J. Tiss. Cult. Meth, 13:24t-246, 1991 © 1991 Tissue Culture Association 0271-8057/91 $01.50+0.00
ALTERING T H E DISSOLVED OXYGEN TENSION IN TISSUE CULTURE MEDIA Loraine H. Anderson, Frank J. Roberts, Bernard Wilson, and William J. Mehm
Division of Altitude and Hyperbaric Physiology, Department of Scientific Laboratories, Armed Forces Institute of Pathology, Washington, DC 20306
SUMMARY: Fast, effective methods to alter the concentration of dissolved oxygen in tissue culture media were desired to study the effect of oxygen tension on the behavior of cells in culture. This report compares the efficacy of two such methods, with the goal to achieve defined oxygen tensions in the liquid phase. In one method, a slight overpressure of defined oxygen tension promoted diffusion from the gas phase to the liquid phase (normal diffusion). In the other method, a vacuum was used to remove dissolved oxygen from the media before exposure of samples to a defined oxygen overpressure (vacuum-assisted diffusion). With normal diffusion, the dissolved oxygen tensions became fixed within 90 min and never reached the target oxygen concentrations. However, with vacuum-assisted diffusion, target oxygen concentrations were reached within 7 min, demonstrating this method to be faster and more complete for altering the dissolved oxygen content in culture media.
Key words:oxygen; culture media; vacuum; method.
I.
INTRODUCTION
normal diffusion (5,6,9) to alter the dissolved oxygen concentration in tissue culture media. Sparging the media is advantageous because it is rapid and effective. However, it does have a disadvantage when used with tissue culture media in that it creates foam. The foam does not readily disperse, suggesting the presence of denatured proteins. Normal diffusion does not cause protein denaturation but is a very slow process. The purpose of this study is to evaluate methods to alter the oxygen concentration in tissue culture media. We have selected two methods to study: a) normal diffusion and b) vacuum-assisted diffusion.
In in vitro culture systems, oxygen is present in two physical phases: the gaseous oxygen in the atmosphere to which the media are exposed (gas phase) and the oxygen dissolved in tissue culture media (dissOlved liquid phase). The oxygen in the dissolved liquid phase represents the cells' actual oxygen environment. To modify the cells' oxygen environment, control of oxygen concentration in both the liquid phase and the gas phase is required. When the oxygen concentration in only one phase is altered, the concentration of oxygen in the other phase changes slowly until the oxygen in the gas and liquid phases reaches equilibrium. Methods to change dissolved oxygen tensions of tissue culture media include: a) exposing the media to a different gas phase (normal diffusion), b) sparging the media with oxygen, and c) removing dissolved gases from the liquid phase with a vacuum, followed by replacement of the dissolved gases with the desired gases from the gas phase (vacuum-assisted diffusion). In normal diffusion, the gases move slowly from the phase of higher concentration to the phase of lower concentration. Sparging, the process of bubbling gas through a liquid, increases the rate of diffusion by increasing the surface area of the gas-liquid interface. Vacuum-assisted diffusion also increases the rate of diffusion but in a different manner. This mechanism involves increasing the diffusion gradient between the gas and the liquid phases by removing the dissolved gases from the liquid phase. Therefore, the increased diffusion gradient promotes more rapid diffusion of oxygen from the gas phase into the liquid phase. Previous investigations have used sparging (1,2,4) or
II.
MATERIALS
A. Equipment 1. Refrigerator-freezer, Hotpoint, Division of General Electric 1 2. Blood gas analyzer, model 170, Corning~ 3. Controlled atmosphere culture chamber, no. 774110005, Bellco 3 4. Pipette Aid, Drummond Scientific 4 5. Gas regulators, no. E12-2-N515B, Air Products 5 6. Carbon dioxide incubator, model 3158, Forma Scientific 6 7. Oxygen-safe vacuum pump, model 5KC47UG1528T 1 8. Controlled atmosphere glove box, Labconco Corp. 7 B. Supplies 1. RPMI 1640, no. 01-123-500, Advanced Biotechnologies s 2. Fetal bovine serum, no. 02-101-1008 3. HEPES buffer, no. 01-508-1008 241
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ANDERSON ET AL. 4. L-Glutamine, no. 01-506-508 5. Complete tissue culture medium--RPMI 1640 supplemented witb 25 mM HEPES, 200 mM L-glutamine, and 10% fetal bovine serum. 6. Six-well microtiter plates, no. 3406, Costar 9 7. Pipette, disposable 10 ml, no. 7075-10 z 8. Glass syringe, 50 ml, no. 2145, Becton-Dickinson 1° 9. Disposable syringe, 10 ml 1° 10. Three-way stopcock, no. K75, Pharmosea111 11. Laboratory grade bubble tubing, no. 8889-224054, Monoject Scientific 12 12. Mixed gases, primary standard grade, 5 (Oxygen concentration was 0, 7.6, 15, 38, 80, or 722 mmHg. All gases contained 38 mmHg of carbon dioxide and balance nitrogen.) 13. Stopper apparatus--composed of glass tubing 10 to 18 cm long inserted through a size 3 stopper (the correct size to fit a 500-ml bottle) 14. Sterile 500-ml bottle
III. PROCEDURE A. Normal diffusion 1. Overview Aliquots of medium are placed in culture vessels located in a small Plexiglas box (controlled atmosphere culture chamber). The chamber is purged with a defined gas phase (Fig. 1) and placed in a 37 ° C incubator. At specific time points, aliquots of medium
are removed for determination of the dissolved oxygen concentration. 2. Setup a. Attach a pressure regulator to the desired gas cylinder. b. Attach a 60-cm length of tubing to the regulator. c. Connect two three-way stopcocks, one at each end, to a 10-cm length of tubing. d. Attach one of these stopcocks to a 50-ml glass syringe. 3. Placing samples of medium in microtiter plate a. Place a 2-rot sample in each well of a six-well microtiter plate. This volume provides a 5-mm depth of medium, the maximum depth that allows adequate gas exchange (3). b. Cover the microtiter plate and place it in the controlled atmosphere culture chamber. c. Place the cover on the controlled atmosphere culture chamber and tighten all damps. 4. Purging the controlled atmosphere culture chamber (Fig. 1) a. Attach the loose end of the 60-cm tubing to the inlet port of the controlled atmosphere culture chamber. b. Turn on the gas cylinder, making sure the regulator is closed. c. Open the valve on the inlet port of the controlled atmosphere culture chamber.
FIC. 1. Controlledatmosphere culture chamber containing culture vessels and media. Gas cylinder is shown configuredto purge the chamber with the defined target gas overpressure.
ANDERSON ET AL. d. Hold open the pressure relief valve on the controlled atmosphere culture chamber. e. Turn on the regulator to 863 mmHg (2 psig) and purge the controlled atmosphere culture chamber for 30 s. This ensures the gas overpressure phase contains the desired target concentrations. f. Release the pressure relief valve and immediately close the inlet valve on the controlled atmosphere culture chamber. Quickly turn off the regulator. (A slight positive pressure, not to exceed 915 mmHg or 3 psig, will remain in the controlled atmosphere culture chamber.) g. Remove the tubing from the controlled atmosphere culture chamber. . Incubation and measurement of dissolved oxygen a. Place controlled atmosphere culture chamber in an incubator at 37 ° C for 15, 30, 60, 90, or 120 rain. (The maximum time selected was based on a report by Sendroy et al. (7). They reported 1.5 to 2.25 h to be more than sufficient time to completely saturate biological solutions with oxygen.) b. Calibrate the blood gas analyzer 15 rain before measuring the samples. This resets the internal calibration, which occurs every 30 min, and prevents interference with sample analysis. c. Remove the controlled atmosphere culture chamber from the incubator and place it next to the blood gas analyzer. (Before measuring the dissolved oxygen concentration of the media samples,
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the oxygen concentration of the gas phase was validated using a blood gas analyzer.) d. To measure the dissolved oxygen concentration of the media sample, open the inlet valve of the controlled atmosphere culture chamber to release the residual internal pressure. e. Open the controlled atmosphere culture chamber and remove the microtiter plate. f. Using a 10-ml disposable syringe, remove all the sample from one microtiter well. Ensure air bubbles are not present in the sample. g. Using a blood gas analyzer, measure and record the partial pressure of oxygen and the pH of the sample. (We monitored the pH for all samples and observed no significant changes.) h. Samples can be analyzed at approximately 2-min intervals. B. Vacuum-assisted diffusion 1. Overview A glove box (Fig. 2) is filled with the desired gas phase. Within the glove box, a glass bottle containing tissue culture medium is connected to a vacuum pump (located outside the glove box) and evacuated. The vacuum created in the bottle removes dissolved gases from the liquid phase, increasing the diffusion gradient. When the vacuum is vented, the components of the gas phase rapidly diffuse into the medium. This vacuum-vent cycle is repeated several times to effect complete gas exchange.
FIG. 2. Vacuum-assisteddiffusionset up. Glovebox is shownwith the vacuumpump and the desired gas cylinder. Vacuumpump is connected to the bottle of medium through a glove box port.
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ANDERSON ET AL. 2. Setup a. Place medium in sterile 500-ml bottle (less than 125 ml of medium). b. Place the bottle of medium, a sterile stopper apparatus, and tubing approximately 30 cm long inside the glove box. 3. Changing the gas phase within the glove box a. Connect the regulator of the desired gas cylinder to the top and side ports of the glove box, using tubing of appropriate length. b. Open the valves at the ports. c. Insert the sampling tubing from the Perkin-Elmer gas analyzer in the glove box to monitor the gas concentrations. Tighten only the clamps at the rear of the door to avoid crimping the tubing. d. Turn on the gas cylinder regulator until the low pressure gauge reads approximately 863 mmHg (2 psig). e. Allow the gas exchange to continue until the desired gas concentrations are achieved, as indicated by the gas analyzer measurements. f. Carefully remove the gas analyzer's sample tubing from the glove box and secure all door clamps. g. Quickly turn off the gas flow regulator to prevent overfilling of the glove box. If the box is overfilled, use the vacuum pump to remove the excess gas (see below). h. If the tubing from the gas cylinder to the ports is removed, ensure that the valves are closed first. 4. Applying the vacuum a. When the glove box contains the desired gas concentrations, carefully place the stopper in the bottle of medium. Connect the tubing to the port inside the glove box and to the glass tubing in the stopper. b. Connect the vacuum pump to the corresponding port outside the glove box. c. Turn on the vacuum pump and open the valve on the glove box port to expose the medium to the vacuum. d. As the medium begins to bubble, rotate the bottle gently to mix the medium. Periodically release the stopper to relieve the vacuum, exposing the medium to the gas in the glove box. e. Five minutes is usually sufficient time to reach the desired oxygen tension in the medium, but the time will vary depending on the vacuum system, the volume of medium, and the target oxygen tension. Using a blood gas analyzer, measure the oxygen tension of the medium at various times to determine the minimum treatment time required to achieve target oxygen concentration in a specific system. f. When the process is complete, close the glove box valve, turn off the pump, and remove the tubing and stopper from the bottle. g. If necessary to facilitate arm movements, more gas may be allowed into the glove box, using the techniques given in section B2. h. The pH and the partial pressures of oxygen and carbon dioxide in the sample of medium are determined using a blood gas analyzer.
i. The pH was monitored for all samples, and no significant changes were observed. IV.
DISCUSSION
A. Normal diffusion When the normal diffusion method was used, the partial pressure of oxygen in the medium became fixed in approximately 90 min (Fig. 3). However, there was not a complete gas exchange and the target-dissolved oxygen concentrations were never achieved. With gaseous (target) oxygen concentrations of 0, 7.6, 15, 38, 80, and 722 mmHg, the dissolved oxygen tensions reached 26.4, 35.6, 65.1, 63.1, 104.5, and 617.8 mmHg, respectively, Data indicate that 90 min was not sufficient time for oxygen to diffuse from the liquid phase to the gas phase. This is not surprising in view of the fact that oxygen is known to diffuse very slowly in to or out of a liquid (10). B. Vacuum-assisted diffusion Vacuum-assisted diffusion was used to change the dissolved oxygen concentration to the following target concentrations: 7.6, 15, 38, and 80 mmHg of oxygen. Within 7 min the dissolved oxygen in the liquid phase reached 8.8, 16.0, 37.6, or 76.3 mmHg, respectively, an almost complete oxygen exchange (Fig 3). However, at the very high (722 mmHg) and the very low (0 mmHg) oxygen concentrations, the exchange was less complete. The former reached only 641.4 mmHg (vs. the target of 722 mmHg) and the latter, 9.4 mmHg (vs. the target of 0). This is not surprising in light of the slow rate of oxygen diffusion. Taylor and Camalier (8) encountered a similar problem when attempting to achieve 0 mmHg of oxygen in tissue culture media. They reported the presence of oxygen-binding proteins in media prepared aerobically and cited the oxygen-binding properties of various proteins as the reason the dissolved oxygen tension never reached 0 mmHg of oxygen. At the opposite end of the spectrum was the difficulty in acl~ieving 722 mmHg of oxygen in the medium after treatmem with 722 mmHg of oxygen. Increasing the time of gas exchange with 722 mmHg of oxygen using either method did not raise the dissolved oxygen in the medium. We suggest that, in addition to the slow diffusion rate of oxygen, oxidative processes may occur in the medium upon exposure to elevated levels of
700 1 600~ E
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Target Oxygen Tension in Gas Phase (mmHg)
FIG. 3, Mean dissolved oxygen tensions _+ SEM for two treatment groups at the target oxygen tensions indicated (n = 6). Note broken scale on the ordinate between 120 and 600 mmHg.
ANDERSON ET AL.
oxygen. Oxidation would utilize the dissolved oxygen and reduce the concentration of free oxygen dissolved in the medium and would thus make it difficult to reach and maintain a level of 722 mmHg of oxygen in the medium. Therefore, of the two methods evaluated here, the vacuum-assisted diffusion method is more effective in altering the dissolved oxygen concentration in liquid culture media. This method is a rapid, simple, and reproducible means of achieving a defined concentration of dissolved oxygen in tissue culture media.
V.
REFERENCES
1. Balin, A. K.; Goodman, D. B. P.; Rasmussen, H., et al. Atmospheric stability in cell culture vessels. In Vitro 12:687-692; 1976. 2. Bankey, P.; Fiegel, V.; Singh, R., et al. Hypoxia and endotoxin induce macrophage-mediated suppression of fibroblast proliferation. J. Trauma 29:972-980; t989.
31 Freshncy, R. I. Culture of animal cells: a manual of basic technique, 2nd ed. New York: Alan R. Liss Inc.; 1987. 4. Horikoshi, T.; Balin, A. K.; Carter, D. M. Effect of oxygen on the growth of human epidermal keratinocytes. J. Invest. Dermatol. 86:424-42; 1986. 5. Mehm, W. J.; Pimsler, M.; Becker, R. L., et al. Effect of oxygen on in vitro fibroblast cell prohferation and collagen biosynthesis. J. Hyperbaric Med. 3:227-234; 1988. 6. Packer, L.; Fuehr, K. Low oxygen concentration extends the lifespan of cultured human diploid cells. Nature 267:423-425; 1977. 7. Sendroy, J., Jr.; Dillon, R. T.; Van Slyke, D. D. Studies of gas and electrolyte equilibria in blood: XIX. The solubihty and physical state of uncombined oxygen in blood. J. Biol. Chem. 105:597-632; 1934-1935. 8. Taylor, W. G.; Camalier, R. F. Modulation of epithelial cell proliferation in culture by dissolved oxygen. J. Cell Physiol. 111:21-27; 1982. 9. Taylor, W. G.; Camalier, R. F.; Sanford, K. K. Density-dependent effects of oxygen on the growth of mammalian fibroblasts in culture. J. Cell Physiol. 95:33-40; 1978. 10. Vander, A. J.; Sherman, J. H.; Luciano, D. S. Human physiology, the mechanisms of body function, 4th ed. New York: McGraw-Hill Book Company; 1985:116.
T h e opinions or assertions c o n t a i n e d h e r e i n are the private views of the a u t h o r s a n d are not to b e c o n s t r u e d as official or as reflecting the views of the D e p a r t m e n t of the Army, the D e p a r t m e n t of the Air Force, or the D e p a r t m e n t of Defense.
1 General Electric, Fort Wayne, IN 2 Corning, Coming, NY 3 Bellco, Vineland, NJ 4 Drummond Scientific, Broomall, PA s Air Products, Hyattsville, MD 6 Forma Scientific, Morietta, OH
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Labconco Corp, Kansas City, MO s Advanced Biotechnologies, Columbia, MD 9 Costar, Cambridge, MA lo Becton-Dickinson, Ruthersford, NJ tl Pharmoseal, Toa Aha, Puerto Rico 12 Monojeet Scientific, St. Louis, MO