (Q'CONNOR, E. R . , % . ~ N K . ~ P., K , and KEESE,C. 11992. Methods for determination of cell volunse in tissue culture. Can. J. Physiol. Pharmacol. 70: S323-S333. In this paper we present an overview of methods for detcrtnining cell vc~lumein both suspension and monolayer cultures. Data from the use of selected methods such as the Coulter counter system for suspension cultures and radiolahellcd intracellular markers for substratum-attached, ta~onolayercultures are presented. The advantages, limitaticms, and conditions under which the different methods can be used are discussed. It is pointed out that there is a need for more direct physical methods for measuring dynamic changes in the cell volume of monolayer cultures without removing the cells from the substratum. Data from a method applicable to such cultures that measures extracellular impedance are presented. KCJJ~ttord.s:cell volume, monolayer cultures. extracellular impedance, radiolabelled intracellular markers, primary astrocyte cultures.
KIMELBERG, H . K., O'CONNOR, E. R.. S ~ N K A IP., P ,et KEE,SE,C. 1992. Methods for determination cdf cell volume in tissue culture. Can. J . Physiol. Pharmacol. 70 : S323-S333. Dans cet article, nous rkvisons Ics mkthodes utilisCes pour determiner He volume celHulaire dans des cultures en couches monocellulaires et dam des cultures dc suspension. On presente des resuitats obtenus selon dcux nakthodes, I'une utilisant le conaptcur Coulaer pour les cultures de suspension et 19autreutilisant les traceurs intracellulaires radiomarques pcaur les cultures en ccduches monocellanlaires liees au substrat. On discute des avantages, dcs limises et des conditions de I'utilisation dcs diffkrentes methodes. On souligne Ha n6cessitC de dkvelopper des methodes physiques plus dircctes pour mesurer le$ variations dynamiques du volume cellulaire des cultures en couches monocellulaires sans lsoler les celllales du substrat. On prescnte les resultats d'une methode applicable 2 de tclles cultures et qui mesure I'impkdance extracellulaire. Mots cC&s : volume cellulaire. cultures en couches monocellulaires, impedance extracellulaire, traccurs intracellulaires radiomarquks. cultures primaires d'astrocytes. [Traduit par la redaction]
Intr~duction Knowledge of cell volume is critically important for the determination of the concentrations of soluble cell constituents such as ions and metabolites. It is important to h o w whether the concentration or activity of a constituent, measured, for example, in the cytoplasrnic space, changes primarily because of transport across the plasma or intracelfular cell membranes or because of changes in cell volume. Also, cell swelling is a common response to many pathological or toxic conditions, and rapid and accurate methods for determining such responses would be very useful. However, determinations of cell volume are not simple and depend on the type of experimental preparation used; satisfactory methods are not readily available for measuring rapid changes (see MacKnight and Leader 11989 for a full discussion of different methods). There is also the question of what is meant by the term cell volume. This is a complex concept. Most cells do not have simple geometric shapes, and have intracellular compartments and both ' "solid" and 'water" spaces, Changes in glid and (or) neuronall cell volume apply to many of the themes covered in this supplement, such as the 'This paper was presented at the satellite sympsiurn of the Iwternational Brain Research Organization meeting held August 10- 14, 1991. University of Saskatchewan, Saskatoon, Sask., Canada, entitled Ions, Water, and Energy in Brain Cells, and has undergone the Journal's usual peer review.
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cellular effects of brain ischemia. The failure of many cell processes or their overactivation may lead to changes in cell volume that must be regulated if pathological situations are to be avoided. Brain tissue, including certain of its constituent cells, expands in volume when the brain is harmed (Kimelberg and Ransom 19861, and is held at the appropriate volume under normal conditions by ion transport processes ultimately fueled by energy-utilizing processes (Siesjo 1984). One may view cell volume as the final common result of the three themes of this symposium, namely energy, ions, and water, and thus cell volume serves as an appropriate connecting theme for this meeting. Historically, relatively static methods were used to determine total tissue space. Tissue space can be divided into intracellular and extracellular spaces by use of a marker for which there is reasonable confidence that it does not cross the cell membrane and is therefore confined to the extracellular space. In earlier studies it was thought that C1- was limited to the extracellular space and could serve this purpose. However, Boyle and Gonway (1941) showed that the intracellular C1concentration in frog skeletal muscle, although very low, has a finite value because it is in equilibrium with the membrane potential. In other cells we now-know that the Cl- concentration can be higher than the value expected from equilibration with the potentialcIn the CNS can be above or below equilibrium in different cells (Kimelberg and Bourke 1982). The simplest way to obtain the total water space is to measure
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CAN. J . PHYSIBL. BHARMACOL. VOL. 70, 1992
the wet weight and then subtract the weight of the totally dried tissue. This may be a problem for small samples such as are obtained from monolayer cultures, so alternative methods are needed. One can use a marker that will diffuse freely in both spaces and achieve the same concentration inside and outside the cell. When this marker is radiolabelled the sensitivity of the determination is greatly increased. However, such a compound needs to be uniformly distributed within the total soluble cell volume for it t o correspond to the water space (equal t o total space minus cell solids). Thus, it is only accurate to talk of the "space" of a particular compound. One exception to this is tritiated water (%IH20), which, of course, diffuses readily into the total water space of both extra- and intra-cellular compartments. The major problem with water, however, is that it diffuses so rapidly that any separation procedure involving washing would immediately cause it to be washed out. Therefore this marker is restricted t o cell suspensions, in which one can separate the cells by centrifugation such that no washing is involved. Corrections are then made for the entrained extracellular space by means of an extracellular marker (see above). Such methods, of course, do not enable one t o distinguish between different cell types within a tissue. This is a general problem, e.g., for metabolic studies; for neural as well as other tissues, investigators have turned t o the use of isolated cell preparations or, more commonly now, cell cultures. Because in these cases the number of cells is quite small and the cells often grow as monolayers rather than in suspension, new problems for determining cell volume have arisen. With this overview of the problem we will now describe some of the methods that are used for individual cells, and will then discuss these results in the context s f the general problem of cell volume measurements in the CNS.
Methods Since much of the data presented in this paper will be a review of material already published, the Methods section will be restricted to the two methods for monolayer cultures currently used in this laboratory: radiolabelled intracellular markers and extracellular impedance measurements.
Radiolabelled inarkers Our previously published method using this approach (Kimelberg and Frangakis 1985; Kimelberg and Walz 1988) was an application to primary astrssyte cultures of the method of Kletzien et al. (l975), namely the transport of '"C-radiolabelled 3-8-methyl-Dglucose (3-OMG) into hepatocytes growing in primary culture. In brief, the growth medium is removed and replaced with a suitable volume (often 1 mL) of medium sf the desired composition. We use a Ringers type solution (mM): NaCl, 122; KC1, 3.3; NaHCO,, 25; CaCl,, I .3; KH,P04, B .2: MgSO,, 0.4, rs-glucose, 10. This medium is first bubbled with 5 % CO, - 95% air for 5 - 18 min to obtain a pH of 7.4 and is then added to the cells, which are then further equilibrated for 30 min at 37'C in a 5 % 'o02- 95 % air atmosphere in a CO, incubator. Hepes can be used instead of HC8,- but it must be remembered that this is a nonphysiological buffer, since if Hepes is imperrneant the cell is deprived of the buffering ability of the intracellular HCO,-. This medium is then replaced with less volume, usually 0.5 mE to conserve radiolabelled material, of the same C02-equilibrated medium with 0.3 pCi (1 Ci = 37 GBq) of ['4CC]3-OMGalong with 1 mM cold 3-OM@ replacing the D-glucose, and usually plus 0.3 pCi of D-[B-3H(N)]mannitol (New England Nuclear, specific activity 19.1 Cilmmol) as an extracellular marker. The cells are then immediately returned to the CO, incubator for 30 min. Medium not buffered with HCO,- does not, of course, need CO,, a practical but not a physiiologi~alconvenience.
In general, most cells respond to an osmotic change in their environment according to classic osmorneter theory, namely cells will swell in hypotonic media and shrink in hypertonic media to dissipate the osmotic gradient. In addition to this passive response many cell types are then able to regulate their volume back to near-normal levels during the continued presence of the osmotic change. Essentially all cell types exhibit a regulatory volume decrease (RVD) in response to hypotonic exposure, but only some cell types show a regulatory volume increase (RQB) in response to hypertonic exposure. To measure volume changes in response to a hypotonic exposure by means of radiolabelled markers, the isotonic medium is replaced with medium made hypotonic by reducing the NaCl, but containing the same concentrations and radioactivity of ['4C]3-OMG and [3M]mannitol. If one does not want to alter ion gradients or medium conductivity when a change is made between isotonic and hypotonic solutions, one can substitute sucrose for part of the NaCl in the original isotonic medium and then simply omit sucrose to obtain a hypotonic medium. After increasing perids of time, with a minimum of approximately 28-30 s between each point, the dishes are removed from the incubator and sets of wells rapidly washed five times (1 mL per well for each wash in ice-cold 0.29 M sucrose solution containing 10 m u Tris nitrate. pH 7.4, and 0.5 mM Ca(N0,)2), which takes about 15 s. Each individual wash volume is rapidly added with an automatic pipette, arnd after all the wash medium is added it is immediately aspirated. After the final washing the cells are solubilized by adding I M NaOH and aliquots taken for determining radioactivity and the protein content of each well. The intraceIlular radioactivity of ['6C]3-8MG and [3W]rnannitol is determined by double-label counting, and the cell spaces for [3H]mannitol and [L4CJ3-OMGare calculated using the specific activity in the medium in cpm/pL (1 cpm = 0.0167 Bq) according to the following formula: 111 space =
cpm - (mg cell protein)cgm . (pL mediunaj-'
'
Because of the extensive washing, [3H]mannitol can be omitted and one can still obtain a good estimate of the intracellular ["C]3QMG space, since the [3H]mannitoI space is usually < 16% of the total.
Ertraceldular impedance nzeasurernerats We have recently developed an impedance method for measuring cell voluine that allows a more direct and dynamic readout of cell volume than the radiolabelled marker methods described above. Hn brief, in this method (see also Mazzoni et al. 1989) the cells are placed in a confined channel separating two chambers containing electrodes. The usual Ringers type solution bathes the cells, and the electrical impedance of this solution above the cells is measured using an applied ac potential at 500 Hz (see Fig. 5A). This method is based on the fact that ionic fluids, like metallic conductors, obey Ohm's law ( R = V/P>.In this measurement the current P is fixed by the external amplifier as it passes through a Iarge external resistance (%,, = 1 MQ), which is much greater than the resistance through the channel (4,).If the current is fixed, then the resistance through the channel is directly proportional to the measured voltage (Ohm's law). If the volume of the cells increases then the voluine of the solution within the channel available for current conduction will decrease by the same amount. resulting in an increase in the measured impedance (resistance for ac field). If the cells shrink this would be recorded as a decrease in measured impedance. This method thus allows for continuous measurement of volume changes in real time. An advantage of this method is that changes in the average cell height of the monolayer can be continuously measured. This can be done becausc &, depends oaa the resistivity (or specific resistance) p of the perfusion media (in J ! . cm), the length of the channel I (cm), and the cross-sectional area A (cm2) above the cells available for current flow. Then
[2] R,, = pl/A
KIMELBERG ET AL.
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TABLE 1. Methods of cell volume determination applicable to different cell and tissue preparations and in vl'k~o Cells it2 vitro In suspension Coulter counter systemN Extracellular impedance (dynamic method for measuring extracellular spaceb Light scattering; visible light range as well as other wavelengthsb,' Total 3M2Q space minus extracellular space markerc Bntracellular marker minus extracellular marker spaces (see Table 2) Substratum-attached Intracellular markers minus extracellular markersd,' Imaging by electron microscopy (EM), high voltage electron microscopy (HVEM), interference rnicro~copy~J~.~ Extracellular impedancef, [h'"p"per Fluorescence emissionj Laser or visible light scatteringk Tissue preparations (slices, intact epithelia, isolated kidney tubules, etc.) Increase in net wet weight with extracellular marker; not cell-specificc
EM^ 'H20 space or intracellular - extracellular markersC Changes in tissue or slice height measured by interference m i c r ~ s c o p y ~ ~ ~ Extracellular impedance; not cell-specificb %on-specificmicroelectrodes (ISMS) sensitive to impermeable ions, e.g., trimethylammonium (TMA+). which can be cell specific if injected into a cell. Also, changes In extracellular space measured by this method can be interpreted as general increases in cell volume' Bra vivo Wet weightldry weight of rapidly removed tissue to obtain swelling but cannot distinguish extracellular or intracellular water gain''
EM^ Radiolabelled intracellular -extracellular markers if can be equilibrated in vivo. Tissue samples can then be rapidly removed and counted for radioactivityC Extracellular impedanceb ISMS for extracellular or intracellular space (see ISM use in tissue preparations above)' "Grinstein et al. (11984). 'Van Harreveld (1966). 'MacKnight and Leader (1989). "Kleleten et al. (1945). 'Kimelberg and Gderie (1988). /Mazzoni et al. (1988). RPa~sons et al. (1989). 'Strange et al. (1991). 'Sene et al. (1988). 'Tanc ea al. (1990). 'Fischbarg et al. (1989); Echevarria and Verkrnan (1992).
This equation can also be written as [3j R~~= p//wll
where w (em) is the width of the channel and h (cm) is the height of eke channel above the cell monolayer available for current conduction- Since P (we make solutions hypo- or h ~ ~ e r - t o nby i c omitting or adding a nonelectrolyte such as sucrose), k, and w are constants for a given experiment, RChis inversely proportional to h. Thus
[4] R,, = k/h where
Thus, by knowing the height of the channel, we can measure the percent change in resistance, and this is a direct measurement of the actual change in height of the cdl monolayer. It is critical to balance each solution to the same conductivity so that p remains the same and thus the impedance differences measured when the solutions are changed are only due to changes in the cell volume reflected as changes in h . The details of this method will be described in a separate publication (E. R. B'Csnnor, C. R. Keese, H. K. Kimelberg? and I. Giaver. in preparation).
Results and discussion To summarize the different methods mentioned in the Introduction and for hrther orientation and completeness we list in Table I the different methods that have been used not only for cells, but d s o for tissue, both in vitr(>and in vivs. We include representative references. We will now present results and discuss selected methods in more detail. Cekks in suspension Cells normally existing in suspension that have been used for volume studies include the different types of blood cells and Ehrlich ascites tumor (EAT) cells. As mentioned in the Introduction, methods for measuring cell volume for these cells are of limited relevance to brain cells, but the Coulter counter technique has been applied to monolayer cultures such as C6 glioma cells or primary astrocyte cultures (see Schneider et al. 1992 and Qlssn and Evers 1992). A potential problem here is that these cells have to be removed from their growing surfaces by either enzymatic treatment (eve.,trypsin) or Ca2+ chelators (with or without enzymes), which can potentially damage or change the cell properties in unknown ways. Ht will
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CAN. J. PHYSIOL. PHARMACOL. VOL. 70, I992
curves shows the increase in the volume after exposure s f the cells to sodium propionate. A clear shift in the modal volume to higher values is seen, and the range of values also increases because the curves now flatten. In Fig. IB it is shown that the entire response is blocked by amiloride, supporting a model of swelling in which the uradissociated propionic acid is expected to freely permeate the cell membrane and then dissociate intracellularly into propionate anion and HT. H C is then thought to exchange for extracellular Naf on the Na+/H9 exchanger leading to accumulation of sodium propionate and swelling, since it is this transporter that is blocked by arnilsride. Furthermore, in Fig. IC, in which the modal cell volume is plotted as a faanction of time, it is shown that the amilorjide block is relieved by monensin, an ionophore that catalyzes Naf /H+ exchange.
I r"
I
0
120
I
180 CELL VOLUME ( p r n j )
I
248
120
FIG. I . Volume increase of neutrophils due to exposure to sodium propionate solution as determined by the Cnulter counter technique. (A) Increase in cell volume after increasing times of exposure to 140 mM sodiunn propionate. Times above each curve indicate minutes after addition. (B) Same experiment except in the presence of I m M arnilloride. ( C ) The time course of the modal cell volume increase is plotted as a function of time; it can be seen that the increase Is totally abolished by I mM amiloride. However, when monensin, a sodium proton ionophore, is also added (at 3 min) swelling resumes because the arniloride block is bypassed by the ionophore. Reproduced with permission from Grinstein and Fumya (1984).
certainly change cell shape and the resulting suspension may not consist of mono-dispersed cells. An example of the Coulter counter technique applied to cells growing in suspension, namely neutrophils, is shown in Fig. 1 (from Grinstein and Furuya 1984). In Fig. I A the shift in the
Subsrratum-amched cells The results from several different methods for measuring cell volume in a gliomal cell line growing as monolayer cultures are shown in Table 2. As can be seen, the values obtained vary according to the method used, by up to 132 5% (range 1.97 - 5.72 p%/mg cell protein). The micrascopic method, of course, measures total cell volume, including solids, and is therefore expected to give the largest value. In this case the volume was obtained by removing cells from the culture dishes and measuring their diameters microscopica8ly, since under these conditions the cells become approximately spherical and the equation for the volume of a sphere can be used. This is necessary because the shapes of cells growing as monolayers are complex (see Fig. 4), but altering the shape of such cells to a sphere will maximize the total volume per unit surface area. 3HH7 measures the total water space. However, 3H2Cl can only be used in these cells after they have been removed from the substratum to form a suspension. The reason, in this case, is that the rapid efflux of q 2 0precludes washing the cells. Thus, the total 3H20 space s f the pelleted cells has to be measured, with an extracellular marker also present to correct for the extracellular volume (see footnotes to Table 2 for a brief description of the centrifbgation methods that need to be used for making such measurements, and note that in this case [E4C]sucrosewas used as the extracellular marker). Radiolahelled infracelbulur- exrrkscellu%arr~rarkers With radiolabelled markers, cell volume is derived from the distribution space of these markers, based on the assumption that the intracellular concentration equals the extracellular concentration when the markers have come to equilibrium. If the intracellular concentration is lower or the marker is excluded from some intracellular compartments, the apparent space will be an underestimate. For [I4C]urea these determinations were usefully made both with and without the cells being removed from the substratum, and gave similar results in each case (Table 2, top line). However, the value obtained with [I4C]3OMG for suspended cells was smaller than for [l4Clurea. Figure 2 shows the changes in cell volume for primary astrocyte monolayer cultures exposed to hypotonic solution as measured with radiolabelled markers. Note there are only two determinations under isotonic conditions (at 0 and 38 min), and the double lines indicate a discontinuity on the time scale. When the cells were exposed to hypotonic solution as described in the Methods section, the marker, in this case [L4C]3-0MG, traced the initial rapid swelling of the cells and then the characteristic decline in volume known as regulatory volume
KIMELBERG ET A&.
TABLE2. Intracellular spaces of glisma cells (rat LRMfifi cells)
Space Method
Space marker --
-
---
-
Attached cellsc Suspended cells, centrifugationd Suspended cells, centrifugationd Suspended cells, centrifugation" Suspended cells, microscopice
-
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(pE/mg proteinjb
9 4
2.47f 0.58 3.07f 0.51
4 4 2
3.93 k0.43 5.72k0.68
-
[I4C]Urea [14C]Urea ['4Ce]3-8-methyBglue~~e Tritiated water --
na
- --
1.97k0.24
-
NOTE: Reproduced from Kimelberg and Walz (1988) with permission "Number of experiments. bValues are means f SD. 'Cells remained attached to the substrate in rnultiwell plates. Urea space was determined from measurements of the radioactivity ('" content) of the medium and the cells by methods used for transport experiments on the same cells (Martin and Shain 1979). dCells were removed from culture dishes enzymatically (mixture of trypsin, collagenase, and chick senrm). Cells were allowed to equilibrate with space markers and then collected by centrifugation through silicone oil. Intracellular space was calculated from radioactivity in the pellet and supernatant. Correction for entrained extracellular fluid was made with ['4~]sucrose. 'Cells were removed from culture dishes enzymatically and photographed on a hernocyesmeter grid under phase contrast optics. Volumes were calculated from cell diameters measured on projected images by using hemocytometer grid lines as the reference distance (D. L. Martin, unpublished work).
decrease QWVB). RVD has been well-studied and various mechanisms have keen proposed (Grinstein et al. 1984; Hoffmann 1987; Hoffmann and Kolb 1991). In the experiments shown in Fig. 2 we also included ["]mannit01 to mark the extracellular space. As can be seen, under isotonic conditions the mannitol is restricted to a much smaller space, so the true intracellular f1%C]3-6MGspace is obtained by subtracting the [3H]mant~itolspace from the total [1"C]3-OMG space. This gives an actual intracellular 3-8MG space of about 4 pElmg protein. Surprisingly, to us at least. when the cells were exposed to hypotonic solution there was a rapid increase in the mannitol space, which essentially came to equilibrium with the ['TC]3-OMG space after 30 min. The simplest explanation of this phenomenon is that when the cells swell they become permeable to mannitol. As we later learned, it is known that when cells swell, the membrane permeability to relatively large intracellular solutes increases as part s f the RVB anechanism, which in plants has been shown to include an increased permeability to rnannitol (Chamberlin and Strange 1989). As shown in Table 3 the uptake of mannitol (measured over 20 min to obtain a final steady-state level) increases with the decreasing osmolality of the medium and thus with the degree of swelling. It begins at a decreased osmolality somewhere between 30 and 70 mmol. The cell then becomes permeable to what was formerly an extracellular marker. So, when using the radiolabelled marker method, one has to ensure that permeability changes to inara- or extra-cellular markers are not taking place. There are several other problems with methods using radiolabelled markers. There is a limit to how rapidly one can make the measurements. One can only make measurements with a minimum time of 15 to 30 s, since effective washing (3 -5 washes) is required. A further limitation is how rapidly the compounds can equilibrate, and also re-equilibrate when the volume rapidly changes. If the rate of equilibration or re-equilibration is slow then one may get an underestimate. 3-OMG enters the cell on the glucose equilibratory transport system (Kletzien et al. 1995) and thus a change in conditions may alter the rate of this transport system. 3-OMG, present as a tracer, also follows the glucose concentration inside and outside the cell and thus anything that changes the intracellular
concentration of glucose will alter the apparent 3-0MG space. One can do the experiments in the absence of glucose, and perhaps over a shore period of time this may not be a problem (Kletzien et al. 1975), but there could be problems associated with extended exposure to a glucose-free mediuna. The problems of changes in a specific transport system do not apply to substances that are thought to diffuse freely, such as urea. However, as with 3-OMG, such markers can also fail to equilibrate in all the different intracellular spaces or they might not equilibrate to the same concentration inside the cell as outside. Thus, different results wiH1 be found for different markers. As can be seen in Table 2. urea gives a larger space than 3-OMG, but in our studies in primary astrocyte cultures we have generally found that [I4C]urea traces a smaller space than ["C]3-8MG (Kimelberg and Walz 1988). Is this related to the fact that for the data shown in Table 2, the 3-BMG space was measured in cells put into suspension or to differences between cell types? Imaging methods Imaging methods are the only way in which the shape changes accompanying cell volume changes can be determined. Other methods will only give an overall volume increase or decrease, treating the cells as though they are a uniform block of space. In substratum-attached cultures the change in volume seems to be usually by a movement upwards of the upper surface, since this is the only unrestricted face. The free edges are flat and adhere to the lower surface of the substratum so that there is little opportunity for sideways movement. Furthermore when the monolayes becomes confluent, neighboring sells will further restrict sideways movement. An unknown factor is the degree to which the increases in surface area are accommodated by membrane unfolding due to smoothing out of surface invaginatioras. The iratracellular cytoskeleton may limit such unfoldings, or be disrupted in the process. Thus, no actual stretching (thinning) of the membrane need occur and consequently, tension increases in the plane of the membrane may be minimal. Inn any case only a 2 -3 % increase in membrane area because of actual swelling can occur without lysis (Hoffmann and KoBb 199 1). Thus it is difficult to compare osmotic pressure forces and volume changes in intact cells with the actual tensions
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CAN. J. PWYSIOE. PHARMACQL. VOL. 90: 1992
Minutes
FIG.2. Effect of exposing primary astrocyte cultures to hypotonic medium (minus I00 mM NaC1) on the ['46]3-OMGand ["lmannitol spaces. The first two values represent measurements at time zero and after 30 rnin in isotonic medium, respectively. Thereafter, hypotonic medium was added as indicated. Each point represents the mean + SE of 4 wells.
TABLE3. Effect of varying medium osmolality on 2 min [3HH]mannitoIspace of primary astrocyte
cultures OsmaolaIity (mosmol!kg)
Time (main)
[3H]Mannitol (pL!mg protein)
NOTE: Spaces were obtained as described in Fig. 2 in isotonic medium immediately after adding isotope (time 0) and after 20 rnin in isotonic (283 waosmollkg) medium. Spaces were then measured 2 wain (time 22) after changing the medium to the osmolalities indicated. Osmolaiity was decreased by removing NaCl and was measured with a vapor pressure osmometer. Values represent means + SEM, n = 4 wells.
generated in the isolated patches of membrane studied in patchclamp experiments (see Sachs 1988; Sackim 1989; Woffmann and Kolb 199 1; Kimelberg 199 1). The upwards expansion of a cell monolayer, as measured by transmission electron microscopy (EM), is illustrated in Fig. 3 for eradothelial cells of the pulmonary aorta exposed to a solution from which BOO mM NaCl was removed. The bottom panel shows the monolayer after 30 rnin of continuous exposure to hypotonic solution, showing that the cells exhibit RVD, since cell height has by now returned to normal. Endothelid cells have a relatively simple shape, and the volume changes are therefore likely to be directly related to the height changes thus visualized. We were careful to avoid fixation artifacts by adjusting the osmolarity of the fixative solution by addition or removal of sucrose to the same osmolaritiy as that of the iso-
tonic or hypotonic medium to which the cells had previously been exposed (see Fig. 3 caption). We have also described volume and shape changes of primary astrwyte cultures exposed to hypotonic solutions using 3-dimensional reconstructions of thick sections viewed by high voltage EM (HVEM) (Parsons et al. 1988). These results are shown in Fig. 4. Again, it can be seen that the predominant change in the cell is an upward movement with no detectable expansion of the attached (bottom) surface area. It can be better appreciated from these 3-dimensional images that the surface changes are complex. Such individual imaging is clearly needed to observe actual shape changes, but these techniques are also very time consuming and thus unsuitable for routine measurements. A visual imaging method applicable to living cells is differential interference contrast (DIC) microscopy coupled with an image-intensified television camera. which allows optical sectioning. Suck a method has been developed and applied to epithelial cells (Ericssn and Spring 1982; Strange et al. 1991), Using ultrathin optical sectioning techniques, 0.2-kcrn sections may be examined, giving very fine resolution in determining changes in cell vdume (Hnoue 1989). Theoretically, a confocaI microscope could also be used to examine changes in cell volume, but the cost and accessibility of such equipment has so far limited its use. Recently, Shain et al. (1992) have shown 8.6-pm optical sections through fixed astrocytoma cells, using confocal microscopy. As for the HVEM technique (see above) this could be used for determining volume if all the individual sections were summed. This could also be applied to living cells but, as stated above, ultratkin video microscopy gives almost the same resolution in sectioning at lower cost. All these imaging methods are, however, quite time consuming in terms of data collection. Inap8csZance measurements To be able to rapidly record volume changes in substratumattached monolayers in a continuous on-line fashion and in a
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KIMELBERG ET AL.
FIG.3. WVD in bovine aorta endothelial cells, growing as monolayers on cover slips, as shown by transmission electronmicroscopy. (A) Control cells. (B) 2 min and ( C ) 30 min after exposure to - BOO mM NaCB mediura-n. Note the marked dispersal of chromatin in B and the suggestion of the beginning of a return to heteroehromatin in C. The cells were fixed in 1e7F glutaraldehyde for 30 min and then 1% 8 s 0 4 for 30 min. These solutions were adjusted to the osmolarity s f the isotonic or hypotonic solutions by addition or removal of sucrose. The cells were then dehydrated by successive acetone steps and imbedded. Sections 0.2 pm thick, perpendicular to the plane of the cell substratum were cut, stained, and viewed with an AIE. HVEM at 10W kV. Final magnification ~ 7 7 5 0 Cells . were kindly provided by Dr. Peter DelVecchio.
direct manner not likely to be dependent on secondary effects such as those likely to occur with radiolabelled 3-OMG, we developed an extracellular impedance method using primary astrocyte cultures. A similar method has also been developed and applied to aortic endothelial cells by Mazzoni et al. (2989). Figure 5A shows a diagram of the apparatus and Fig. 5B an RVD response for primary astrocyte cultures. In this versionl
of the model the solution is changed in both chambers such that the solution in one chamber is higher than in the other, producing a constant flow of solution through the chamber. This avoids any possibility of changes in the composition of the solution in the channel owing to efflux or influx of ions from or into the cells. The glass cover slip (cassette) on which the cells are growing is first equilibrated in an isotonic solution and then rapidly inserted into the chamber (within 10 s). This
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CAN. J . PHYSIOL. PHARMACOE. VOL. 70, 1992
FIG.4. Tilted stereo pairs of computer recorastnactions from serial thick sections of primary astrocyte cultures. ( a ) Control. (b) One minute after exposure to hypoosmotic solution (- 100 mM NaCl). (c) Thirty minutes after suck exposure. The solid white intracellular structure is the nucleus. The contour lines, one drawn every 0.15 pm, show the shape and elevation of the cells as in a contour map. As can be seen the expansion in b is by an upwards movement of the cell, indicated by an increased number s f contour lines and steepness (the space between the lines lessens). Reproduced with permission from Parsons et al. (1989).
is a current limitation of the method and ideally a constant perfusion system that uses a closed system needs to be developed. Depending on the relative resistance of the chambers and the channel, the measurement is also dependent on the absolute volume of the solutions in the chambers, and of course, as mentioned in the Methods section, the conductivities of the different solutions have to be exactly balanced. The sensitivity limit of the measurement is almost 0.001 %, as can be seen from Fig. 5. If the monolayer is 5 pm high and the chamber 108 prn high then this is a 8.1 pm decrease in the chamber height or 2% of the cell height. This is better than the sentivities of radiolabelled markers, which are never better than 10%. Also, sensitivity could be increased by reducing the chamber height. Figure 6 shows an RVD response for both primary astrocytes and fibroblast monolayer cultures. As was expected, both cells showed an RVD response, since RVD appears to be an almost ubiquitous response of cells following exposure to hypotonic solutions (Hoffman and Kolb 1991). The difference in the magnitude of the initial swelling is more likely to be a result of different cell densities than actual differences in swelling. On the other hand, physical restraints in the cell might limit the initial swelling, the cells may have different initial heights, or the rapidity of the RVD response may attenuate the swelling to different degrees, since as soon as the cell swells such swelling presumably activates a compensating WVD. The relative responses of the cells in terms of percent change in resistance are given as the increase of the resistance
normalized to 1.00 at time zero. Thus, 1.03 represents a 3 % increase. Since resistance is proportional to the volume of the solution above the cells, which is proportional to the average cross-sectional area, one can calculate, based on the resistivity of the solution, the average decrease or increase in the height s f the cells (see Methods). By then carefully wiping off the cells and finding the resistance when cells are not present one can find the initial height of the cell and thus calculate the actual average increase. Note that what is always being measured is the average decrease in volume of the solution owing to the increase in height of the entire cell monolayer. Since the monolayes has an uneven surface topography, one is measuring an average change. Figure 7 shows the response of primary astrocyte cultures to increased medium potassium as compared with the response of fibroblasts. As can be seen, the potassium response is much slower in onset than the hypotonic-induced swelling and reaches a plateau, with no suggestion of RVD. The initial slow dip in resistance may be due to the high K + solution perhaps being slightly hyperosmotic so the cells initially shrink. Since the fibroblast cultures are the same as used in Fig. 6 , it seems that the astrocyte response in the case of raised medium K + is truly greater than the fibroblast response. Other dynamic methods Dynamic changes in substratum-attached cells can also be obtained by means of lases light scattering. Here the laser light provides a highly collimated source whose scattering depends
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.-..-Be........
.........Q.D.D.............
a~~.~........~.o.~~
0. 99 0
400
I260
800
time (s)
Cells on g k s s cover slip
FIG. 6. WVD response of astrocytes (broken line) and fibroblasts (solid line) as measured by the impedance method. The lower broken line shows the effect s f a hypotonic solution when a blank glass cover slip was inserted. The cells were exposed to - 100 rnM NaCl solution at time zero and show the characteristic RVB response. The resistance (normres) (see Fig. 5) was initially normalized to 1.00.
IMQ
(Not to Scale)
0.94 1 6
1
500
I
I
1000 1500 time (s)
L
I
2308
2500
FIG. 7 . The response of (a) astrocytes and (6) fibroblasts in an isotonic high potassium solution. (c) The uncorrected fibroblast response in which the early rapid drop in the resistance was due to a movement sf the cassette. This was simply normalized to give the plot shown in b. In these experiments the 200 naM sucrose normally in the solution was replaced with 100 mM KCH. norm.res, normalized resistance.
0.WS 8
2w
400
608
80Q
trme (8%
FIG. 5. Electrical impedance methods showing RVD in primary astrocyte cultures. (A) Model of chamber. The total height of fluid in the chamber is 1BO pm with the glass cassette without cells, which will contribute about 3-4 ym. (B) Swelling and RVB of cells measured as an increase in the impedance of the fluid. At zero time, cells growing on a no. B .5 glass cover slip were inserted into the chamber in hypotonic medium from which BOO mM sucrose had been removed. A 5 V, 580 Hz signal is applied across the 2 gold (Au) electrodes and the impedance or resistance changes, initially normalized to 1.680, are measured by the phase-sensitive detector. As can be seen, there is a 2.5% increase in the resistance of the solution above the cells. At 500 Hz there should be no current flow through the cells (Van Harreveld 1966). See Methods for further details. norm.res, normalized resistance.
on the concentration s f light-scattering molecules in the cell. Thus, as the cell expands the concentration of these compounds decreases and the reverse occurs when the cell shrinks, altering the laser light scattering accordingly (Fischbarg et al.
11989). This technique has been recently successful%yapplied to C6 glioma cells and astrocytes growing in primary culture (K. Strange, personal communication). Recently, 45 " Bight scattering has been used (Eschervarria and V e r h a n 1992) to measure volume changes in macrophages growing on cover slips, but unlike the results of Fischbarg et al. (1989) no evidence was found for water moving through the glucose transporter. In addition it should be possible to measure relative volume changes using fluorescence from a fluorescence probe trapped in the intracellular space. For example, one could use the fluorescence after excitation at the Ca2+-independent isosbestic point of h r a 2 at 360 nm, the pH-independent isosbestic point of 2,7-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) at 450 nm, or the @aH-independent emission isosbestic point of indo 1 at 447 ram (Grynkiewicz et al. 1985), or other suitable fluorescent probes. Recent work has utilized such a technique, and shown that it can be applied to monolayer cultures, including astrocytes, to measure RVD (Tauc et al. 1998; Eriksson et al. 1992). However, the field of measurement will
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have to be limited to a portion of the cytop1Jasm in order to measure a dilution of the probe. If an entire cell or group of cells is measured the total fluorescence is not likely to change. Also, it is essential that the cell membrane does not become leaky to the probe during volume changes, since changes in the intracellular cl~ncentraticsnof the probe must only be due to volume changes.
Conclusions As can be seen, there are a number of problems in measuring cell swelling by the different techniques summarized in Table 1, even in homogene
of
A'k.urosurg and Depcarbnzenb of P I ~ u n n ~ ~ c ~and l o g yToxicology, Albany Medical Coblege, ACbciray, NY 12.208, U.S.A. AND
C. KEESE Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by Iowa State University on 12/17/14 For personal use only.
Rensseluea Po/ytecknic lt;lstibutc., Troy, ArY 12180, U.S. A.
Rcccivcd Noven-nber '7. 1991
KIME,LBP,RG, H.K . , (Q'CONNOR, E. R . , % . ~ N K . ~ P., K , and KEESE,C. 11992. Methods for determination of cell volunse in tissue culture. Can. J. Physiol. Pharmacol. 70: S323-S333. In this paper we present an overview of methods for detcrtnining cell vc~lumein both suspension and monolayer cultures. Data from the use of selected methods such as the Coulter counter system for suspension cultures and radiolahellcd intracellular markers for substratum-attached, ta~onolayercultures are presented. The advantages, limitaticms, and conditions under which the different methods can be used are discussed. It is pointed out that there is a need for more direct physical methods for measuring dynamic changes in the cell volume of monolayer cultures without removing the cells from the substratum. Data from a method applicable to such cultures that measures extracellular impedance are presented. KCJJ~ttord.s:cell volume, monolayer cultures. extracellular impedance, radiolabelled intracellular markers, primary astrocyte cultures.
KIMELBERG, H . K., O'CONNOR, E. R.. S ~ N K A IP., P ,et KEE,SE,C. 1992. Methods for determination cdf cell volume in tissue culture. Can. J . Physiol. Pharmacol. 70 : S323-S333. Dans cet article, nous rkvisons Ics mkthodes utilisCes pour determiner He volume celHulaire dans des cultures en couches monocellulaires et dam des cultures dc suspension. On presente des resuitats obtenus selon dcux nakthodes, I'une utilisant le conaptcur Coulaer pour les cultures de suspension et 19autreutilisant les traceurs intracellulaires radiomarques pcaur les cultures en ccduches monocellanlaires liees au substrat. On discute des avantages, dcs limises et des conditions de I'utilisation dcs diffkrentes methodes. On souligne Ha n6cessitC de dkvelopper des methodes physiques plus dircctes pour mesurer le$ variations dynamiques du volume cellulaire des cultures en couches monocellulaires sans lsoler les celllales du substrat. On prescnte les resultats d'une methode applicable 2 de tclles cultures et qui mesure I'impkdance extracellulaire. Mots cC&s : volume cellulaire. cultures en couches monocellulaires, impedance extracellulaire, traccurs intracellulaires radiomarquks. cultures primaires d'astrocytes. [Traduit par la redaction]
Intr~duction Knowledge of cell volume is critically important for the determination of the concentrations of soluble cell constituents such as ions and metabolites. It is important to h o w whether the concentration or activity of a constituent, measured, for example, in the cytoplasrnic space, changes primarily because of transport across the plasma or intracelfular cell membranes or because of changes in cell volume. Also, cell swelling is a common response to many pathological or toxic conditions, and rapid and accurate methods for determining such responses would be very useful. However, determinations of cell volume are not simple and depend on the type of experimental preparation used; satisfactory methods are not readily available for measuring rapid changes (see MacKnight and Leader 11989 for a full discussion of different methods). There is also the question of what is meant by the term cell volume. This is a complex concept. Most cells do not have simple geometric shapes, and have intracellular compartments and both ' "solid" and 'water" spaces, Changes in glid and (or) neuronall cell volume apply to many of the themes covered in this supplement, such as the 'This paper was presented at the satellite sympsiurn of the Iwternational Brain Research Organization meeting held August 10- 14, 1991. University of Saskatchewan, Saskatoon, Sask., Canada, entitled Ions, Water, and Energy in Brain Cells, and has undergone the Journal's usual peer review.
:Author for correspondence. Printed in Canada i Imprime
ail
Canada
cellular effects of brain ischemia. The failure of many cell processes or their overactivation may lead to changes in cell volume that must be regulated if pathological situations are to be avoided. Brain tissue, including certain of its constituent cells, expands in volume when the brain is harmed (Kimelberg and Ransom 19861, and is held at the appropriate volume under normal conditions by ion transport processes ultimately fueled by energy-utilizing processes (Siesjo 1984). One may view cell volume as the final common result of the three themes of this symposium, namely energy, ions, and water, and thus cell volume serves as an appropriate connecting theme for this meeting. Historically, relatively static methods were used to determine total tissue space. Tissue space can be divided into intracellular and extracellular spaces by use of a marker for which there is reasonable confidence that it does not cross the cell membrane and is therefore confined to the extracellular space. In earlier studies it was thought that C1- was limited to the extracellular space and could serve this purpose. However, Boyle and Gonway (1941) showed that the intracellular C1concentration in frog skeletal muscle, although very low, has a finite value because it is in equilibrium with the membrane potential. In other cells we now-know that the Cl- concentration can be higher than the value expected from equilibration with the potentialcIn the CNS can be above or below equilibrium in different cells (Kimelberg and Bourke 1982). The simplest way to obtain the total water space is to measure
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CAN. J . PHYSIBL. BHARMACOL. VOL. 70, 1992
the wet weight and then subtract the weight of the totally dried tissue. This may be a problem for small samples such as are obtained from monolayer cultures, so alternative methods are needed. One can use a marker that will diffuse freely in both spaces and achieve the same concentration inside and outside the cell. When this marker is radiolabelled the sensitivity of the determination is greatly increased. However, such a compound needs to be uniformly distributed within the total soluble cell volume for it t o correspond to the water space (equal t o total space minus cell solids). Thus, it is only accurate to talk of the "space" of a particular compound. One exception to this is tritiated water (%IH20), which, of course, diffuses readily into the total water space of both extra- and intra-cellular compartments. The major problem with water, however, is that it diffuses so rapidly that any separation procedure involving washing would immediately cause it to be washed out. Therefore this marker is restricted t o cell suspensions, in which one can separate the cells by centrifugation such that no washing is involved. Corrections are then made for the entrained extracellular space by means of an extracellular marker (see above). Such methods, of course, do not enable one t o distinguish between different cell types within a tissue. This is a general problem, e.g., for metabolic studies; for neural as well as other tissues, investigators have turned t o the use of isolated cell preparations or, more commonly now, cell cultures. Because in these cases the number of cells is quite small and the cells often grow as monolayers rather than in suspension, new problems for determining cell volume have arisen. With this overview of the problem we will now describe some of the methods that are used for individual cells, and will then discuss these results in the context s f the general problem of cell volume measurements in the CNS.
Methods Since much of the data presented in this paper will be a review of material already published, the Methods section will be restricted to the two methods for monolayer cultures currently used in this laboratory: radiolabelled intracellular markers and extracellular impedance measurements.
Radiolabelled inarkers Our previously published method using this approach (Kimelberg and Frangakis 1985; Kimelberg and Walz 1988) was an application to primary astrssyte cultures of the method of Kletzien et al. (l975), namely the transport of '"C-radiolabelled 3-8-methyl-Dglucose (3-OMG) into hepatocytes growing in primary culture. In brief, the growth medium is removed and replaced with a suitable volume (often 1 mL) of medium sf the desired composition. We use a Ringers type solution (mM): NaCl, 122; KC1, 3.3; NaHCO,, 25; CaCl,, I .3; KH,P04, B .2: MgSO,, 0.4, rs-glucose, 10. This medium is first bubbled with 5 % CO, - 95% air for 5 - 18 min to obtain a pH of 7.4 and is then added to the cells, which are then further equilibrated for 30 min at 37'C in a 5 % 'o02- 95 % air atmosphere in a CO, incubator. Hepes can be used instead of HC8,- but it must be remembered that this is a nonphysiological buffer, since if Hepes is imperrneant the cell is deprived of the buffering ability of the intracellular HCO,-. This medium is then replaced with less volume, usually 0.5 mE to conserve radiolabelled material, of the same C02-equilibrated medium with 0.3 pCi (1 Ci = 37 GBq) of ['4CC]3-OMGalong with 1 mM cold 3-OM@ replacing the D-glucose, and usually plus 0.3 pCi of D-[B-3H(N)]mannitol (New England Nuclear, specific activity 19.1 Cilmmol) as an extracellular marker. The cells are then immediately returned to the CO, incubator for 30 min. Medium not buffered with HCO,- does not, of course, need CO,, a practical but not a physiiologi~alconvenience.
In general, most cells respond to an osmotic change in their environment according to classic osmorneter theory, namely cells will swell in hypotonic media and shrink in hypertonic media to dissipate the osmotic gradient. In addition to this passive response many cell types are then able to regulate their volume back to near-normal levels during the continued presence of the osmotic change. Essentially all cell types exhibit a regulatory volume decrease (RVD) in response to hypotonic exposure, but only some cell types show a regulatory volume increase (RQB) in response to hypertonic exposure. To measure volume changes in response to a hypotonic exposure by means of radiolabelled markers, the isotonic medium is replaced with medium made hypotonic by reducing the NaCl, but containing the same concentrations and radioactivity of ['4C]3-OMG and [3M]mannitol. If one does not want to alter ion gradients or medium conductivity when a change is made between isotonic and hypotonic solutions, one can substitute sucrose for part of the NaCl in the original isotonic medium and then simply omit sucrose to obtain a hypotonic medium. After increasing perids of time, with a minimum of approximately 28-30 s between each point, the dishes are removed from the incubator and sets of wells rapidly washed five times (1 mL per well for each wash in ice-cold 0.29 M sucrose solution containing 10 m u Tris nitrate. pH 7.4, and 0.5 mM Ca(N0,)2), which takes about 15 s. Each individual wash volume is rapidly added with an automatic pipette, arnd after all the wash medium is added it is immediately aspirated. After the final washing the cells are solubilized by adding I M NaOH and aliquots taken for determining radioactivity and the protein content of each well. The intraceIlular radioactivity of ['6C]3-8MG and [3W]rnannitol is determined by double-label counting, and the cell spaces for [3H]mannitol and [L4CJ3-OMGare calculated using the specific activity in the medium in cpm/pL (1 cpm = 0.0167 Bq) according to the following formula: 111 space =
cpm - (mg cell protein)cgm . (pL mediunaj-'
'
Because of the extensive washing, [3H]mannitol can be omitted and one can still obtain a good estimate of the intracellular ["C]3QMG space, since the [3H]mannitoI space is usually < 16% of the total.
Ertraceldular impedance nzeasurernerats We have recently developed an impedance method for measuring cell voluine that allows a more direct and dynamic readout of cell volume than the radiolabelled marker methods described above. Hn brief, in this method (see also Mazzoni et al. 1989) the cells are placed in a confined channel separating two chambers containing electrodes. The usual Ringers type solution bathes the cells, and the electrical impedance of this solution above the cells is measured using an applied ac potential at 500 Hz (see Fig. 5A). This method is based on the fact that ionic fluids, like metallic conductors, obey Ohm's law ( R = V/P>.In this measurement the current P is fixed by the external amplifier as it passes through a Iarge external resistance (%,, = 1 MQ), which is much greater than the resistance through the channel (4,).If the current is fixed, then the resistance through the channel is directly proportional to the measured voltage (Ohm's law). If the volume of the cells increases then the voluine of the solution within the channel available for current conduction will decrease by the same amount. resulting in an increase in the measured impedance (resistance for ac field). If the cells shrink this would be recorded as a decrease in measured impedance. This method thus allows for continuous measurement of volume changes in real time. An advantage of this method is that changes in the average cell height of the monolayer can be continuously measured. This can be done becausc &, depends oaa the resistivity (or specific resistance) p of the perfusion media (in J ! . cm), the length of the channel I (cm), and the cross-sectional area A (cm2) above the cells available for current flow. Then
[2] R,, = pl/A
KIMELBERG ET AL.
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TABLE 1. Methods of cell volume determination applicable to different cell and tissue preparations and in vl'k~o Cells it2 vitro In suspension Coulter counter systemN Extracellular impedance (dynamic method for measuring extracellular spaceb Light scattering; visible light range as well as other wavelengthsb,' Total 3M2Q space minus extracellular space markerc Bntracellular marker minus extracellular marker spaces (see Table 2) Substratum-attached Intracellular markers minus extracellular markersd,' Imaging by electron microscopy (EM), high voltage electron microscopy (HVEM), interference rnicro~copy~J~.~ Extracellular impedancef, [h'"p"per Fluorescence emissionj Laser or visible light scatteringk Tissue preparations (slices, intact epithelia, isolated kidney tubules, etc.) Increase in net wet weight with extracellular marker; not cell-specificc
EM^ 'H20 space or intracellular - extracellular markersC Changes in tissue or slice height measured by interference m i c r ~ s c o p y ~ ~ ~ Extracellular impedance; not cell-specificb %on-specificmicroelectrodes (ISMS) sensitive to impermeable ions, e.g., trimethylammonium (TMA+). which can be cell specific if injected into a cell. Also, changes In extracellular space measured by this method can be interpreted as general increases in cell volume' Bra vivo Wet weightldry weight of rapidly removed tissue to obtain swelling but cannot distinguish extracellular or intracellular water gain''
EM^ Radiolabelled intracellular -extracellular markers if can be equilibrated in vivo. Tissue samples can then be rapidly removed and counted for radioactivityC Extracellular impedanceb ISMS for extracellular or intracellular space (see ISM use in tissue preparations above)' "Grinstein et al. (11984). 'Van Harreveld (1966). 'MacKnight and Leader (1989). "Kleleten et al. (1945). 'Kimelberg and Gderie (1988). /Mazzoni et al. (1988). RPa~sons et al. (1989). 'Strange et al. (1991). 'Sene et al. (1988). 'Tanc ea al. (1990). 'Fischbarg et al. (1989); Echevarria and Verkrnan (1992).
This equation can also be written as [3j R~~= p//wll
where w (em) is the width of the channel and h (cm) is the height of eke channel above the cell monolayer available for current conduction- Since P (we make solutions hypo- or h ~ ~ e r - t o nby i c omitting or adding a nonelectrolyte such as sucrose), k, and w are constants for a given experiment, RChis inversely proportional to h. Thus
[4] R,, = k/h where
Thus, by knowing the height of the channel, we can measure the percent change in resistance, and this is a direct measurement of the actual change in height of the cdl monolayer. It is critical to balance each solution to the same conductivity so that p remains the same and thus the impedance differences measured when the solutions are changed are only due to changes in the cell volume reflected as changes in h . The details of this method will be described in a separate publication (E. R. B'Csnnor, C. R. Keese, H. K. Kimelberg? and I. Giaver. in preparation).
Results and discussion To summarize the different methods mentioned in the Introduction and for hrther orientation and completeness we list in Table I the different methods that have been used not only for cells, but d s o for tissue, both in vitr(>and in vivs. We include representative references. We will now present results and discuss selected methods in more detail. Cekks in suspension Cells normally existing in suspension that have been used for volume studies include the different types of blood cells and Ehrlich ascites tumor (EAT) cells. As mentioned in the Introduction, methods for measuring cell volume for these cells are of limited relevance to brain cells, but the Coulter counter technique has been applied to monolayer cultures such as C6 glioma cells or primary astrocyte cultures (see Schneider et al. 1992 and Qlssn and Evers 1992). A potential problem here is that these cells have to be removed from their growing surfaces by either enzymatic treatment (eve.,trypsin) or Ca2+ chelators (with or without enzymes), which can potentially damage or change the cell properties in unknown ways. Ht will
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CAN. J. PHYSIOL. PHARMACOL. VOL. 70, I992
curves shows the increase in the volume after exposure s f the cells to sodium propionate. A clear shift in the modal volume to higher values is seen, and the range of values also increases because the curves now flatten. In Fig. IB it is shown that the entire response is blocked by amiloride, supporting a model of swelling in which the uradissociated propionic acid is expected to freely permeate the cell membrane and then dissociate intracellularly into propionate anion and HT. H C is then thought to exchange for extracellular Naf on the Na+/H9 exchanger leading to accumulation of sodium propionate and swelling, since it is this transporter that is blocked by arnilsride. Furthermore, in Fig. IC, in which the modal cell volume is plotted as a faanction of time, it is shown that the amilorjide block is relieved by monensin, an ionophore that catalyzes Naf /H+ exchange.
I r"
I
0
120
I
180 CELL VOLUME ( p r n j )
I
248
120
FIG. I . Volume increase of neutrophils due to exposure to sodium propionate solution as determined by the Cnulter counter technique. (A) Increase in cell volume after increasing times of exposure to 140 mM sodiunn propionate. Times above each curve indicate minutes after addition. (B) Same experiment except in the presence of I m M arnilloride. ( C ) The time course of the modal cell volume increase is plotted as a function of time; it can be seen that the increase Is totally abolished by I mM amiloride. However, when monensin, a sodium proton ionophore, is also added (at 3 min) swelling resumes because the arniloride block is bypassed by the ionophore. Reproduced with permission from Grinstein and Fumya (1984).
certainly change cell shape and the resulting suspension may not consist of mono-dispersed cells. An example of the Coulter counter technique applied to cells growing in suspension, namely neutrophils, is shown in Fig. 1 (from Grinstein and Furuya 1984). In Fig. I A the shift in the
Subsrratum-amched cells The results from several different methods for measuring cell volume in a gliomal cell line growing as monolayer cultures are shown in Table 2. As can be seen, the values obtained vary according to the method used, by up to 132 5% (range 1.97 - 5.72 p%/mg cell protein). The micrascopic method, of course, measures total cell volume, including solids, and is therefore expected to give the largest value. In this case the volume was obtained by removing cells from the culture dishes and measuring their diameters microscopica8ly, since under these conditions the cells become approximately spherical and the equation for the volume of a sphere can be used. This is necessary because the shapes of cells growing as monolayers are complex (see Fig. 4), but altering the shape of such cells to a sphere will maximize the total volume per unit surface area. 3HH7 measures the total water space. However, 3H2Cl can only be used in these cells after they have been removed from the substratum to form a suspension. The reason, in this case, is that the rapid efflux of q 2 0precludes washing the cells. Thus, the total 3H20 space s f the pelleted cells has to be measured, with an extracellular marker also present to correct for the extracellular volume (see footnotes to Table 2 for a brief description of the centrifbgation methods that need to be used for making such measurements, and note that in this case [E4C]sucrosewas used as the extracellular marker). Radiolahelled infracelbulur- exrrkscellu%arr~rarkers With radiolabelled markers, cell volume is derived from the distribution space of these markers, based on the assumption that the intracellular concentration equals the extracellular concentration when the markers have come to equilibrium. If the intracellular concentration is lower or the marker is excluded from some intracellular compartments, the apparent space will be an underestimate. For [I4C]urea these determinations were usefully made both with and without the cells being removed from the substratum, and gave similar results in each case (Table 2, top line). However, the value obtained with [I4C]3OMG for suspended cells was smaller than for [l4Clurea. Figure 2 shows the changes in cell volume for primary astrocyte monolayer cultures exposed to hypotonic solution as measured with radiolabelled markers. Note there are only two determinations under isotonic conditions (at 0 and 38 min), and the double lines indicate a discontinuity on the time scale. When the cells were exposed to hypotonic solution as described in the Methods section, the marker, in this case [L4C]3-0MG, traced the initial rapid swelling of the cells and then the characteristic decline in volume known as regulatory volume
KIMELBERG ET A&.
TABLE2. Intracellular spaces of glisma cells (rat LRMfifi cells)
Space Method
Space marker --
-
---
-
Attached cellsc Suspended cells, centrifugationd Suspended cells, centrifugationd Suspended cells, centrifugation" Suspended cells, microscopice
-
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(pE/mg proteinjb
9 4
2.47f 0.58 3.07f 0.51
4 4 2
3.93 k0.43 5.72k0.68
-
[I4C]Urea [14C]Urea ['4Ce]3-8-methyBglue~~e Tritiated water --
na
- --
1.97k0.24
-
NOTE: Reproduced from Kimelberg and Walz (1988) with permission "Number of experiments. bValues are means f SD. 'Cells remained attached to the substrate in rnultiwell plates. Urea space was determined from measurements of the radioactivity ('" content) of the medium and the cells by methods used for transport experiments on the same cells (Martin and Shain 1979). dCells were removed from culture dishes enzymatically (mixture of trypsin, collagenase, and chick senrm). Cells were allowed to equilibrate with space markers and then collected by centrifugation through silicone oil. Intracellular space was calculated from radioactivity in the pellet and supernatant. Correction for entrained extracellular fluid was made with ['4~]sucrose. 'Cells were removed from culture dishes enzymatically and photographed on a hernocyesmeter grid under phase contrast optics. Volumes were calculated from cell diameters measured on projected images by using hemocytometer grid lines as the reference distance (D. L. Martin, unpublished work).
decrease QWVB). RVD has been well-studied and various mechanisms have keen proposed (Grinstein et al. 1984; Hoffmann 1987; Hoffmann and Kolb 1991). In the experiments shown in Fig. 2 we also included ["]mannit01 to mark the extracellular space. As can be seen, under isotonic conditions the mannitol is restricted to a much smaller space, so the true intracellular f1%C]3-6MGspace is obtained by subtracting the [3H]mant~itolspace from the total [1"C]3-OMG space. This gives an actual intracellular 3-8MG space of about 4 pElmg protein. Surprisingly, to us at least. when the cells were exposed to hypotonic solution there was a rapid increase in the mannitol space, which essentially came to equilibrium with the ['TC]3-OMG space after 30 min. The simplest explanation of this phenomenon is that when the cells swell they become permeable to mannitol. As we later learned, it is known that when cells swell, the membrane permeability to relatively large intracellular solutes increases as part s f the RVB anechanism, which in plants has been shown to include an increased permeability to rnannitol (Chamberlin and Strange 1989). As shown in Table 3 the uptake of mannitol (measured over 20 min to obtain a final steady-state level) increases with the decreasing osmolality of the medium and thus with the degree of swelling. It begins at a decreased osmolality somewhere between 30 and 70 mmol. The cell then becomes permeable to what was formerly an extracellular marker. So, when using the radiolabelled marker method, one has to ensure that permeability changes to inara- or extra-cellular markers are not taking place. There are several other problems with methods using radiolabelled markers. There is a limit to how rapidly one can make the measurements. One can only make measurements with a minimum time of 15 to 30 s, since effective washing (3 -5 washes) is required. A further limitation is how rapidly the compounds can equilibrate, and also re-equilibrate when the volume rapidly changes. If the rate of equilibration or re-equilibration is slow then one may get an underestimate. 3-OMG enters the cell on the glucose equilibratory transport system (Kletzien et al. 1995) and thus a change in conditions may alter the rate of this transport system. 3-OMG, present as a tracer, also follows the glucose concentration inside and outside the cell and thus anything that changes the intracellular
concentration of glucose will alter the apparent 3-0MG space. One can do the experiments in the absence of glucose, and perhaps over a shore period of time this may not be a problem (Kletzien et al. 1975), but there could be problems associated with extended exposure to a glucose-free mediuna. The problems of changes in a specific transport system do not apply to substances that are thought to diffuse freely, such as urea. However, as with 3-OMG, such markers can also fail to equilibrate in all the different intracellular spaces or they might not equilibrate to the same concentration inside the cell as outside. Thus, different results wiH1 be found for different markers. As can be seen in Table 2. urea gives a larger space than 3-OMG, but in our studies in primary astrocyte cultures we have generally found that [I4C]urea traces a smaller space than ["C]3-8MG (Kimelberg and Walz 1988). Is this related to the fact that for the data shown in Table 2, the 3-BMG space was measured in cells put into suspension or to differences between cell types? Imaging methods Imaging methods are the only way in which the shape changes accompanying cell volume changes can be determined. Other methods will only give an overall volume increase or decrease, treating the cells as though they are a uniform block of space. In substratum-attached cultures the change in volume seems to be usually by a movement upwards of the upper surface, since this is the only unrestricted face. The free edges are flat and adhere to the lower surface of the substratum so that there is little opportunity for sideways movement. Furthermore when the monolayes becomes confluent, neighboring sells will further restrict sideways movement. An unknown factor is the degree to which the increases in surface area are accommodated by membrane unfolding due to smoothing out of surface invaginatioras. The iratracellular cytoskeleton may limit such unfoldings, or be disrupted in the process. Thus, no actual stretching (thinning) of the membrane need occur and consequently, tension increases in the plane of the membrane may be minimal. Inn any case only a 2 -3 % increase in membrane area because of actual swelling can occur without lysis (Hoffmann and KoBb 199 1). Thus it is difficult to compare osmotic pressure forces and volume changes in intact cells with the actual tensions
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CAN. J. PWYSIOE. PHARMACQL. VOL. 90: 1992
Minutes
FIG.2. Effect of exposing primary astrocyte cultures to hypotonic medium (minus I00 mM NaC1) on the ['46]3-OMGand ["lmannitol spaces. The first two values represent measurements at time zero and after 30 rnin in isotonic medium, respectively. Thereafter, hypotonic medium was added as indicated. Each point represents the mean + SE of 4 wells.
TABLE3. Effect of varying medium osmolality on 2 min [3HH]mannitoIspace of primary astrocyte
cultures OsmaolaIity (mosmol!kg)
Time (main)
[3H]Mannitol (pL!mg protein)
NOTE: Spaces were obtained as described in Fig. 2 in isotonic medium immediately after adding isotope (time 0) and after 20 rnin in isotonic (283 waosmollkg) medium. Spaces were then measured 2 wain (time 22) after changing the medium to the osmolalities indicated. Osmolaiity was decreased by removing NaCl and was measured with a vapor pressure osmometer. Values represent means + SEM, n = 4 wells.
generated in the isolated patches of membrane studied in patchclamp experiments (see Sachs 1988; Sackim 1989; Woffmann and Kolb 199 1; Kimelberg 199 1). The upwards expansion of a cell monolayer, as measured by transmission electron microscopy (EM), is illustrated in Fig. 3 for eradothelial cells of the pulmonary aorta exposed to a solution from which BOO mM NaCl was removed. The bottom panel shows the monolayer after 30 rnin of continuous exposure to hypotonic solution, showing that the cells exhibit RVD, since cell height has by now returned to normal. Endothelid cells have a relatively simple shape, and the volume changes are therefore likely to be directly related to the height changes thus visualized. We were careful to avoid fixation artifacts by adjusting the osmolarity of the fixative solution by addition or removal of sucrose to the same osmolaritiy as that of the iso-
tonic or hypotonic medium to which the cells had previously been exposed (see Fig. 3 caption). We have also described volume and shape changes of primary astrwyte cultures exposed to hypotonic solutions using 3-dimensional reconstructions of thick sections viewed by high voltage EM (HVEM) (Parsons et al. 1988). These results are shown in Fig. 4. Again, it can be seen that the predominant change in the cell is an upward movement with no detectable expansion of the attached (bottom) surface area. It can be better appreciated from these 3-dimensional images that the surface changes are complex. Such individual imaging is clearly needed to observe actual shape changes, but these techniques are also very time consuming and thus unsuitable for routine measurements. A visual imaging method applicable to living cells is differential interference contrast (DIC) microscopy coupled with an image-intensified television camera. which allows optical sectioning. Suck a method has been developed and applied to epithelial cells (Ericssn and Spring 1982; Strange et al. 1991), Using ultrathin optical sectioning techniques, 0.2-kcrn sections may be examined, giving very fine resolution in determining changes in cell vdume (Hnoue 1989). Theoretically, a confocaI microscope could also be used to examine changes in cell volume, but the cost and accessibility of such equipment has so far limited its use. Recently, Shain et al. (1992) have shown 8.6-pm optical sections through fixed astrocytoma cells, using confocal microscopy. As for the HVEM technique (see above) this could be used for determining volume if all the individual sections were summed. This could also be applied to living cells but, as stated above, ultratkin video microscopy gives almost the same resolution in sectioning at lower cost. All these imaging methods are, however, quite time consuming in terms of data collection. Inap8csZance measurements To be able to rapidly record volume changes in substratumattached monolayers in a continuous on-line fashion and in a
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KIMELBERG ET AL.
FIG.3. WVD in bovine aorta endothelial cells, growing as monolayers on cover slips, as shown by transmission electronmicroscopy. (A) Control cells. (B) 2 min and ( C ) 30 min after exposure to - BOO mM NaCB mediura-n. Note the marked dispersal of chromatin in B and the suggestion of the beginning of a return to heteroehromatin in C. The cells were fixed in 1e7F glutaraldehyde for 30 min and then 1% 8 s 0 4 for 30 min. These solutions were adjusted to the osmolarity s f the isotonic or hypotonic solutions by addition or removal of sucrose. The cells were then dehydrated by successive acetone steps and imbedded. Sections 0.2 pm thick, perpendicular to the plane of the cell substratum were cut, stained, and viewed with an AIE. HVEM at 10W kV. Final magnification ~ 7 7 5 0 Cells . were kindly provided by Dr. Peter DelVecchio.
direct manner not likely to be dependent on secondary effects such as those likely to occur with radiolabelled 3-OMG, we developed an extracellular impedance method using primary astrocyte cultures. A similar method has also been developed and applied to aortic endothelial cells by Mazzoni et al. (2989). Figure 5A shows a diagram of the apparatus and Fig. 5B an RVD response for primary astrocyte cultures. In this versionl
of the model the solution is changed in both chambers such that the solution in one chamber is higher than in the other, producing a constant flow of solution through the chamber. This avoids any possibility of changes in the composition of the solution in the channel owing to efflux or influx of ions from or into the cells. The glass cover slip (cassette) on which the cells are growing is first equilibrated in an isotonic solution and then rapidly inserted into the chamber (within 10 s). This
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CAN. J . PHYSIOL. PHARMACOE. VOL. 70, 1992
FIG.4. Tilted stereo pairs of computer recorastnactions from serial thick sections of primary astrocyte cultures. ( a ) Control. (b) One minute after exposure to hypoosmotic solution (- 100 mM NaCl). (c) Thirty minutes after suck exposure. The solid white intracellular structure is the nucleus. The contour lines, one drawn every 0.15 pm, show the shape and elevation of the cells as in a contour map. As can be seen the expansion in b is by an upwards movement of the cell, indicated by an increased number s f contour lines and steepness (the space between the lines lessens). Reproduced with permission from Parsons et al. (1989).
is a current limitation of the method and ideally a constant perfusion system that uses a closed system needs to be developed. Depending on the relative resistance of the chambers and the channel, the measurement is also dependent on the absolute volume of the solutions in the chambers, and of course, as mentioned in the Methods section, the conductivities of the different solutions have to be exactly balanced. The sensitivity limit of the measurement is almost 0.001 %, as can be seen from Fig. 5. If the monolayer is 5 pm high and the chamber 108 prn high then this is a 8.1 pm decrease in the chamber height or 2% of the cell height. This is better than the sentivities of radiolabelled markers, which are never better than 10%. Also, sensitivity could be increased by reducing the chamber height. Figure 6 shows an RVD response for both primary astrocytes and fibroblast monolayer cultures. As was expected, both cells showed an RVD response, since RVD appears to be an almost ubiquitous response of cells following exposure to hypotonic solutions (Hoffman and Kolb 1991). The difference in the magnitude of the initial swelling is more likely to be a result of different cell densities than actual differences in swelling. On the other hand, physical restraints in the cell might limit the initial swelling, the cells may have different initial heights, or the rapidity of the RVD response may attenuate the swelling to different degrees, since as soon as the cell swells such swelling presumably activates a compensating WVD. The relative responses of the cells in terms of percent change in resistance are given as the increase of the resistance
normalized to 1.00 at time zero. Thus, 1.03 represents a 3 % increase. Since resistance is proportional to the volume of the solution above the cells, which is proportional to the average cross-sectional area, one can calculate, based on the resistivity of the solution, the average decrease or increase in the height s f the cells (see Methods). By then carefully wiping off the cells and finding the resistance when cells are not present one can find the initial height of the cell and thus calculate the actual average increase. Note that what is always being measured is the average decrease in volume of the solution owing to the increase in height of the entire cell monolayer. Since the monolayes has an uneven surface topography, one is measuring an average change. Figure 7 shows the response of primary astrocyte cultures to increased medium potassium as compared with the response of fibroblasts. As can be seen, the potassium response is much slower in onset than the hypotonic-induced swelling and reaches a plateau, with no suggestion of RVD. The initial slow dip in resistance may be due to the high K + solution perhaps being slightly hyperosmotic so the cells initially shrink. Since the fibroblast cultures are the same as used in Fig. 6 , it seems that the astrocyte response in the case of raised medium K + is truly greater than the fibroblast response. Other dynamic methods Dynamic changes in substratum-attached cells can also be obtained by means of lases light scattering. Here the laser light provides a highly collimated source whose scattering depends
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.-..-Be........
.........Q.D.D.............
a~~.~........~.o.~~
0. 99 0
400
I260
800
time (s)
Cells on g k s s cover slip
FIG. 6. WVD response of astrocytes (broken line) and fibroblasts (solid line) as measured by the impedance method. The lower broken line shows the effect s f a hypotonic solution when a blank glass cover slip was inserted. The cells were exposed to - 100 rnM NaCl solution at time zero and show the characteristic RVB response. The resistance (normres) (see Fig. 5) was initially normalized to 1.00.
IMQ
(Not to Scale)
0.94 1 6
1
500
I
I
1000 1500 time (s)
L
I
2308
2500
FIG. 7 . The response of (a) astrocytes and (6) fibroblasts in an isotonic high potassium solution. (c) The uncorrected fibroblast response in which the early rapid drop in the resistance was due to a movement sf the cassette. This was simply normalized to give the plot shown in b. In these experiments the 200 naM sucrose normally in the solution was replaced with 100 mM KCH. norm.res, normalized resistance.
0.WS 8
2w
400
608
80Q
trme (8%
FIG. 5. Electrical impedance methods showing RVD in primary astrocyte cultures. (A) Model of chamber. The total height of fluid in the chamber is 1BO pm with the glass cassette without cells, which will contribute about 3-4 ym. (B) Swelling and RVB of cells measured as an increase in the impedance of the fluid. At zero time, cells growing on a no. B .5 glass cover slip were inserted into the chamber in hypotonic medium from which BOO mM sucrose had been removed. A 5 V, 580 Hz signal is applied across the 2 gold (Au) electrodes and the impedance or resistance changes, initially normalized to 1.680, are measured by the phase-sensitive detector. As can be seen, there is a 2.5% increase in the resistance of the solution above the cells. At 500 Hz there should be no current flow through the cells (Van Harreveld 1966). See Methods for further details. norm.res, normalized resistance.
on the concentration s f light-scattering molecules in the cell. Thus, as the cell expands the concentration of these compounds decreases and the reverse occurs when the cell shrinks, altering the laser light scattering accordingly (Fischbarg et al.
11989). This technique has been recently successful%yapplied to C6 glioma cells and astrocytes growing in primary culture (K. Strange, personal communication). Recently, 45 " Bight scattering has been used (Eschervarria and V e r h a n 1992) to measure volume changes in macrophages growing on cover slips, but unlike the results of Fischbarg et al. (1989) no evidence was found for water moving through the glucose transporter. In addition it should be possible to measure relative volume changes using fluorescence from a fluorescence probe trapped in the intracellular space. For example, one could use the fluorescence after excitation at the Ca2+-independent isosbestic point of h r a 2 at 360 nm, the pH-independent isosbestic point of 2,7-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) at 450 nm, or the @aH-independent emission isosbestic point of indo 1 at 447 ram (Grynkiewicz et al. 1985), or other suitable fluorescent probes. Recent work has utilized such a technique, and shown that it can be applied to monolayer cultures, including astrocytes, to measure RVD (Tauc et al. 1998; Eriksson et al. 1992). However, the field of measurement will
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have to be limited to a portion of the cytop1Jasm in order to measure a dilution of the probe. If an entire cell or group of cells is measured the total fluorescence is not likely to change. Also, it is essential that the cell membrane does not become leaky to the probe during volume changes, since changes in the intracellular cl~ncentraticsnof the probe must only be due to volume changes.
Conclusions As can be seen, there are a number of problems in measuring cell swelling by the different techniques summarized in Table 1, even in homogene
