SPECIAL COMMUNICATION
A method for measuring the oxygen consumption of intact cell
monolayers
Kamel
Mamchaoui and
Georges
Saumon
Institut National de la Santé et de la Recherche
Médicale Unité 82, Faculté Xavier Bichat, 75018 Paris, France
 |
ABSTRACT |
This report describes an open-air method
for measuring the O2 consumption
(
O2) of intact monolayers
of cultured cells. This method is based on Fick's second law of
diffusion. It requires only a micromanipulator and a miniature
O2 electrode to measure the
PO2 gradient in the culture medium in
the well. It was compared with the conventional oxygraph chamber
method. Both methods gave the same value for
O2 in freshly isolated rat
type II cells: 166 ± 15.3 nmol · h
1 · 106
cells
1 for the open-air method and 151 ± 11.6 nmol · h
1 · 106
cells
1 for the oxygraph chamber method (n = 11 experiments). But the open-air method gave significantly larger
values for
O2 in cells cultured for 2 days (236 ± 8.8 nmol · h
1 · 106
cells
1) than the oxygraph method (71 ± 15.2 nmol · h
1 · 106
cells
1; P < 0.001; n = 12 experiments). This suggests that the way cells are detached from their
substratum to be placed in the oxygraph chamber affects their
O2. The open-air method may
be useful for studies on the metabolic properties of monolayers because the cells do not risk being damaged.
metabolism; type II pneumocytes; cell differentiation
 |
INTRODUCTION |
THE INFLUENCE OF ENVIRONMENTAL FACTORS on the metabolic
properties of alveolar type II cells has been the subject of numerous studies (2, 11, 19, 20, 27, 28). The preservation or deterioration of
cellular integrity is often assessed by measuring the cell ATP content
and/or O2 consumption
(
O2) (26).
O2 is almost always estimated
by the oxygraph cell method, which consists of a Clark electrode fitted
to an airtight chamber. Cell suspensions are placed in the chamber and
the decrease in PO2 in the bathing
medium is used to calculate
O2. Despite its widespread use, this method is far from perfect. The chamber is not always perfectly sealed, and some bubbles may remain trapped. Perhaps more
importantly, monolayers of cultured cells must be detached before they
are placed in the oxygraph. Isolation by enzymatic treatment followed
by mechanical detachment stresses cells (1, 33) and may affect
their metabolic properties and
O2 (8). The means employed to
detach cultured cells from their substratum may produce similar effects
(10). The wide range of type II cell
O2 values published
to date (from 14 to 260 nmol · h
1 · 106
cells
1) may reflect the degree of damage produced by
different cell isolation procedures as well as the difficulty of
obtaining reproducible interlaboratory measurements with the oxygraph.
Another important point is the resting
O2 value of type II cells. It
is often overlooked that cell monolayers are covered with a layer of
liquid, the thickness of which may greatly affect O2
delivery depending on the cell oxidative metabolism rate. For example,
some cell types are, in fact, anoxic under conventional culture
conditions (32). Alveolar type II cells cultured at an air-liquid
interface maintain differentiation, but they lose these properties when
immersed in liquid (5). It is unclear what part O2
provision plays in these changes. We have therefore designed a simple
method for measuring the
O2
of intact cell monolayers that may also be applied to freshly isolated
cells. This method only requires a simple micromanipulator and a
commercially available O2 microelectrode.
 |
THEORY |
The theory that underlies the open-air method of
mea- suring
O2 is
classic and found in all treatises on epithelial transport (for
example, Ref. 29). Briefly, the diffusion of O2 from
ambient air to the cell monolayer is given by Fick's second law
|
(1)
|
in
which C is O2 concentration, t is time, D
is the O2 diffusion coefficient in water (3.3 × 10
5 cm2/s), and x is the
distance (in cm) from the surface of the culture medium.
In the steady state (when the medium is left to stabilize for
sufficient time; this point will be considered below), the
O2 concentration profile is stationary, and
C/
t = 0. Thus (D × d2C)/dx2 = 0, and dC/dx = a
constant. The general solution of this equation is
|
(2)
|
where
a and b are constants that satisfy the boundary
conditions: C(0) =
PO2 (x = 0) at the medium surface and C(h) =
PO2 (x = h), where
h is depth of the medium to the bottom of the well, in which
PO2 is in millimeters of mercury and
is the O2 solubility coefficient at 37°C
[
= 0.94 (µmol · cm
3 · atm
1)/PB,
where PB is the barometric pressure]. This gives
|
(3)
|
The O2 flux (JO2) along the
x-axis is given by
|
(4)
|
O2 = JO2 · S, the
area covered by the monolayer (assuming that cells are uniformly spread
over the bottom of the well). Thus
|
(5)
|
Because the O2 gradient is linear (Eq. 2), it is
not necessary to measure the PO2 in
the vicinity of the monolayer (x = h) and so risk
damaging cells. Measuring PO2 at different depths within the well can be used to verify the linearity of
the PO2 gradient, and hence the
steady state, and to calculate this gradient (a) by linear
regression of the PO2 values against
the displacement of the O2 electrode from its initial position.
O2 is given by the
simple equation
|
(6)
|
It may be of interest to determine how long it takes to establish a
stable gradient. An analytic solution for an unsteady PO2 profile is difficult to find, but
the profile can be computed with the finite-difference method. An
identical problem was treated and solved graphically by Eckert (6) for
the temperature changes in a wall. The O2 gradient is zero
when the culture medium is renewed because the medium O2
concentration is equal everywhere to that of the environment with which
it was equilibrated (for example, the incubator atmosphere). Let us
consider a thin layer of liquid of thickness
x. The
finite-difference equation is
|
(7)
|
If the cell
O2 is constant, a
steady state will be achieved in the
x adjacent to
the cells after a time interval 
and the
PO2 gradient within this
x will be the same as that within the entire medium
depth (h) at steady state (Eq. 4). Thus the boundary
conditions are a =
C/
t(t,h) and P0 = PO2(t,0), where
P0 is the initial condition
[PO2(0, x)].
The unsteady O2 concentration profile within the well can
be calculated with conventional numerical methods (22). It is thus
possible to approximately evaluate the time necessary to reach
quasi-steady-state conditions in the well.
 |
MATERIALS AND METHODS |
Isolation of alveolar type II cells. Rat alveolar type II cells
were obtained from the lungs of male Sprague-Dawley (200 g) rats
(Charles River, Saint-Aubin lès Elbeuf, France) as described by
Dobbs et al. (4). Blood was cleared from the lung by perfusion through
the pulmonary artery, and alveolar macrophages were removed by
bronchoalveolar lavage. Fifteen milliliters of a digestion solution
containing 3 U/ml of porcine elastase (Worthington Biochemical, Freehold, NJ) were injected into the trachea twice at 10-min intervals while the lungs were immersed in normal saline at 37°C. The
enzymatic reaction was stopped 20 min after the first elastase
instillation by lavage with fetal bovine serum (GIBCO BRL, Cergy,
France) in DMEM (Sigma, Saint-Quentin Fallavier, France). The
peripheral parenchyma was dissected from the large bronchi and vessels
and chopped with small scissors in DMEM and 1 mg/ml of DNase I
(Boehringer Mannheim, Meylan, France). The fragments were filtered
twice through nylon tissue (150- and 30-µm mesh). The cells were
centrifuged, and the pellets were suspended in DMEM. They were plated
on petri dishes covered with rat IgG and left at 37°C for 2 h in a
humidified incubator in 5% CO2-20% O2-75%
N2 (PO2 = 143 mmHg).
Nonadherent cells were harvested, centrifuged, and suspended in
DMEM, 10% fetal bovine serum, and 0.05 mg/ml of gentamicin at
106 cells/ml. Purity was
90% as assessed by modified
Papanicolaou staining, and viability was >95% by trypan blue
exclusion. The protein content of the cells was obtained with the
Bio-Rad (Munich, Germany) protein assay. The cells were used
immediately or were cultured (0.5 ml/well) for 2 days in
1.94-cm2 wells (Costar, Brumath, France). Cells are
confluent and homogeneously distributed in the well with this technique
(Fig. 1). For conventional
O2 measurement, cultured
type II cells were detached by incubating them for 15 min with Versene
(GIBCO BRL) followed by scraping with a rubber policeman. They were
centrifuged, and the pellet was suspended in DMEM at different
concentrations. The number of cells in suspension and those adhering to
the wells were standardized, with the assumption that the protein
content of 106 cells was 0.13 mg (8). Cell integrity was
verified by adding succinate (final concentration 10 mM) to the
chamber.

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Fig. 1.
Type II cells form homogeneous monolayers after 2 days of culture under
conventional conditions as shown by phase-contrast microscopy. Original
magnification, ×100.
|
|
Conventional
O2
measurement. A YSI (Yellow Springs, OH) model 5300 oxygraph was
used. It consisted of a semiminiature Clark electrode (Instech,
Plymouth Meeting, PA) fitted to a small (600-µl) airtight chamber.
The O2 electrode was calibrated by immersion in dithionite
and DMEM equilibrated with room air. The cell suspension was slowly
introduced through an opening in the bottom of the chamber so as to
drive out microbubbles through a corresponding opening in the top. The
openings were closed simultaneously once the chamber was filled. The
cell suspension was continuously stirred with a magnetic bar. The
number of viable cells present in the suspension was determined with a
hemocytometer. The signal from the O2 electrode was
digitized (National Instruments, Austin, TX) and stored in a
PC-compatible computer (Elonex, Gennevilliers, France).
O2 was calculated from the
linear regression of PO2 vs. time
|
(8)
|
where
V is the chamber volume.
Open-air
O2
measurement. This measurement was done directly in the cell culture
incubator. A glass O2 microelectrode (737GC, Diamond
General, Ann Arbor, MI) with a 100- to 150-µm-diameter tip was used.
It was connected to a nanoammeter (Tacussel, Paris, France). The
electrode was attached to a micromanipulator (World Precision
Instruments, Stevenage, UK) in the strict vertical position. The
electrode was calibrated by immersion in culture medium equilibrated with pure nitrogen and air. The tip of the electrode was brought into
contact with the culture medium surface (easily visible by tangential
illumination) and slowly immersed to 1.8 mm. It was left to equilibrate
for 30-60 min, PO2 readings were made while the electrode was slowly raised in 200-µm steps (1 step/10
min) to 1 mm, and two more measurements were made at 0.5 mm and at the
surface. We obtained much better results (i.e., linear gradients) with
this procedure than by measuring PO2 during the descent. This is perhaps due to the hydrodynamic profile of
the electrode. Regression of PO2
versus depth was used to calculate
O2.
Statistical methods. Results are given as means ± SE.
O2 values were compared by
Student's paired and unpaired t-test. Regression was done with
the least mean squares method. Significance was accepted at the
P < 0.05 level.
 |
RESULTS |
Figure 2A shows the
PO2 gradient in a well containing no
cells. There was no significant variation in
PO2 along the vertical axis,
attesting to negligible
O2 by
the electrode. Figure 2B shows data from an experiment during
which the PO2 gradient was measured
under steady-state conditions. Unsteady PO2 profiles with
JO2 set at 36 nmol · h
1 · cm
2,
to approximately match the PO2
gradient observed during the experiment shown in Fig. 2B, are
shown in Fig. 3. Because PO2 profiles change rather rapidly,
their determination, and thus that of
O2, cannot be very precise
under unsteady conditions. A quasi steady state (quasi-linear profile)
is obtained after 60 min, with the depth of the medium being 0.24 cm.
O2 is biased by ~5% if the
unsteady 60-min PO2 values are used
and by <1% if the measurement is made at 120 min (when the gradient is obtained as described in MATERIALS AND METHODS). The
95% confidence interval for the correlation coefficient of the linear
regressions of the profiles was 0.992-0.998 (n = 23 experiments). Figure 2B shows that the
PO2 gradient completely disappeared
120 min after the addition of 10
6 M rotenone.

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Fig. 2.
Representative PO2 profiles in wells.
A: well containing only medium equilibrated with room air.
B: well with type II cells cultured for 2 days in a humidified
incubator (20% O2) under control conditions ( ) and in
presence of rotenone ( ). Depth, distance from surface of culture
medium in which PO2 measurement was
made.
|
|

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Fig. 3.
Theoretical unsteady PO2 profiles at
indicated times after culture medium was changed in a well containing a
cell monolayer consuming O2 at 33 nmol · h 1 · cm 2.
A quasi-linear O2 gradient was obtained after 60 min.
|
|
The
O2 values of different
concentrations of freshly isolated type II cells were simultaneously
measured by the two methods (10 paired values and 1 unpaired value).
The chamber method was used with more cells per milliliter than in the
open-air method to minimize the potential depressing effect of small
air bubbles on PO2 changes. The
chamber cell suspensions contained 0.7-3 × 106
cells/ml. They were diluted threefold before being plated in the wells.
The correlation between the
dPO2/dx slope in the well
(Eq. 5) and the
dPO2/dt slope in the chamber (Eq. 8) in the 10 paired experiments is shown in Fig.
4. These slopes were correlated with the
number of cells in the chamber (r = 0.98; P < 0.001)
and in the well (r = 0.95; P < 0.001). There was no
significant difference between the
O2 values obtained with the
two methods (Table 1). In contrast, the
O2 of cells cultured for 2 days was lower when measured on detached cells in the chamber than on
intact cells in the wells (Table 1). Adding succinate (n = 6 experiments) to the chamber medium increased
O2 of detached cells by
~70% in two experiments but was without effect in the other four.
The
O2 measured in the intact
cells in these four experiments with the open-air method was
significantly larger than that in the detached cells (242 ± 12.5 vs.124 ± 21.2 nmol · h
1 · 106
cells
1; P < 0.05).

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Fig. 4.
Relationship between PO2 changes in
oxygraph chamber (dPO2/dt,
where t is time) and PO2
gradient in well (dPO2/dx,
where x is distance from surface of culture medium) of
different concentrations of freshly isolated type II cells. Correlation
reflects their proportionality (see text for details: Eqs. 5 and 8).
|
|
 |
DISCUSSION |
The main advantage of the open-air method is that it is simple to
implement and allows measurement of
O2 on intact monolayer of
cells. The only assumptions are that cell
O2 does not change during the
measurement and that the cells are uniformly distributed on the bottom
of the well. A nonstable
O2
or nonuniform cell dispersal would result in nonlinear
PO2 gradients that can be easily
recognized. The uniformity of the cell distribution may also be
verified by simple visual or microscopic inspection (Fig. 1).
Several methods have been designed to measure
O2 in intact adherent cells
(10, 12, 34). All are based on the changes in
PO2 in a sealed volume and require a
specific apparatus with a well-defined geometry. They thus have the
same drawbacks as the conventional oxygraph chamber method: the need
for an absolute seal and the avoidance of trapped bubbles. In contrast,
the open-air method is flexible and requires a minimal investment: an
unsophisticated micromanipulator and a glass microelectrode. We used a
commercially available electrode, but there are many reports that
describe how to make miniature glass O2 electrodes (see,
for example, Refs. 13, 21, 30, 31), saving cost when extensive studies
are planned.
The sensitivity of the open-air method depends on the O2
electrode response and the resolution of the amplifier. Electrodes with
150-µm tips have a dynamic range of ~100 pA (between pure N2 and air). Resolution of modern ammeters is 0.1 pA, which
allows reliable measurement of PO2
differences of <1 mmHg. It is thus conceivable to estimate gradients
of 5 mmHg/cm by successive measurements within 0.2 cm. This corresponds
to an
O2 of ~7 nmol · h
1 · 106
cells
1, assuming a cell density of
105/cm2. This is about one-fourth the
consumption of cells with low consumption rates (30 nmol · h
1 · 106
cells
1 in carcinoma cells; see Ref. 25). Indeed,
increasing the depth of the culture medium and the distance over which
PO2 measurements are made can
increase this resolution.
The open-air method and the conventional oxygraph chamber method yield
similar values for freshly isolated cells (Fig. 4, Table 1). However, a
greater cell density was required in chamber measurements to obtain
O2 values comparable to those
measured in the wells, probably because a few microbubbles remained in the chamber despite careful manipulation. Good agreement between the
two methods was not found for cultured cells. The need to detach the
cells is a major inconvenience of the oxygraph cell method. Cells are
submitted to a number of stresses (enzymatic treatment, decreased
extracellular Ca2+ concentration, and mechanical stress)
that may affect their viability and metabolic properties. This obstacle
to measuring
O2 of
cultured cells may explain the scarcity of such measurements, except
for the pioneering study of Fisher et al. (8) and the work of Simon et
al. (28) on cloned cells. Our detachment procedure (which is widely
used) significantly affected
O2, and this may also be the
case in hands more expert than ours [however, Fisher et al. (8)
did not find that the
O2 of
cultured, detached cells was lower than that of freshly isolated
cells]. The dispersion of
O2 measured in these cells
was rather large, which may reflect different degrees of cell stress.
The reduced
O2 was
not always accompanied by major membrane damage because the addition of
succinate did not significantly increase
O2 in the majority of the
experiments. Cells cultured for 2 days had a higher
O2 than freshly isolated cells when measured by the open- air method. This has also been observed by Fisher et al., who suggested that this may be due to some
damage produced while isolating the cells or to a higher metabolic rate
caused by the culture conditions.
The disadvantage of the open-air method is the need to wait for a
steady state before making a measurement. This precludes the study of
transients or the sequential addition of inhibitors. But the main
advantage, in addition to leaving cells intact, is that iterative
measurements can be made on the same monolayer as the culture is
continued, which may be of value for assessing chronic cell treatments.
The time required to establish a steady PO2 gradient can be estimated if the
O2 is roughly known. If the gradient is already established, quiet positioning of the
electrode in the well produces minimum perturbation and the measurement
may be much faster. Trials should be done to optimize this step,
depending on individual culture conditions. The time needed to detach
cells from their substratum, collect them by centrifugation, and place
them in the oxygraph chamber is about the same as that needed to make
an open-air measurement, which makes the open-air method no more
tedious than the conventional one.
Published values of
O2 for
type II cells are given in Table
2. The wide dispersion of
these values may be due to varying amounts of damage produced during
isolation or to the difficulty in obtaining reproducible oxygraph
measurements or precisely determining the number of cells in the
chamber. Knowledge of the precise value of the
O2 of cells is not
inconsequential. With the assumption that type II cells in culture are
at 0.20 × 106/cm2 [which is usual
in most studies (17) but which may be several times greater (8)]
and a medium depth of 0.26 cm (500 µl of medium in a
1.94-cm2 well), Eq. 4 shows that cells are anoxic
if the JO2
80 nmol · h
1 · cm
2
(
O2 of 400 nmol · h
1 · 106
cells
1). Thus with a
O2 of 236 nmol · h
1 · 106
cells
1, the PO2 in
the immediate vicinity of a cell will be zero if the medium height
exceeds 0.45 cm (which corresponds to a medium volume of 865 µl/well
when 24-well plates were used). This is worth considering, especially
because it has been shown that hypoxic (anoxic?) conditions affect the
expression of important type II cell membrane enzymes such as
Na+-K+-ATPase and Na+ channels (23,
24). Our measurements of the
O2 of intact type II cell
monolayers also suggest that it is unlikely that a restriction of
JO2 during conventional (500 µl/1.94-cm2 well) culture conditions can explain the
phenotype of type II cells cultured at an air-liquid interface (5).
 |
ACKNOWLEDGEMENTS |
We thank Etienne Delavault (Commissariat à l'Energie
Atomique, Saclay, France) for the Eckert reference.
 |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: G. Saumon,
INSERM U82, Faculté Xavier Bichat, BP 416, 75870 Paris Cedex 18, France (E-mail: saumon{at}bichat.inserm.fr).
Received 14 July 1999; accepted in final form 3 November 1999.
 |
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