Department of Pediatrics, National Jewish Medical and Research Center, Denver, Colorado 80206
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ABSTRACT |
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Exposure of cultured cells to changing gaseous environments is used as a model for understanding both the immediate and long-term effects of such exposures on lung cells in vivo. We conducted experiments with polystyrene tissue culture flasks and plates to determine the time course of changes in oxygen concentration occurring under in vitro conditions. Only a few minutes were required for the concentration of oxygen in the environmental chamber to reach equilibrium with that of the flushing gas. However, >3 h were required for the oxygen content in the medium in the tissue culture flasks and plates to achieve equilibrium. The low solubility of oxygen in aqueous solutions and the limited diffusion of oxygen through a boundary layer of gas above the medium are the major barriers to rapid oxygen transport into the culture medium. The delay in achieving the desired PO2 within the culture medium limits the temporal precision of any assessment of the correlation of cellular events with the concentration of oxygen to which those cells are exposed.
permeability; polystyrene
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INTRODUCTION |
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CULTURED MAMMALIAN CELLS are widely used to study the effects of environmental factors on cellular function. The investigator not only can tightly regulate the environmental conditions to which the cells are exposed but also can study the responses of single cell types. Research efforts in our laboratory have begun addressing rapid changes in cellular function in response to abrupt changes in PO2. It is important in studies of this nature to fully characterize the relationship between the concentration of oxygen to which the cells are exposed along with the duration of that exposure and the cellular responses that are actually being studied. In the preparation for tissue culture studies in this area, we performed the experiments described here to better understand the kinetics of oxygen loading and unloading in our system. We determined that limited exchange of oxygen across the gas-medium boundary along with slow diffusion of oxygen across a boundary layer of gas just above the medium presented the predominant hindrance to oxygen transport into the tissue culture medium. As a result, the time that was required to fully equilibrate the medium with the external oxygen concentration was >3 h in most experiments. This inability to achieve rapid changes in PO2 within the tissue culture medium limits the precision with which cellular events can be correlated in time with changes in environmental oxygen concentration.
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METHODS |
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The 100-mm plates used in this experiment were purchased from BD Biosciences (Bedford, MA), and the T-75 flasks were purchased from Corning (Corning, NY). A hole ~1.2 cm in diameter was melted through the lid of the 100-mm plates and the top surface of the T-75 flasks. A 2-cm length of polypropylene tubing ~1 cm in inner diameter was hot-glued vertically through the hole to provide support for a YSI model 5331 oxygen electrode. A Viton O-ring 0.8 cm in inner diameter was slipped over the shaft of the model 5331 electrode, and its position was adjusted to hold the tip of the electrode ~2 mm above the bottom surface of the tissue culture plate or flask. Twenty milliliters of 10 mM phosphate-buffered saline (PBS), pH 7.4, were added to each plate or flask. PBS was used rather than cell culture medium to avoid protein fouling of the Clark electrode. In another set of dishes and plates, most of the polystyrene comprising the top of the culture vessel was removed by cutting away expanses of polystyrene around the upright polypropylene tubing. This perforation of the lid was done to provide an experimental platform with which to study oxygen transport into the medium without the effect of diffusion impairment across the lid material. Another set of experiments was performed with the electrodes suspended 2 mm above the surface of the PBS in the culture vessels with either intact or perforated tops. In this fashion, temporal changes in the oxygen concentration in the chamber and in the tissue culture dishes and flasks could be compared with those changes occurring in the PBS. Teflon membranes 0.002 inch thick were installed on the model 5331 electrodes rather than the standard 0.001-inch-thick Teflon membranes. This was done to reduce the contribution of electrode oxygen consumption to the overall oxygen depletion observed in the hypoxia experiments.
One assembly with an intact top and one with a perforated top were
placed in a polystyrene environmental chamber with an internal volume
of 5.72 liters (Billups-Rothenberg, Del Mar, CA), and the electrical
connections for the electrodes were passed out through the gas access
ports. A diagram of the exposure chamber with the culture plates and
Clark oxygen electrodes is presented in Fig. 1. The electrode- tissue culture
plate-environmental chamber ensemble was maintained at 37°C inside a
tissue culture incubator (Shel-Lab model 1820, Sheldon Manufacturing,
Cornelius, OR). The electrode wires were passed out of the incubator
and connected to a 10-channel multiplexer (Diamond General Development,
Ann Arbor, MI). Signals from the multiplexer were passed to a chemical
microsensor II (Diamond General) that performed current-to-voltage
signal conversion and amplification and maintained electrode
polarization at 800 mV. An analog voltage proportional to the Clark
electrode current then was passed to an analog-to-digital conversion
card (PCI-MIO-16E-4, National Instruments, Austin, TX) installed on a
Macintosh G3 computer. Data was collected, displayed, and saved
digitally with a program written by one of us (C. B. Allen) with
LabVIEW software (National Instruments). Once the current in
both electrodes had stabilized, gas flow was started to the chamber at
5 l/min. In the hyperoxia experiments, this gas consisted of 95%
O2 and 5% CO2, whereas in the hypoxia
experiments, the flushing gas was 100% N2. Gas flow was
maintained through the chamber for at least 3 h or until the
current through the electrodes had reached a semipermanent plateau,
which we defined as at least 10 min with no increase in electrode
current. The current achieved at this plateau was accepted as 100%
saturation with the flushing gas. In cases where no plateau was
achieved during the time of the experiment, the electrode was removed
from the tissue culture apparatus and submerged ~2 mm in unstirred
PBS equilibrated with the flushing gas by bubbling. The electrode
current measured in this fashion was then used as the 100% saturation
value. In the hyperoxia experiments, the electrode current at 21%
O2 (air) was taken to be 0% saturation, whereas the
current at 95% O2 was taken to be 100% saturation. In the
hypoxia experiments, the electrode current at 21% O2 (air)
was taken to be 0% saturation, whereas the electrode current at 0%
O2 (100% N2) was taken to be 100% saturation.
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An additional series of experiments was performed to determine whether the results found in the tissue culture plates containing PBS were similar to those from plates containing tissue culture medium, fetal bovine serum (FBS), and growing cells. To avoid difficulties in the interpretation of data from the Clark electrodes due to protein fouling and pH effects in the serum- and bicarbonate-containing tissue culture medium, a fiber-optic oxygen-measuring system was employed in these studies. This system consists of six fiber-optic oxygen probes (model FOXY OR125, Ocean Optics, Dunedin, FL). Each of these was connected through bifurcated fiber-optic cables to a six-channel fluorescence spectrometer (S2000 series, Ocean Optics) and a 450-nm pulsed blue LED light source (model R-LS-450, Ocean Optics). The spectrometer and light source were connected through custom wiring and circuitry to a PCI-MIO-16-E4 data acquisition card (National Instruments) installed on a Macintosh G3 desktop computer. Software written in LabVIEW (National Instruments) by one of us (C. B. Allen) was used to control the data acquisition and data storage process.
The lids of the 100-mm tissue culture plates were modified to hold the sensing end of the fiber-optic oxygen probes ~1.5 mm from the bottom of the plates. The plates containing the A549 cells were initially seeded with 2 × 106 cells/plate and allowed to grow to confluence in Ham's F-12 medium, Kaighn's modification (F12-K; GIBCO BRL, Life Technologies, Grand Island, NY) with 10% FBS, 100 U/ml of penicillin, and 100 µg/ml of streptomycin added.
Five experimental groups were studied in this series of experiments: a group of three plates containing 20 ml of 10 mM PBS (pH 7.4) flushed with 100% N2, a group of three plates containing 20 ml of 10 mM PBS (pH 7.4) flushed with 95% N2-5% CO2, a group of three plates containing 20 ml of serum-free F12-K flushed with 95% N2-5% CO2, a group of three plates containing 20 ml of F12-K containing 10% FBS flushed with 95% N2-5% CO2, and a group of three plates containing 20 ml of F12-K containing 10% FBS overlying confluent A549 cells and flushed with 95% N2-5% CO2. The experimental plates with the modified lids were placed in a rectangular Plexiglas chamber with a volume of 3.9 liters that had been modified for passage of the fiber-optic cables into the chamber. The fiber-optic oxygen probes were placed in each plate, and the chamber was then closed.
The fiber-optic probes were allowed to stabilize in their respective plates while the chamber was flushed with 21% O2-5% CO2-74% N2 for the experiments flushed with 5% CO2-containing gas or with room air for the 100% N2-flushed experiments. Once the signals from the oxygen probes were stable, the flushing gas was changed to the anoxic mixture. This gas was flushed through the chamber for at least 3 h at 3.9 l/min.
At the end of each experimental run, the signal intensities at 0, 21, and 95% O2 were determined by exposing the fiber-optic probes to the respective medium sequentially equilibrated with each gas mixture. These values were used to generate the second-order polynomial coefficients used to convert the signal intensities into PO2 in millimeters of mercury according to the method described by the manufacturer of the probes (Ocean Optics).
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RESULTS |
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Typical Clark electrode current profiles for 100-mm plates and
T-75 flasks containing PBS exposed to hyperoxia are presented in Fig.
2. The rate at which the oxygen in the
gas phase achieved equilibrium within the 100-mm plate with the intact
lid was slightly slower than that in the 100-mm plate with the
perforated lid. This suggests that the design of the venting structure
of the 100-mm plates allows rather rapid equilibration of oxygen across the lid. In contrast, the rate at which the oxygen levels in the gas in
T-75 flasks with intact tops equilibrated with that of the flushing gas
was substantially slower than that in the T-75 flasks where the tops
had been perforated with large holes. From this, it can be concluded
that the filter material of the vented cap is a substantial barrier to
the equilibration of oxygen. In both the intact 100-mm plates and T-75
flasks, >3 h of incubation in the hyperoxic gas mixture was required
before the concentration of oxygen in the PBS approached saturation.
Interestingly, the rate at which oxygen equilibrated in the PBS in the
100-mm plates with perforated lids was accelerated to a greater extent
than would have been expected from the increased rate of oxygen
transport into the gas phase in the perforated plates. The rate of
equilibration of the PBS in the perforated T-75 flasks was similar to
that of the PBS in the perforated 100-mm plates despite the disparity in the rates of equilibration of oxygen in the gas phase in these plates.
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Typical electrode current profiles for the 100-mm plates and T-75
flasks exposed to hypoxic conditions are presented in Fig. 3. As in hyperoxia, it is clear that the
venting structure of the 100-mm plates allowed more rapid equilibration
of oxygen than did the vented cap of the T-75 flask. Furthermore, a
similar disparity between the rates of oxygen equilibration in the gas
and liquid phases was observed in the hypoxia experiments in the 100-mm
plates.
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The time required to achieve 50, 75, and 90% saturation with the
flushing gas was calculated for the gas and liquid phases to provide
more quantitative comparisons of the kinetics of oxygen transport. The
timing indexes for the hyperoxic exposures in the 100-mm plates and
T-75 flasks are presented in Tables 1 and
2, respectively, and the timing indexes
for the hypoxic exposures in the 100-mm plates and T-75 flasks
are presented in Tables 3 and
4, respectively. It is clear that the
intact top of either the 100-mm plates or T-75 flasks provided a
significant impediment to the dissolution of oxygen into the medium.
Although it only required ~4 min for the oxygen concentration of the
gas in the perforated 100-mm plates and T-75 flasks to achieve 90%
equilibration with the flushing gas, it still required nearly 1 h
for the PBS in the perforated vessels to achieve 90% O2
saturation. In comparison, equilibration of the gas phase to 90%
O2 saturation in the intact 100-mm plates and T-75 flasks
required 13.50 ± 0.95 and 46.67 ± 2.54 min, respectively.
The PBS in the intact 100-mm plates and T-75 flasks required
134.67 ± 17.68 and 161.00 ± 17.21 min, respectively, to
achieve 90% saturation.
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A pattern of timing similar to that seen in hyperoxia was noted in the hypoxia experiments. Only ~3 min was required for the gas within the perforated 100-mm plates and T-75 flasks to achieve 90% O2 saturation. The PBS within the perforated 100-mm plates and T-75 flasks required 73.00 ± 4.50 and 60.50 ± 2.02 min, respectively, to achieve 90% saturation with O2. With an intact top, however, these times were substantially increased. The gas phase in the 100-mm plates and T-75 flasks required 13.25 ± 1.13 and 55.67 ± 3.96 min, respectively, to achieve 90% O2 saturation. The PBS within the intact 100-mm plates and T-75 flasks required ~3 h (197.92 ± 17.13 and 172.67 ± 14.33 min, respectively) to achieve 90% O2 saturation.
Typical PO2 profiles in the 100-mm plates
studied with the fiber-optic oxygen probes are presented in Fig.
4. In this experimental apparatus,
equilibration of oxygen in the experimental liquid required >3 h to
decline to 5% of the PO2 present in the
air-equilibrated medium. After 3 h of being flushed with anoxic
gas, the PBS flushed with 95% N2-5% CO2 had
achieved a PO2 of 22.5 ± 0.6 (SE) mmHg, whereas the PBS-containing plates flushed with 100% N2 had
achieved a PO2 of 23.0 ± 1.3 (SE) mmHg.
At this 3-h time point, the plates flushed with 95% N2-5%
CO2 containing serum-free medium and serum-containing medium had achieved a PO2 of 19.0 ± 0.6 and 22.4 ± 0.7 (SE) mmHg, respectively. There was no statistical
difference between the oxygen concentrations achieved at 3 h in
any of the cell-free groups (P 0.05 by Tukey-Kramer
test). However, the oxygen concentration in the plates holding
confluent A549 cells in serum-containing medium declined much more
rapidly, achieving 5% of the full range (6.7 mmHg) in 72.1 ± 0.3 (SE) min.
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DISCUSSION |
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Over 30 years ago, Chapman et al. (5) wrote "The justification for publishing a detailed examination of the minutiae of the oxygen-diffusion process in plastic Petri dishes is the insidious nature of the artifacts which can arise and the need to demonstrate quantitatively as well as qualitatively that the proposed explanations are adequate." It is our impression from the current literature that although the issue of oxygen diffusion into and out of tissue culture plates has been the subject of several studies over the years, the practical consequences of these diffusion issues remain poorly appreciated by many investigators. The majority of publications (8, 12, 14) addressing oxygen diffusion issues have been directed toward maintaining stable oxygen concentrations in the medium in the face of rapid cellular oxygen consumption. As such, oxygen concentrations have been measured after periods of days rather than on the scale of minutes and hours studied here.
Oxygen must travel a tortuous path before reaching the cell surface in the tissue culture plate. Oxygen molecules entering the environmental chamber are first transported across the environmental chamber interior by convective mixing during the flushing period. Then, the oxygen must pass either through the polystyrene material of the tissue culture plate by a slow process of diffusion or through the vent feature of the plate by further convection. Once inside the tissue culture plate, the gas moves across the unmoving gas toward the tissue culture medium by diffusion. At the gas-medium interface, the rate at which oxygen enters the medium is limited by the relatively low solubility of oxygen in aqueous medium. Diffusion of oxygen through the full thickness of the culture medium layer is the final step in the oxygen transport process.
The oxygen concentration to which cells in a monolayer culture are exposed is a result of the balance between oxygen delivery across the full medium thickness and the rate of oxygen consumption by the culture cells. Metzen et al. (12) have described an equation in which specific oxygen consumption, plate protein content, medium thickness, and medium surface area are used to estimate pericellular PO2 values for a number of cell types. They found good agreement between the calculated pericellular PO2 values and those measured with a Clark oxygen electrode. However, when the environmental oxygen concentration is changing, such calculations will become more challenging. Rather than attempt to develop detailed mathematical models to estimate the oxygen-loading kinetics in an idealized cell culture system, we elected to use empirical methods to gain a better appreciation of the kinetics of oxygen transport in the tissue culture systems we currently use.
We report here that it required nearly 30 min for the PO2 of the gas inside the 100-mm tissue culture plates to equilibrate with that of the gas outside them. Furthermore, it required >3 h for the PO2 of the medium inside the tissue culture plates to equilibrate with the PO2 of the gas outside the plates. This would indicate that unless the period of chamber flushing is extended to several hours, the oxygen concentration to which cells are exposed is likely not the same as that of the flushing gas. In our experiments, we found that the presence of perforations in the lids of 100-mm plates accelerates equilibration of the gas phase to 90% saturation by ~9 min. However, perforation of the lids accelerated equilibration of the medium to 90% saturation by almost 100 min. This suggests that perforation of the lids has an effect on equilibration of the medium beyond just accelerating the rate at which the gas above the medium gains oxygen content. We believe that the perforations in the tops of both the 100-mm plates and T-75 flasks allow convective mixing of the gas immediately overlying the medium. We further speculate that this convective mixing of the gas just above the medium breaks down a local oxygen gradient produced in a thin boundary layer of gas above the medium. As oxygen moves across this gas-liquid interface, the difference between the oxygen concentrations on the opposite sides of the interface declines. Without mixing, we believe that this diminished gradient of oxygen between the gas and liquid phases slows the rate at which oxygen dissolves in the medium. When the presence of lid perforations allows this boundary layer of gas to be mixed by convective movement, the concentration of oxygen at the gas-liquid interface is effectively maintained at that of the flushing gas. As a result, the rate at which the oxygen moves across the gas-liquid boundary is accelerated over that in plates with the intact lids.
Hypoxia experiments conducted with fiber-optic oxygen probes in a smaller rectangular exposure chamber revealed that changes in chamber size and shape can affect the rate of oxygen equilibration. We also found that the presence of cells affects oxygen equilibration. Although we observed no differences in the rate of equilibration of oxygen in PBS, serum-free medium, and serum-containing medium in the square chambers, we did observe a striking acceleration in the decline in oxygen concentration in those experiments in which A549 cells were present. This accelerated decline in PO2 is probably due to the additional oxygen consumed by the cultured cells. It might be expected that in contrast to the situation in hypoxia, the presence of cells could slow the equilibration of culture medium under hyperoxic conditions. Many experimental protocols result in changes in both cell proliferation and respiration and, as a result, in the changing rates of oxygen consumption by the cell monolayer. In such cases, assumptions about the rate of oxygen equilibration into the medium may be erroneous.
In a previous report, Allen and White (2) found that glucose consumption is strikingly increased in A549 cells exposed to hyperoxia. Our finding suggested that unless extraordinary measures were taken in terms of either increasing medium volume or increasing the frequency of medium changes, glucose depletion would likely occur. However, daily feeding with fresh medium returns the ambient PO2 of the tissue culture medium to that of room air. The failure to achieve instantaneously the desired experimental PO2 values in the tissue culture medium may provide the cells with a degree of "rest" from hyperoxic or hypoxic treatment. Such a rest period was shown by Frank et al. (6) to protect rats against lethal hyperoxia. In their study, a reduction in oxygen concentrations to 50-75% during the rest period was just as protective as a rest period in room air. Although the relief from high PO2 that cells experience during the flushing period would be of shorter duration than that given the rats by Frank et al., it may still limit the magnitude of the cellular damage or other responses to the hyperoxic challenge.
In recent years, a wide variety of cell signaling events have been described that occur within minutes of cellular stress such as that occurring with a sudden change in PO2. The delay in achieving the desired PO2 within the tissue culture medium limits the temporal precision of any assessment of correlation between the cellular events with the concentration of oxygen to which they are exposed. It also suggests caution in acceptance of the immediacy (or lack thereof) of the cellular responses reported throughout the literature. Continuous gas flow exposure systems will not circumvent the problems described in this report. As long as the medium is changed on a frequent basis, as it must be to replace energy substrates such as glucose (2) and glutamine (1), antioxidant vitamins such as vitamin E (9), and other nutrients required by stressed cells, there will continue to be periods of rest or recovery provided to the cells. Such periods of recovery can cause marked changes in the injury phenotype. For example, Maniscalco et al. (11) have reported that vascular endothelial growth factor (VEGF) mRNA increases in rabbit alveolar epithelial cells in vivo after only 1 day of recovery in air after 64 h of oxygen exposure. Because VEGF has been shown to be a survival factor for endothelial cells exposed to hyperoxia and other toxic challenges (3, 7, 13), temporary relief of the hyperoxic inhibition of VEGF production (10, 15) might result in decreased endothelial cell injury and death.
Some simple approaches have been used to increase oxygen transport in tissue culture conditions. Balin et al. (4) used gentle horizontal shaking to minimize gas diffusion gradients created in the medium as a result of oxygen consumption by the WI-38 cells under study. Unfortunately, oxygen concentrations in the shaken and stationary culture plates were assessed only after a period of several hours so that no information is available concerning the rate of oxygen equilibration in that system. Another approach was used by Yang and Wang (16) in which pressurization of the headspace air over the tissue culture medium to 2,067 mmHg resulted in increased bacterial and algal growth. Although increasing oxygen transport into the cell culture medium, such pressurization is inconsistent with oxygen exposure protocols in which the desired PO2 values are those maximally achievable at sea-level pressure (760 mmHg).
In summary, we have found that several hours are required for the oxygen concentration in the medium within traditional tissue culture plates and flasks to achieve equilibrium with the oxygen concentration in the gas flushing the environmental chamber when the oxygen concentration within that flushing gas is changed to either 0 or to 100% O2 from 21% O2. Furthermore, we have found that the architectural features of the exposure chamber can significantly affect this equilibration time. We also found that the presence of respiring cells can accelerate this equilibration in the case of hypoxic exposures. From our findings with "perforated" tissue culture plates and flasks, we propose that the greatest impediment to oxygen equilibration occurs at the gas-medium interface and that disruption of a gas (and perhaps medium) boundary layer at that interface by turbulent gas flow above the medium can accelerate equilibration.
We conclude from our findings that the in vitro cellular responses to hyperoxic or hypoxic exposure described in many of the published reports may be a consequence of a change in oxygen concentration smaller in magnitude than that which was made in the flushing gas concentration. It is also likely that experiments conducted with sequential medium changes may introduce a degree of adaptation to the experimental oxygen environment being studied because of the slow oxygen equilibration. Based on our findings, future experiments addressing the rapid changes in cells occurring during signaling events must take into account this slow rate of oxygen equilibration. Investigators using environmental chambers to expose cultured cells to changing gaseous environments need to characterize gas equilibration kinetics in their specific system with their specific cell type to more fully understand and correctly interpret the results of their experiments.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-57144, HL-52732, and HL-56263.
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FOOTNOTES |
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Address for reprint requests and other correspondence: C. B. Allen, National Jewish Medical and Research Center, 1400 Jackson St., Denver, CO 80206 (E-mail: allenc{at}njc.org).
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. Section 1734 solely to indicate this fact.
Received 28 November 2000; accepted in final form 17 May 2001.
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