NMR IN BIOMEDICINE
NMR Biomed 11, 11–18 (1998)
Monitoring of cell volume and water exchange time in
perfused cells by diffusion-weighted 1H NMR spectroscopy
Josef Pfeuffer, Ulrich FloÈgel and Dieter Leibfritz*
Fachbereich Biologie/Chemie, UniversitaÈt Bremen, D-28334, Bremen, Germany
Received 12 May 1997; revised 14 August 1997; accepted 26 August 1997
ABSTRACT: Diffusion of intracellular water was measured in perfused cells embedded in basement membrane gel
threads. F98 glioma cells, primary astrocytes, and epithelial KB cells were used and were exposed to osmotic stress,
immunosuppressiva, the water channel blocker p-chloromercuriobenzenesulfonate (pCMBS), and apoptotic
conditions. With diffusion-weighted 1H NMR spectroscopy changes in the intracellular signal could be monitored
and quantified with single signal (ss), constant diffusion time (ct), and constant gradient strength (cg) experiments.
The temporal resolution of the ss monitoring was 3.5 s with a standard deviation of 0.5% of the signal intensity and 32
s (3%) with ct monitoring, respectively. A mean intracellular residence time of water was determined with the cg
experiment to about 50 ms. Changes of this exchange time from (51.9 1.0) to (59.0 1.1) ms were observed during
treatment with pCMBS. The changes in the diffusion attenuated signal could be simulated analytically varying the
intracellular volume fraction and exchange time by combination of restricted diffusion (Tanner model) and water
exchange (Kärger model). This sensitive and noninvasive NMR method on perfused cells allows to determine
changes in the intracellular volume and residence time in a simple and accurate manner. Many further applications as
anoxia, volume regulation, ischemia and treatment with various pharmaceuticals are conceiveable to follow up their
effect on the cell volume and the exchange time of intracellular water.  1998 John Wiley & Sons, Ltd.
KEYWORDS: 1H NMR; cell volume; exchange time; intracellular water; F98 glioma cells; primary astrocytes; KB
cells; basement membrane gel
INTRODUCTION
Cell cultures are widely used to study metabolic
pathways and serve as an in vitro model which allows
much better experimental control of physiological
parameters than tissue or in vivo studies. Various cell
preparations are known depending upon the aim of the
investigation, e.g. cells in suspension or immobilized
cells grown on microcarrier beads, in hollow fiber
bioreactors or in gel threads.1 Specific interest exists in
the potential selection of intracellular information
regarding metabolite, ion and water exchange. In order
to get knowledge about selected compartments of a
biological system2,3 different physical properties as
relaxation times, refraction index, impedance or diffusion
constants are used. This study focuses on NMR methods
which are capable to quantify changes in the intracellular
water and its exchange across the cell membrane.
Cell volume measurements are mostly carried out by
flow cytometry based on light scattering and conductiv*Correspondence to: D. Leibfritz, Universität Bremen, FB2-NW2, D28334, Bremen, Germany.
E-mail: [email protected]
Abbreviations used: BMG, basement membrane gel; DMEM,
Dulbecco’s modified Eagle medium; pCMBS, p-chloromercuriobenzenesulfonate.
 1998 John Wiley & Sons, Ltd.
ity.4–8 With fluorescence or NMR spectroscopic methods
one is able to quantify the intracellular signal by addition
of intracellular or extracellular marker,9,10 relaxation11,12
or chemical shift13 reagents. Accurate changes of the cell
diameter can also be detected by sophisticated video
imaging.14 The intracellular water exchange time and the
permeability of the cell membrane are assessable by
influx or efflux techniques (using e.g. deuterated or
tritiated water15–17 or Li‡, Na‡, Cs‡18–20) and spectroscopic fluorescence or NMR techniques relying on
different relaxation21–26 or diffusion properties.27–29
Permeability changes can also be monitored by scattered
light intensity measurements.30–32 Some of these methods have the disadvantage to be either applicable to cells
in suspension only, which may affect the natural response
of normally adherently growing cells, or to require the
addition of chemicals to obtain the requested information, which also may exert undesired side effects.
Recently, a noninvasive NMR method of acquiring
intracellular water and metabolite signals has been
described.33–35 The separation of extracellular and
intracellular information is achieved using the different
diffusion properties of the molecules in the compartments
undergoing free and restricted diffusion. Intracellular
water diffusion is restricted by the cell membrane and
hereby the intracellular diffusion coefficient is apparently
CCC 0952–3480/98/010011–08 $17.50
12
J. PFEUFFER, U. FLÖGEL AND D. LEIBFRITZ
Table 1. Variation of the physiological parameters p2 and t2 in the analytical model and their effects on the relative intracellular
signal intensity Srel.
(a)a
p2b
Sreld
(b)
t 2 / msc
Sreld
50
1.0
55
1.06
60
1.12
(c)
p2
t 2 / ms
Sreld
0.05
50
1.0
0.055
51.6
1.12
0.06
53.1
1.25
a
0.05
1.0
0.055
1.1
0.06
1.2
0.065
1.3
0.07
1.4
0.10
2.0
65
1.17
70
1.21
100
1.40
0.065
54.6
1.38
0.07
55.9
1.50
0.10
63.0
2.30
the data are plotted in Fig. 1; b t2 = 50 ms = Const; c p2 = 0.05 = Const; d Srel is calculated at b = 20000 s/mm2.
lowered up to two orders of magnitude. At high fieldgradient strengths the intracellular signal component can
be selected by diffusion-weighted 1H NMR spectroscopy.
In this study diffusion-weighted 1H NMR spectroscopy
is used to measure the intracellular water signal and to
monitor alterations in cell volume and water exchange
time on perfused cells. For the NMR measurements, the
cells are embedded in basement membrane gel (BMG)
threads, a substrate which permits the continuous
monitoring of perfused cells over a couple of days under
physiological conditions.36 The validity and evidence of
the methods is confirmed and restrictions are discussed
based on an analytical two-compartment model.
MODEL
The diffusion attenuation of the water signal measured by
the pulsed-field-gradient spin echo technique was
recently investigated in extended analytical simulations
and experimental studies.37,38 In perfused glioma cells
the time-dependence of the intracellular signal gave clear
evidence for restricted diffusion at permeable boundaries.
The cellular microstructure was characterized by a
distribution of propagation lengths, and the mean
residence time for intracellular water was determined to
about 50 ms. A combination of the Tanner formula39 for
restricted diffusion in a box and the Kärger equations40,41
for a two-compartment system with exchange was used
for the analytical model calculations. The signal
attenuation S = S (q̃2, tD, D1, D2, p2, t2, a, a) was
dependent on the experimental parameters q̃2 and tD (q
value and diffusion time), on the physiological parameters D1, D2, p2, t2 (extracellular/intracellular diffusion
constant, intracellular volume fraction and exchange
time), and on the morphological parameter a (box
length). A Gaussian distribution of box lengths with
mean a and width a was used to account for the complex
cellular morphology with regard to cell shape and size.
This model is able to describe the effects of
physiological changes on the signal attenuation curves
during cell swelling. p2 and t2 are varied in the Table 1
(a)–(c) and the resulting relative signal intensity Srel is
calculated at b = 20 000 s/mm2.
Cell volume
Figure 1. Model calculation of the effects of cellular swelling
on the diffusion attenuation (ct data) at large b values. The
intracellular volume fraction is linearly varied by
p2 = [0.05,0.06,¼,0.1]. Model parameters: diffusion constants D1,2 = 3.0/1.010ÿ3 mm2/s, intracellular exchange
time t2 = 50 ms, box lengths' distribution a = 5 mm/
a = 10 mm, = 10 ms, tD = 35 ms
 1998 John Wiley & Sons, Ltd.
p2 Is increased by 100% from p2 = 0.05–0.1 (Table 1(a)
and Fig. 1), while t2 = 50 ms and a = 5 mm / a = 10 mm is
kept constant (the parameters = 10 ms, tD = 35 ms, and
Gmax = 333 mT/m in Fig. 1 correspond to a typical ct
experiment as shown in the next section). Srel correlates
exactly with the intracellular volume fraction p2 for
b > 4000 s/mm2, e.g. increasing p2 from 0.05 to 0.1
increases by 100% S(b > 4000). At small b values the
extracellular signal component (free diffusion) mainly
has control of the diffusion attenuation. At larger b values
the intercept of the second signal component changes
with the intracellular volume fraction (restricted intracellular diffusion) whereas the appertaining slope and
curvature of the curves remain constant. The apparent,
intracellular
diffusion
coefficient
Dapp,intra
= 0.027110ÿ3 mm2/s is determined from the negative
slopes at b = 20000 s/mm2, which remains constant for all
used pp
The inherent apparent displacement
2.
rapp = 6 Dapp tD is 2.39 mm.
NMR IN BIOMEDICINE, VOL. 11, 11–18 (1998)
MONITORING OF CELL VOLUME AND WATER EXCHANGE TIME
Intracellular exchange time
Another point of interest regarding cell physiology are
changes in the membrane permeability. The corresponding parameter in the model is the intracellular exchange
time which is related to the permeability by the volumeto-surface ratio of the cells.16 As simulated previously37,38 the mean intracellular residence time tintra
can be calculated from the negative reciprocal slope of a
cg experiment (see below) at larger q values and diffusion
times.
An increase of t2 from 50 to 100 ms also increases Srel
up to 1.4 [Table 1(b)]. Dapp,intra decreases hereby slightly
to 0.026510ÿ3 mm2/s (rapp to 2.36 mm). Variations of
both parameters p2 and t2 are summarized in Table 1(c).
The relative changes of the exchange time t2 are
supposed to be proportional to the cubic root of the
relative changes in p2 assuming a constant membrane
permeability.16 Therefore the effect of t2 becomes much
less dominant than the effect of p2, but is not neglegible.
Cell swelling simulated by an 100% increase of p2
elevates the relative signal intensity Srel by 130%, which
depends furthermore on the b value. Dapp,intra decreases
slightly to 0.026910ÿ3 mm2/s and rapp to 2.38 mm.
Different to the proposal of van Zijl et al.33 the
intracellular volume fraction can not be calculated from
the intercept of the second intracellular signal component
in a straightforward manner. This model would hold only
for completely impermeable cell walls. As intracellular
water exchanges, the measured intracellular signal is
lowered and the inherent intercept always underestimates
the real intracellular volume fraction. Furthermore, as
seen in Fig. 1 the extrapolated intercept depends on the
chosen b range because the distribution of propagation
lengths leads to a curvature of the intracellular signal
component.
Considering the signal attenuation S (q̃2, tD) as a
function of the two independent experimental parameters
q value q̃2 and diffusion time tD, leads to the two types of
experiments (ct and cg), from which a different Dapp can
be calculated:
1 @ ln S…~q2 ; tD †
ct
Dapp :ˆ ÿ …1†
tD
@~q2
tD ˆconst
Dcg
app
1 @ ln S…~q2 ; tD †
1
:ˆ ÿ 2 ˆÿ 2
~
~q intra
q
@tD
~
q2 ˆconst
The interpretation of the data is more difficult because
of the physiological situation which is a combination of
extracellular free diffusion, intracellular restricted diffusion and exchange. To summarize, analytical modeling
shows that the separated intracellular water signal at high
diffusion-weighting can be used to monitor changes in
intracellular volume and exchange time. Apparent
intracellular diffusion coefficients and the mean intracel 1998 John Wiley & Sons, Ltd.
13
lular residence time can be evaluated independently from
the slopes of ct and cg experiments, respectively.
EXPERIMENTAL
Cell cultures and materials
The preparation of primary astrocytes, F98 rat glioma42,43 and human epidermoid KB cells for the NMR
experiments, the used materials, pulse sequences and the
data analysis were previously described.34,37,38,44 Standard culture medium was Dulbecco’s modified Eagle
medium (DMEM) supplemented with 40 mm taurine and
5% fetal calf serum. For the NMR measurements, the
cells were embedded in BMG threads, transferred under
steril conditions into an 8-mm NMR tube with a
perfusion insert and were perfused at 310 K with culture
medium (1 mL/min) oxygenated with 95% O2/5% CO2.
About 108 cells (determined by cell counting) were used
in each experiment. An intracellular volume fraction of
0.05 was estimated based on an average cell diameter of
6–8 mM and a sample volume of 0.5 mL.
The osmolarity of the media (hypotonic 180 mosm/L,
isotonic 300 mosm/L, hypertonic 420 mosm/L) was
modified by changing the NaCl concentration. The
concentration of the immunosuppressiva (Cyclosporin
A, Rapamycin) were 5 mg/mL, of hexadecylphosphocholine (Miltefosine) 70 mM, and of the water channel
blocker p-chloromercuriobenzenesulfonate (pCMBS)
0.5 mM.
Spectroscopy
The diffusion-weighted 1H NMR experiments were
conducted on a BRUKER AMX 360 MHz (8.4 T)
system. The maximal available gradient strength with
the 8-mm probe was 333 mT/m in z-direction. The
diffusion-weighted spin echo signals were obtained by a
90°-t-180°-t sequence with unipolar gradients during t.
The experimental parameters determining the diffusion
attenuation were the gradient duration , the gradient
strength G and the separation D of the leading edges of
the gradients, which defines the q value as q̃2 = (g G)2,
the diffusion time as tD = Dÿ/3, and the b value as b = q̃2
tD. While keeping the RF pulse interval t constant, T2
effects were excluded.
Experiments were performed to observe the signal
attenuation S dependent on G and D , i.e. the q value and
the diffusion time. For ss experiments (single signal)
successive signals at a constant G and D were acquired.
For ct experiments (constant diffusion time) G was varied
and D was kept constant, for cg experiments (constant
gradient strength) correspondingly D was varied and G
kept constant. To obtain the signal intensity S(G,
D) = S(q̃2, tD) we performed a Fourier transformation,
NMR IN BIOMEDICINE, VOL. 11, 11–18 (1998)
14
J. PFEUFFER, U. FLÖGEL AND D. LEIBFRITZ
an automatic phase correction of the spectra and an
integration of the water peak, using a five-point Newton–
Cotes integration formula.
Monitoring
Three methods were used to monitor the intracellular
water signal: ct monitoring (constant diffusion time
experiment), ss monitoring (single signal at large b
value), and cg monitoring (constant gradient experiment).
ct Monitoring. In order to monitor the intracellular
signal component, consecutive ct data were taken at high
b values in the linear range b [15 000…25 000] s/mm2.
One ct data set could be acquired in 32 s using 16 points
with TR = 1.5 s (including the time for starting the
sequence and saving the data). The apparent diffusion
ct
was calculated from the negative slope of
coefficient Dapp
ln S vs b value by linear regression. The inherent intercept
then was proportional to the changes of the intracellular
signal and could be plotted vs experimental time.
ss Monitoring. The time resolution while monitoring the
intracellular signal could be optimized by recording only
a single signal at one singular large b value (b = 15000 s/
mm2) with a time resolution of 3.5 s. Hereby a Dapp could
not be calculated, as only one signal was measured at a
fixed b value.
cg Monitoring. Consecutive cg data were taken at high
diffusion times in the range tD = [42.3 …98.3] ms to
monitor the mean intracellular exchange time tintra.
Using 16 points with TR = 1.5 s the time resolution of 32
s was the same as with ct monitoring. tintra could be
calculated from the negative reciprocal slope of ln S vs tD.
ct/cg Monitoring. Simultaneous dynamical information
about changes of the intracellular volume fraction and of
the intracellular exchange time could be received with an
alternated ct/cg monitoring. Hereby ct and cg experiments were performed alternately with a total time
resolution of 70 s.
RESULTS AND DISCUSSION
A typical ct experiment on perfused F98 cells embedded
in BMG threads is shown in Fig. 2. An apparent diffusion
ct
can be calculated from the negative
coefficient Dapp
slope of ln S vs b value. At small b values the signal is
due to extracellular, free diffusing water and
ct
= (2.7 0.2)10ÿ3 mm2/s. At larger b values the
Dapp
ct
=
negative slope decreases rapidly to Dapp
ÿ3
2
ct
(0.060 0.002) 10 mm /s. The low Dapp is caused
by the restricted diffusion of water inside the cells, which
 1998 John Wiley & Sons, Ltd.
Figure 2. Diffusion attenuated signal (ct experiment) vs b
value measured on F98 glioma cells ( = 15 ms, tD = 15 ms,
T = 306 K). The (apparent) diffusion coef®cients are evaluated at small and large b values from the negative slope by
linear regression. The experimental data can not be
explained by a simple bi-exponential two-compartment
model as indicated by the continuous (linear regression at
the limits) and dotted (bi-exponential ®t) lines
provides the basis to separate intracellular from extracellular signal contributions and to detect the intracellular
signal separately at large b values. As shown by the
extrapolated lines at small and large b values in Fig. 2 the
experimental data can neither be sufficiently described by
the superposition of the two exponential curves (continuous line in Fig. 2), nor by a bi-exponential fit (dotted
line).
Changes of the intracellular signal component will be
considered in the following part as a probe for the
intracellular volume and exchange time during exposure
to various cellular constraints such as osmotic stress,
apoptotic conditions, immunosuppressive stress, and
mercury reagents. Preliminary applications of these
methods (ct/ss/cg monitoring) have been reported elsewhere.45–48 Other investigations have been carried out
during oxygen deprivation, chronical osmotic stress,
halted perfusion, hyperammonemia (on F98 cells)49–51,
and swell behavior of oocytes (Xenopus laevis) with
expressed water channels52.
Monitoring changes in the intracellular volume
Figure 3(a) shows ct experiments on primary astrocytes
under diverse osmotic conditions. After exposure to
hypoosmotic medium the astrocytes swell and the signal
at large b values is increased. In hyperosmotic medium
the cells shrink and signal is decreased.
ct Monitoring. In Fig. 3(b) the intercepts of the
consecutive ct data are normalized to 100% of the
control values in isotonic medium. The standard deviation in control medium is 3%. After exposure to
NMR IN BIOMEDICINE, VOL. 11, 11–18 (1998)
MONITORING OF CELL VOLUME AND WATER EXCHANGE TIME
15
hypoosmotic conditions cell swelling of the F98 glioma
cells is visible within minutes as well as the two-step
regulatory volume decrease of the cells.53–57 These cells
show a high overshoot of the intracellular signal (10–15
min) immediately after exposure to hypoosmotic medium
and a regulatory volume decrease from 165 to 130%
within 5 h of exposure to hypoosmotic stress. The
calculated apparent intracellular diffusion coefficient
remains constant within the experimental signal deviact
= (0.0626 0.0005)10ÿ3 mm2/s (control) and
tion, Dapp
(0.061 0.002)10ÿ3 mm2/s (averaged over 6 h).
ss Monitoring. Osmotic swelling and recovery of F98
cells is shown in Fig. 3(c) with a time resolution of 3.5 s.
Swelling of the ensemble is achieved within 3 min. The
standard deviation of the signal intensity in control
medium is 0.53%, which is more than 5-fold better than
the ct monitoring in Fig. 3(b).
With this sensitive method the temperature dependence of the intracellular water signal has been examined
at b = 15000 s/mm2. A decrease in temperature of a few
Kelvin only leads to a significant increase of the
intracellular signal intensity of 5% / K (measured at
310 K). Therefore the experimental setup demands an
accurate temperature control.
Figure 4 shows two further applications of ct
monitoring, namely astrocyte swelling after exposure to
the immunosuppressiva Cyclosporin A and Rapamycin
[Fig. 4(a)] and shrinking of KB cells during Miltefosineinduced apoptosis [Fig. 4(b)]. Cyclosporin A induces a
significantly larger cell swelling than Rapamycin on
ct
= (0.051 0.002)10ÿ3 mm2/s).
primary astrocytes (Dapp
This may reflect the observed secondary neurotoxic
effects of these drugs on human patients.58 Apoptosis, the
programmed cell death, induced by Miltefosine in KB
cells, leads to a considerable volume decrease in the
initial stage during the first 4 h upon induction
ct
= (0.046 0.002)10ÿ3 mm2/s).59,60,61
(Dapp
Exchange of intracellular water
Figure 3. Monitoring of changes in cell volume under
osmotic stress on primary astrocytes (a) and F98 glioma cells
(b,c). (a) ct experiments (constant diffusion time) under
isoosmotic, hypoosmotic, and hyperosmotic conditions. (b)
ct monitoring in the range b = [15108¼24270] s/mm2. (c) ss
monitoring (single signal) at b = 15000 s/mm2 with a time
resolution of 3.5 s ( = 10 ms, tD = 31.7 ms)
 1998 John Wiley & Sons, Ltd.
Figure 5 shows a cg experiment on F98 cells with
gradient strength G = 250 mT/m. The mean intracellular
residence time tintra = (48.6 0.3) ms is calculated from
the negative reciprocal slope by linear regression in the
range tD = [42.3…100] ms. According to the simulations
only the data at high gradient strengths, i.e. large q
values, are suitable to determine tintra correctly without
extracellular signal contributions.37,38 The apparent
intracellular diffusion coefficient is evaluated to
ÿ3
mm2/s.
Dcg
app = (0.046 0.001)10
cg Monitoring. In Fig. 6. an alternated ct / cg monitoring
is shown to receive information about cell volume and
exchange time of F98 cells under treatment with pCMBS.
The ct data of Fig. 6(a) and the cg data of Fig. 6(b) have
NMR IN BIOMEDICINE, VOL. 11, 11–18 (1998)
16
J. PFEUFFER, U. FLÖGEL AND D. LEIBFRITZ
Figure 4. ct monitoring of changes in cell volume
(b = [15108¼24270] s/mm2, = 10 ms, tD = 31.7 ms). (a)
Exposition of the immunosuppressiva Cyclosporin A (◊) and
Rapamycin (&) to primary astrocytes. (b) Apoptosis, induced
in epidermoid KB cells with Miltefosine
Figure 5. cg experiment (constant gradient) with F98 glioma
cells at gradient strength G = 250 mT/m qÄ2 = 448103 mmÿ2,
tD = [17.3¼100] ms, = 10 ms). The mean intracellular
residence time is calculated to tintra = (48.6 0.3) ms from
the negative, reciprocal slope in the linear range at long
diffusion times
 1998 John Wiley & Sons, Ltd.
Figure 6. Changes in cell volume and intracellular residence
time of water upon treatment of F98 with the water channel
blocker pCMBS as monitored by alternated ct/cg experiments (ct data: b = [15108¼24270] s/mm2, = 10 ms,
tD = 31.7 ms. cg data: tD = [42.3¼98.3] ms, = 8 ms,
G = 250 mT/m)
been measured alternately. The standard deviation of the
ct control is 0.9% and 1.0 ms of the cg control,
respectively. Addition of 0.5 mM pCMBS to F98 cells
leads to a biphasic swell process, i.e. an initial signal
increase to 115% of the control value during the first
hour and a further increase to 180% after the second hour
of exposure. The intracellular exchange time increases
hereby from (51.9 1.0) ms to (59.0 1.1) ms (standard
deviation of 20 points). With higher concentrations
(2mM) of pCMBS the intracellular signal increases up
to 400% which has been observed by ct monitoring and
light microscopic image analysis (data not shown).37
pCMBS binds selectively to sulfhydryl groups and is
known to block a.o. mercury sensitive water channels,62–
68
e.g. in erythrocytes.25,27 As pCMBS shows several
toxic effects onto the cells,69–73 not only the membrane
permeability is changed. Initial swelling is probably
caused by cell depolarization and inhibition of the Na‡,
NMR IN BIOMEDICINE, VOL. 11, 11–18 (1998)
MONITORING OF CELL VOLUME AND WATER EXCHANGE TIME
K‡-ATPase. The second stage of the biphasic process
may be explained by a dissociation of cytoskeletal
elements permitting the massive swelling under osmotic
forces.71
In conclusion, the results show that diffusion-weighted
1
H NMR spectroscopy is capable to monitor changes in
cell volume with a time resolution in the order of seconds
and can determine the mean residence time of intracellular water directly and noninvasively. The apparent
intracellular diffusion coefficient is significantly lowered
by the restricted diffusion of the intracellular water which
is used to detect the intracellular signal separately. This
signal component is very sensitive (i) to changes in the
intracellular volume fraction caused by cell swelling or
shrinking, (ii) to changes of the intracellular exchange
time, and (iii) also to changes in temperature which
demands an accurate temperature control. Many further
applications such as anoxia, volume regulation, ischemia
and treatment with various pharmaceuticals are conceiveable to follow up their effect on the cell volume and the
membrane permeability to water.
11.
12.
13.
14.
15.
16.
17.
18.
19.
Acknowledgments
The authors thank Dr N. Serkova and J. Henke for the
cultivation of the primary astrocytes and the KB cells,
respectively, and Dr W. Dreher for stimulating discussions regarding theory and methods.
20.
21.
22.
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