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
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ABSTRACT
INTRODUCTION
THEORY
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This report describes an open-air method for measuring the O2 consumption (QO2) 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 QO2 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 QO2 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 QO2. 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
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ABSTRACT
INTRODUCTION
THEORY
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 (QO2) (26). QO2 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 QO2. 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 QO2 (8). The means employed to detach cultured cells from their substratum may produce similar effects (10). The wide range of type II cell QO2 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 QO2 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 QO2 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
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ABSTRACT
INTRODUCTION
THEORY
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The theory that underlies the open-air method of mea- suring QO2 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
&dgr;C/&dgr;<IT>t</IT> = <IT>D</IT>(&dgr;<SUP>2</SUP>C/&dgr;<IT>x</IT><SUP>2</SUP>) (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 delta C/delta t = 0. Thus (D × d2C)/dx2 = 0, and dC/dx = a constant. The general solution of this equation is
C(<IT>x</IT>) = <IT>ax</IT> + <IT>b</IT> (2)
where a and b are constants that satisfy the boundary conditions: C(0) = alpha PO2 (x = 0) at the medium surface and C(h) = alpha 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 alpha  is the O2 solubility coefficient at 37°C [alpha  = 0.94 (µmol · cm-3 · atm-1)/PB, where PB is the barometric pressure]. This gives
C(<IT>x</IT>) = &agr; ⋅ [P<SC>o</SC><SUB>2</SUB>(<IT>h</IT>) − P<SC>o</SC><SUB>2</SUB>(0)]/<IT>h</IT> + &agr; ⋅ P<SC>o</SC><SUB>2</SUB>(0) (3)
The O2 flux (JO2) along the x-axis is given by
<IT>J</IT><SC>o</SC><SUB>2</SUB>  =  −<IT>D</IT>(dC/d<IT>x</IT>) or <IT>J</IT><SC>o</SC><SUB>2</SUB> = −<IT>D</IT> ⋅ &agr; ⋅ [P<SC>o</SC><SUB>2</SUB>(<IT>h</IT>) − P<SC>o</SC><SUB>2</SUB>(0)]/<IT>h</IT> (4)
QO2 = JO2 · S, the area covered by the monolayer (assuming that cells are uniformly spread over the bottom of the well). Thus
<A><AC>Q</AC><AC>˙</AC></A><SC>o</SC><SUB>2</SUB> = −(<IT>D</IT> ⋅ <IT>S</IT> ⋅ &agr;)[P<SC>o</SC><SUB>2</SUB>(<IT>h</IT>) − P<SC>o</SC><SUB>2</SUB>(0)]/<IT>h</IT> (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. QO2 is given by the simple equation
<A><AC>Q</AC><AC>˙</AC></A><SC>o</SC><SUB>2</SUB> = −<IT>D</IT> ⋅ <IT>S</IT> ⋅ &agr; ⋅ <IT>a</IT> (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 Delta x. The finite-difference equation is
&Dgr;C/&Dgr;<IT>t</IT>  =  −<IT>D</IT>[&Dgr;<SUP>2</SUP>C/(&Dgr;<IT>x</IT>)<SUP>2</SUP>] or &Dgr;P<SC>o</SC><SUB>2</SUB>/&Dgr;<IT>t</IT> = −<IT>D</IT>[&Dgr;<SUP>2</SUP>P<SC>o</SC><SUB>2</SUB>/(&Dgr;<IT>x</IT>)<SUP>2</SUP>] (7)

If the cell QO2 is constant, a steady state will be achieved in the Delta x adjacent to the cells after a time interval Delta tau and the PO2 gradient within this Delta x will be the same as that within the entire medium depth (h) at steady state (Eq. 4). Thus the boundary conditions are a = delta C/delta 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
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ABSTRACT
INTRODUCTION
THEORY
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 approx 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 QO2 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 QO2 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). QO2 was calculated from the linear regression of PO2 vs. time
<A><AC>Q</AC><AC>˙</AC></A><SC>o</SC><SUB>2</SUB> = &agr; ⋅ V ⋅ (dP<SC>o</SC><SUB>2</SUB>/d<IT>t</IT>) (8)
where V is the chamber volume.

Open-air QO2 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 QO2.

Statistical methods. Results are given as means ± SE. QO2 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
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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 QO2 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 QO2, 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. QO2 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 (open circle ). 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 QO2 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 QO2 values obtained with the two methods (Table 1). In contrast, the QO2 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 QO2 of detached cells by ~70% in two experiments but was without effect in the other four. The QO2 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).


                              
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Table 1.   QO2 of alveolar type II cells measured by the 2 methods


    DISCUSSION
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ABSTRACT
INTRODUCTION
THEORY
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The main advantage of the open-air method is that it is simple to implement and allows measurement of QO2 on intact monolayer of cells. The only assumptions are that cell QO2 does not change during the measurement and that the cells are uniformly distributed on the bottom of the well. A nonstable QO2 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 QO2 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 QO2 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 QO2 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 QO2 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 QO2, and this may also be the case in hands more expert than ours [however, Fisher et al. (8) did not find that the QO2 of cultured, detached cells was lower than that of freshly isolated cells]. The dispersion of QO2 measured in these cells was rather large, which may reflect different degrees of cell stress. The reduced QO2 was not always accompanied by major membrane damage because the addition of succinate did not significantly increase QO2 in the majority of the experiments. Cells cultured for 2 days had a higher QO2 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 QO2 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 QO2 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 QO2 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 (QO2 of 400 nmol · h-1 · 106 cells-1). Thus with a QO2 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 QO2 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).

                              
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Table 2.   QO2 of alveolar type II cells


    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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
THEORY
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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