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Dissolved oxygen concentration in culture medium: assumptions and pitfalls
Placenta (2005), 26, 353e357 doi:10.1016/j.placenta.2004.07.002
SHORT COMMUNICATION Dissolved Oxygen Concentration in Culture Medium: Assumptions and Pitfalls D. Newby, L. Marks and F. Lyall* Maternal and Fetal Medicine Section, Institute of Medical Genetics, Yorkhill, Glasgow G3 8SJ, UK Paper accepted 8 July 2004
Oxygen is a key factor in the regulation of cytotrophoblast differentiation, proliferation and invasion in early pregnancy. Abnormalities in oxygen concentration have also been linked to a number of pregnancy disorders. Cell culture models have been used to study the effect of oxygen on cytotrophoblast behaviour in vitro, however, there is often little or no validation of oxygen levels in these cell culture systems. In this study, dissolved oxygen levels in culture medium maintained in standard culture conditions (18% O2) measured 18%. On transfer to a low oxygen environment (2% O2), oxygen levels decreased to 6e8% after 4 h and reached 2% only after 24 h in culture. Culture medium pre-gassed with nitrogen to remove dissolved oxygen quickly absorbed oxygen when exposed to ambient air during dispensing and required further incubation in a 2% oxygen environment before dissolved oxygen levels equilibrated to 2%. Thus, cultured cells placed in a low oxygen environment would be exposed to varying levels of oxygen before the desired level of oxygen exposure is reached. This study highlights the importance of validation of oxygen levels and potential problems associated with in vitro studies on the regulatory effects of oxygen. Placenta (2005), 26, 353e357 Ó 2004 Elsevier Ltd. All rights reserved.
INTRODUCTION A number of important clinical conditions such as preeclampsia, fetal growth restriction and spontaneous miscarriage have been linked to the placenta being exposed to abnormal oxygen concentrations [1]. Women living at high altitude are exposed to chronic hypoxia and they too have increased risk of complications of pregnancy [2], underscoring the involvement of oxygen. Cytotrophoblast proliferation and differentiation appears, at least in part, to be regulated by oxygen tension. These observations have been the driving force for investigators to develop in vitro models to determine the effects of different oxygen concentrations on cell culture models such as primary trophoblast cultures and placental explant culture [3e7]. One of the key issues in studies of this nature is the actual oxygen levels which cultured cells are exposed to during incubation in different oxygen environments. Some authors report specific methods to control the oxygen environments to which the cells are exposed and/or the measurement of oxygen in culture medium [3,5,7,8], while * Corresponding author. Tel.: C44 141 201 0657; fax: C44 141 357 4277. E-mail address: [email protected] (F. Lyall). 0143e4004/$esee front matter
others make no mention of oxygen levels in their culture systems other than in the incubator environment [6,9,10]. The aim of this study was to define the oxygen environments encountered in our cell culture systems and to address some of the issues and potential problems associated with studies of this nature. METHODS Cell culture incubators Studies were carried out using two Forma Scientific waterjacketed incubators (Forma Scientific, Inc. USA), one set at 5% CO2 in air for standard culture conditions with the sensor on the incubator reading approximately 18.2% O2 and one set at 5% CO2/93% N2 with the oxygen sensor reading approximately 2% O2 for low oxygen conditions. Oxygen levels in each incubator environment were measured using a Fyrite Gas Analyser. Measurement of dissolved oxygen levels There are many commercially available oxygen probes and meters, of varying specifications, for the measurement of dissolved oxygen in a liquid environment but few are ideal for the measurement of dissolved oxygen in cell culture systems Ó 2004 Elsevier Ltd. All rights reserved.
Placenta (2005), Vol. 26
354
Experiment 1
20
Dissolved oxygen (%)
such as those used in trophoblast and explants cultures. A Jenway 970 Portable Dissolved Oxygen/(C Meter and Electrode (Jencons Scientific Ltd, UK) was chosen for the measurement of dissolved oxygen in this study due to the cost and the suitability of the technical specifications, in particular the integral temperature compensation. Issues relating to the measurement of oxygen in cell culture systems and the factors that influenced the choice of oxygen probe used in this study are discussed in more detail later in this communication. The oxygen meter was calibrated, according to the manufacturer’s instructions, to 21% oxygen in water saturated air and to 0% oxygen using the zero salts supplied with the meter. In order to satisfy the requirements of the oxygen electrode, experiments were carried out using approximately 125 ml of M199 (supplemented with 10% fetal bovine serum and 1% antimycotic/antibiotic solution) (GibcoBRL Life Technologies, UK) in a T75 vented-lid cell culture flask. This was necessary so that the depth of the culture medium in the upright flask was sufficient to cover the temperature compensating element of the electrode and allowed stirring of the culture medium during measurement. The flask was placed flat in the incubators to allow maximum surface area for gaseous exchange. The surface area/volume in the T75 flask was 75 cm2/125 ml and this compares to 8 cm2/4 ml in 35 mm culture dishes, 1.9 cm2/0.4e1 ml in 24 well plates and 0.6 cm2/ 200 ml in the Millicell inserts used by many investigators for trophoblast and explant culture experiments. All experiments were performed in triplicate and repeated three times.
15 10 5
0
5
15
10
20
25
Time (h)
Figure 1. Dissolved oxygen concentration (% O2) in test culture medium incubated at 37 (C in hypoxic conditions for up to 24 h. Oxygen levels were measured using a Jenway Model 970 Dissolved Oxygen Meter and Electrode (Jencons Scientific Ltd, UK).
then dispensed into smaller volumes (5 ! 25 ml) to mimic culture medium changes (total dispensing time 4 min) and then returned to the T75 flask. Dissolved oxygen in the culture medium was measured immediately in the 125 ml of culture medium and the flask placed in the low oxygen incubator at 37 (C. Dissolved oxygen levels were measured at intervals up to 16 h. RESULTS Oxygen levels, measured using a Fyrite Gas Analyser, were 18e20% in the standard incubator and 2e3% in the low oxygen incubator. Experiment 1
Culture medium was warmed to 37 (C in T75 flasks in standard culture conditions (5% CO2 in air). Dissolved oxygen levels were measured and the flasks were then transferred to low oxygen conditions at 37 (C for up to 24 h. Dissolved oxygen levels in the culture medium were measured at intervals during the incubation period. Separate flasks were used for each time point to prevent the medium being affected by atmospheric oxygen.
The dissolved oxygen concentration in culture medium maintained in standard culture conditions measured approximately 18% (pO2 140 mmHg). When culture medium was transferred to the low oxygen incubator, oxygen levels decreased gradually to measure 6e8% (46e61 mmHg) after 4 h, 7% (53 mmHg) after 8 h, 3% (23 mmHg) after 16 h and 2e3% (15e23 mmHg) after 24 h of incubation (Figure 1). The pH was unaffected by the changes in oxygen concentration (pH 7.2e7.4).
Experiment 2
Experiment 2
Nitrogen gas (Cryoservice, UK) was bubbled through culture medium (2e3 psi) (125 ml) in a T75 flask for 30 min to eliminate dissolved oxygen in the culture medium. The opening to the flask was sealed with parafilm through which there was an inlet for the pipette delivering the nitrogen gas and an outlet to allow gas to escape. The flask was immediately transferred to a 2% oxygen environment at 37 (C. Dissolved oxygen levels in the culture medium were measured after 1 h and following overnight incubation.
When nitrogen gas was bubbled through culture medium, dissolved oxygen levels fell from 18e20% to approximately 1.5% (11 mmHg) after 15 min and to 0% after 30 min. When pre-gassed culture medium (0%) was placed directly into the incubator with the 2% oxygen environment, oxygen levels in the culture medium equilibrated to approximately 2% after 1 h and remained at 2e3% (15e23 mmHg) following overnight incubation. Experiment 3
Experiment 3 Nitrogen gas was bubbled through culture medium (125 ml) in a T75 flask for 30 min to eliminate dissolved oxygen in the culture medium, as described above. The culture medium was
When culture medium pre-gassed with nitrogen to eliminate oxygen (0%) had been dispensed in ambient air, dissolved oxygen levels increased to 8.5% (65 mmHg). Further incubation of the cell culture medium in a hypoxic environment
Newby et al.: Dissolved Oxygen Concentration in Culture Medium
of 2% oxygen for approximately 16 h was required for dissolved oxygen levels in the culture medium to decrease to 2% (15 mmHg). DISCUSSION There are many factors that must be given careful consideration when measuring dissolved oxygen levels in a cell culture system. Although many oxygen electrodes and meters are available, not all are suitable or ideal for use with cell cultures. Many probes are simply too large for the direct measurement of oxygen in cell cultures maintained in, for example, 35 mm dishes, 24-well plates or Millicell inserts. Smaller probes, such as the ISO2 electrode (World Precision Instruments, Inc, USA) with a 2 mm diameter tip, are available but often at much greater expense. Temperature is of vital importance as temperature effects the concentration of dissolved oxygen in a liquid. The MI-730 electrode (Microelectrodes, Inc, USA) is quoted by the manufacturers as having a change in probe response of 2.2% per degree change in temperature. Thus, unless an electrode has an integral temperature compensation function (e.g. Jenway 970, Jencons Scientific Ltd, UK), care must be taken to ensure that all measurements, including calibration, should be carried out at the same temperature (i.e. 37 (C in the case of cultured cells). This is not always possible for a variety of reasons depending on the method used for calibration. For example, many manufacturers recommend calibration in ambient air, while pre-gassing liquids, either for calibration or to establish defined oxygen levels, results in changes in liquid temperature that are difficult to control. Trophoblast cells and villous explants are most commonly cultured in a static environment. However, dissolved oxygen measurements cannot accurately be carried out in a static system due to oxygen starvation at the membrane of the electrode. This would result in false low oxygen readings, while agitation of cultures may dislodge cells or explants. Thus it would be difficult to obtain accurate measurements in many cell culture systems. Some authors have reported using a blood gas analyser to measure dissolved oxygen in culture medium sampled from cell cultures [7]. We initially used a Bayer 288 blood gas analyser (Bayer, Germany) in our experiments. However, this was not suitable as the analyser could only accurately measure oxygen levels in whole blood (correspondence with manufacturer) and gave false readings with culture medium. Kilani et al. [11] have reported their experience using a blood gas analyser and the requirement for addition of whole blood to the medium to improve the accuracy of readings. In order to characterise oxygen levels within the oxygen controlled cell culture environments used in our laboratory in a way that best compensated for the factors discussed above, we chose an oxygen electrode with automatic temperature compensation and measured dissolved oxygen levels in test culture medium maintained within the same environments as our cultured cells, rather than directly in cell cultures themselves. Dissolved oxygen levels in culture medium incubated in a hypoxic environment of 2% oxygen following
355
prior exposure to atmospheric oxygen or standard culture conditions decreased gradually to 2% over an incubation period of 24 h. During this time cultured cells would be exposed to varying oxygen concentrations and degrees of hypoxia at the cell/culture medium interface depending on the length of time in culture and before the desired oxygen concentration, in this case 2%, is reached. These experiments were carried out using a large volume of culture medium due to the requirements of the oxygen electrode in use. Presumably, with the much smaller volumes of culture medium used in our actual cell culture experiments the rate of gaseous exchange between the culture medium and the environment would be greater and hypoxic conditions would be established more rapidly. However, problems associated with culturing cells in a static environment, which may limit the rate of gaseous exchange, still remain. A rocking platform placed inside the incubator may be useful in instances where cells are firmly attached to substrate to allow diffusion. However, the authors should ensure that effects observed are not due to flow/shear stress per se. One way of circumventing the delay in establishing the appropriate culture conditions may be to pre-gas the culture medium, either with nitrogen to eliminate oxygen from the medium or with a gas of the desired mix and oxygen level. As we have shown, after pre-gassing with nitrogen to 0% oxygen, dissolved oxygen levels will equilibrate to 2% and remain at that level when maintained within a 2% oxygen environment. However, manipulation and microscopic monitoring of cultured cells is often carried out in ambient air, thus exposing the culture medium and cells to atmospheric oxygen. We have demonstrated that culture medium that has been pre-gassed with nitrogen to eliminate oxygen quickly absorbs atmospheric oxygen on exposure to ambient air. Further incubation in a 2% oxygen environment for 16 h was required for dissolved oxygen levels in the culture medium to equilibrate back to 2%, thus limiting the advantage of pregassing the culture medium. Gassing cultures directly to avoid uptake of atmospheric oxygen also has inherent difficulties particularly when millicell dishes are used. Such difficulties include dislodging the cells or explants during gassing, the cooling effect of the gas on the culture medium and the time involved in gassing individual cultures in an experiment. Furthermore, cultures maintained in a low oxygen environment would be exposed to increased oxygen levels during handling and during the associated time delay before the cell culture environment equilibrates to appropriate levels on return to the incubator, thus disrupting the exposure of the cells to the required oxygen concentration. The only way to avoid uptake of atmospheric oxygen would be to use a chamber flushed with the appropriate gas mix to allow manipulation of cultures in the same oxygen environment as the incubator atmosphere [3] and then to be able to transfer the dishes to the incubator without exposure to any atmospheric air. This work highlights the importance of, and difficulties associated with, the validation of oxygen levels in vitro cell
Placenta (2005), Vol. 26
356
culture studies investigating the effects of oxygen on placental development and function. Our studies focused on plastic dishes since this is what most workers use. However, studies with glass dishes may, because of differences in oxygen retention, reveal other issues. The issues highlighted are particularly important with regard to reproducibility and the comparison of data between different research laboratories. Based on our experiences we propose a number of recommendations when investigators perform culture experiments using varying oxygen conditions. Clearly, different groups will be asking different questions in their model systems and therefore the recommendations can only be used as a guideline. These have been sub-divided into two categories; Group 1: what this paper’s results have provided evidence to support and Group 2: what the authors’ ‘‘expert opinion’’ adds to the recommendations.
4. If the culture dishes are to be removed for examination under the microscope, authors should state how this aected the oxygen concentration.
Group 2 recommendations
Group 1 recommendations 1. The oxygen concentration in the incubator or chamber should be validated using a suitable device such as the Fyrite Gas Analyser used in the present study. 2. Authors should state the volume of medium per area of culture dish and how long it took to reach the required oxygen concentration before the experiment in question started. 3. Authors should specify if the culture medium was changed during the course of the experiment and how this aected the oxygen concentration. Ideally medium should be changed in a chamber flushed with the desired oxygen concentration and with medium that has been pre-bubbled to the required oxygen concentration. We do not recommend bubbling directly into culture dishes where cells are growing. A gloved box cabinet would be required with the pipette and pre-gassed medium placed inside the box before the box was flushed to obtain the desired oxygen concentration. Pipettes or tips should be flushed once with medium to remove air trapped inside the pipette. At this point the lid of the culture dish can be removed. However, authors should bare in mind that even with the lid on air exchange will still be taking place, albeit more slowly.
5. Measurement of the oxygen concentration in the culture medium should be made using an oxygen electrode with a tip size which can be fully immersed in the culture medium. A number of companies including Jencons (http:// www.jenway.com) and Diamond General (http://www. diamondgeneral.com) sell a range of electrodes with differing tip sizes. The smaller tip electrodes are more expensive. These must be calibrated before use according to the manufacturer’s instructions. The electrodes must have an integral temperature compensation function as temperature affects the dissolved oxygen in the culture medium. 6. We do not recommend shaking the culture dish containing cells to measure oxygen concentration when villous explants are cultured on matrigel since this may dislodge them slightly or completely thus altering their invasive potential. However, gentle shaking of a control dish which contains medium but no explant will give the researcher an idea of how the oxygen concentration has changed as a result of the electrode being in a static environment. 7. Authors should specify the oxygen concentration of the culture medium that the placental villous tissue was collected into after delivery of the placenta. The culture medium should be gassed to the appropriate level in the laboratory in a universal container and then the sample could be quickly dropped into it at collection. They should state how long it took from collection to preparation and the conditions used to dissect the piece of tissue. Ideally all procedures should be performed in the starting oxygen concentration of the experiment. We feel it would be unrealistic and technically very dicult to isolate single trophoblast cells in a low oxygen environment because of the many steps involved. 8. After consideration of the above manipulations in their experiment it would be helpful to other readers if the authors then defined whether, on balance, their culture system represents a model of hypoxia or a model or re-perfusion.
ACKNOWLEDGEMENT We are grateful to Action Research and the British Heart Foundation for funding.
REFERENCES [1] Kingdom JCP, Kaufmann P. Oxygen and placental villous development: origins of fetal hypoxia. Placenta 1997;18:623e4. [2] Zamudio S, Palmer SK, Regensteiner JG, Moore LG. High altitude and hypertension during pregnancy. Am J Human Biol 1995;7:182e93. [3] Genbacev O, Joslin R, Damsky CH, Polliotti BM, Fisher SJ. Hypoxia alters early gestation human cytotrophoblast differentiation/invasion in
vitro and models the placental defects that occur in preeclampsia. J Clin Invest 1996;97:540e50. [4] Genbacev O, Zhou Y, Ludlow JW, Fisher SJ. Regulation of human placental development by oxygen tension. Science 1997;277:1669e72. [5] Watson AL, Skepper JN, Janiaux E, Burton GJ. Susceptibility of human placenta syncytiotrophoblastic mitochondria to oxygen-mediated damage in relation to gestational age. J Clin Endocrinol Metab 1998;83: 1697e705.
Newby et al.: Dissolved Oxygen Concentration in Culture Medium [6] Caniggia I, Homa M, Winter J, Gassmann M, Lye SJ, Kuliszewski M, et al. Hypoxia-inducible factor-1 mediates the biological effects of oxygen on human trophoblast differentiation through TGFb3. J Clin Invest 2000;105:577e87. [7] Huppertz B, Kingdom J, Caniggia I, Desoye G, Black S, Korr H, et al. Hypoxia favours necrotic versus apoptotic shedding of placental syncytiotrophoblast into the maternal circulation. Placenta 2003;24: 181e90. [8] Fitzpatrick TE, Graham CH. Stimulation of plasminogen activator inhibitor-1 expression in immortalized human trophoblast cells cultured under low levels of oxygen. Exp Cell Res 1998;245:155e62.
357 [9] Kudo Y, Boyd CAR, Sargent IL, Redman CWG. Hypoxia alters expression and function of syncytin and its receptor during trophoblast cell fusion of human placental BeWo cells: implications for impaired trophoblast syncytialisation in pre-eclampsia. Biochim Biophys Acta 2003;1638: 63e71. [10] Nelson DM, Smith SD, Furesz TC, Sadovsky Y, Ganapathy V, Parvin CA, et al. Hypoxia reduces expression and function of system amino acid transporters in cultured term human trophoblasts. Am J Physiol Cell Physiol 2003;284:C310e5. [11] Kilani RT, MacKova M, Davidge ST, Guilbert LJ. Placenta 2003;24: 826e34.
SHORT COMMUNICATION Dissolved Oxygen Concentration in Culture Medium: Assumptions and Pitfalls D. Newby, L. Marks and F. Lyall* Maternal and Fetal Medicine Section, Institute of Medical Genetics, Yorkhill, Glasgow G3 8SJ, UK Paper accepted 8 July 2004
Oxygen is a key factor in the regulation of cytotrophoblast differentiation, proliferation and invasion in early pregnancy. Abnormalities in oxygen concentration have also been linked to a number of pregnancy disorders. Cell culture models have been used to study the effect of oxygen on cytotrophoblast behaviour in vitro, however, there is often little or no validation of oxygen levels in these cell culture systems. In this study, dissolved oxygen levels in culture medium maintained in standard culture conditions (18% O2) measured 18%. On transfer to a low oxygen environment (2% O2), oxygen levels decreased to 6e8% after 4 h and reached 2% only after 24 h in culture. Culture medium pre-gassed with nitrogen to remove dissolved oxygen quickly absorbed oxygen when exposed to ambient air during dispensing and required further incubation in a 2% oxygen environment before dissolved oxygen levels equilibrated to 2%. Thus, cultured cells placed in a low oxygen environment would be exposed to varying levels of oxygen before the desired level of oxygen exposure is reached. This study highlights the importance of validation of oxygen levels and potential problems associated with in vitro studies on the regulatory effects of oxygen. Placenta (2005), 26, 353e357 Ó 2004 Elsevier Ltd. All rights reserved.
INTRODUCTION A number of important clinical conditions such as preeclampsia, fetal growth restriction and spontaneous miscarriage have been linked to the placenta being exposed to abnormal oxygen concentrations [1]. Women living at high altitude are exposed to chronic hypoxia and they too have increased risk of complications of pregnancy [2], underscoring the involvement of oxygen. Cytotrophoblast proliferation and differentiation appears, at least in part, to be regulated by oxygen tension. These observations have been the driving force for investigators to develop in vitro models to determine the effects of different oxygen concentrations on cell culture models such as primary trophoblast cultures and placental explant culture [3e7]. One of the key issues in studies of this nature is the actual oxygen levels which cultured cells are exposed to during incubation in different oxygen environments. Some authors report specific methods to control the oxygen environments to which the cells are exposed and/or the measurement of oxygen in culture medium [3,5,7,8], while * Corresponding author. Tel.: C44 141 201 0657; fax: C44 141 357 4277. E-mail address: [email protected] (F. Lyall). 0143e4004/$esee front matter
others make no mention of oxygen levels in their culture systems other than in the incubator environment [6,9,10]. The aim of this study was to define the oxygen environments encountered in our cell culture systems and to address some of the issues and potential problems associated with studies of this nature. METHODS Cell culture incubators Studies were carried out using two Forma Scientific waterjacketed incubators (Forma Scientific, Inc. USA), one set at 5% CO2 in air for standard culture conditions with the sensor on the incubator reading approximately 18.2% O2 and one set at 5% CO2/93% N2 with the oxygen sensor reading approximately 2% O2 for low oxygen conditions. Oxygen levels in each incubator environment were measured using a Fyrite Gas Analyser. Measurement of dissolved oxygen levels There are many commercially available oxygen probes and meters, of varying specifications, for the measurement of dissolved oxygen in a liquid environment but few are ideal for the measurement of dissolved oxygen in cell culture systems Ó 2004 Elsevier Ltd. All rights reserved.
Placenta (2005), Vol. 26
354
Experiment 1
20
Dissolved oxygen (%)
such as those used in trophoblast and explants cultures. A Jenway 970 Portable Dissolved Oxygen/(C Meter and Electrode (Jencons Scientific Ltd, UK) was chosen for the measurement of dissolved oxygen in this study due to the cost and the suitability of the technical specifications, in particular the integral temperature compensation. Issues relating to the measurement of oxygen in cell culture systems and the factors that influenced the choice of oxygen probe used in this study are discussed in more detail later in this communication. The oxygen meter was calibrated, according to the manufacturer’s instructions, to 21% oxygen in water saturated air and to 0% oxygen using the zero salts supplied with the meter. In order to satisfy the requirements of the oxygen electrode, experiments were carried out using approximately 125 ml of M199 (supplemented with 10% fetal bovine serum and 1% antimycotic/antibiotic solution) (GibcoBRL Life Technologies, UK) in a T75 vented-lid cell culture flask. This was necessary so that the depth of the culture medium in the upright flask was sufficient to cover the temperature compensating element of the electrode and allowed stirring of the culture medium during measurement. The flask was placed flat in the incubators to allow maximum surface area for gaseous exchange. The surface area/volume in the T75 flask was 75 cm2/125 ml and this compares to 8 cm2/4 ml in 35 mm culture dishes, 1.9 cm2/0.4e1 ml in 24 well plates and 0.6 cm2/ 200 ml in the Millicell inserts used by many investigators for trophoblast and explant culture experiments. All experiments were performed in triplicate and repeated three times.
15 10 5
0
5
15
10
20
25
Time (h)
Figure 1. Dissolved oxygen concentration (% O2) in test culture medium incubated at 37 (C in hypoxic conditions for up to 24 h. Oxygen levels were measured using a Jenway Model 970 Dissolved Oxygen Meter and Electrode (Jencons Scientific Ltd, UK).
then dispensed into smaller volumes (5 ! 25 ml) to mimic culture medium changes (total dispensing time 4 min) and then returned to the T75 flask. Dissolved oxygen in the culture medium was measured immediately in the 125 ml of culture medium and the flask placed in the low oxygen incubator at 37 (C. Dissolved oxygen levels were measured at intervals up to 16 h. RESULTS Oxygen levels, measured using a Fyrite Gas Analyser, were 18e20% in the standard incubator and 2e3% in the low oxygen incubator. Experiment 1
Culture medium was warmed to 37 (C in T75 flasks in standard culture conditions (5% CO2 in air). Dissolved oxygen levels were measured and the flasks were then transferred to low oxygen conditions at 37 (C for up to 24 h. Dissolved oxygen levels in the culture medium were measured at intervals during the incubation period. Separate flasks were used for each time point to prevent the medium being affected by atmospheric oxygen.
The dissolved oxygen concentration in culture medium maintained in standard culture conditions measured approximately 18% (pO2 140 mmHg). When culture medium was transferred to the low oxygen incubator, oxygen levels decreased gradually to measure 6e8% (46e61 mmHg) after 4 h, 7% (53 mmHg) after 8 h, 3% (23 mmHg) after 16 h and 2e3% (15e23 mmHg) after 24 h of incubation (Figure 1). The pH was unaffected by the changes in oxygen concentration (pH 7.2e7.4).
Experiment 2
Experiment 2
Nitrogen gas (Cryoservice, UK) was bubbled through culture medium (2e3 psi) (125 ml) in a T75 flask for 30 min to eliminate dissolved oxygen in the culture medium. The opening to the flask was sealed with parafilm through which there was an inlet for the pipette delivering the nitrogen gas and an outlet to allow gas to escape. The flask was immediately transferred to a 2% oxygen environment at 37 (C. Dissolved oxygen levels in the culture medium were measured after 1 h and following overnight incubation.
When nitrogen gas was bubbled through culture medium, dissolved oxygen levels fell from 18e20% to approximately 1.5% (11 mmHg) after 15 min and to 0% after 30 min. When pre-gassed culture medium (0%) was placed directly into the incubator with the 2% oxygen environment, oxygen levels in the culture medium equilibrated to approximately 2% after 1 h and remained at 2e3% (15e23 mmHg) following overnight incubation. Experiment 3
Experiment 3 Nitrogen gas was bubbled through culture medium (125 ml) in a T75 flask for 30 min to eliminate dissolved oxygen in the culture medium, as described above. The culture medium was
When culture medium pre-gassed with nitrogen to eliminate oxygen (0%) had been dispensed in ambient air, dissolved oxygen levels increased to 8.5% (65 mmHg). Further incubation of the cell culture medium in a hypoxic environment
Newby et al.: Dissolved Oxygen Concentration in Culture Medium
of 2% oxygen for approximately 16 h was required for dissolved oxygen levels in the culture medium to decrease to 2% (15 mmHg). DISCUSSION There are many factors that must be given careful consideration when measuring dissolved oxygen levels in a cell culture system. Although many oxygen electrodes and meters are available, not all are suitable or ideal for use with cell cultures. Many probes are simply too large for the direct measurement of oxygen in cell cultures maintained in, for example, 35 mm dishes, 24-well plates or Millicell inserts. Smaller probes, such as the ISO2 electrode (World Precision Instruments, Inc, USA) with a 2 mm diameter tip, are available but often at much greater expense. Temperature is of vital importance as temperature effects the concentration of dissolved oxygen in a liquid. The MI-730 electrode (Microelectrodes, Inc, USA) is quoted by the manufacturers as having a change in probe response of 2.2% per degree change in temperature. Thus, unless an electrode has an integral temperature compensation function (e.g. Jenway 970, Jencons Scientific Ltd, UK), care must be taken to ensure that all measurements, including calibration, should be carried out at the same temperature (i.e. 37 (C in the case of cultured cells). This is not always possible for a variety of reasons depending on the method used for calibration. For example, many manufacturers recommend calibration in ambient air, while pre-gassing liquids, either for calibration or to establish defined oxygen levels, results in changes in liquid temperature that are difficult to control. Trophoblast cells and villous explants are most commonly cultured in a static environment. However, dissolved oxygen measurements cannot accurately be carried out in a static system due to oxygen starvation at the membrane of the electrode. This would result in false low oxygen readings, while agitation of cultures may dislodge cells or explants. Thus it would be difficult to obtain accurate measurements in many cell culture systems. Some authors have reported using a blood gas analyser to measure dissolved oxygen in culture medium sampled from cell cultures [7]. We initially used a Bayer 288 blood gas analyser (Bayer, Germany) in our experiments. However, this was not suitable as the analyser could only accurately measure oxygen levels in whole blood (correspondence with manufacturer) and gave false readings with culture medium. Kilani et al. [11] have reported their experience using a blood gas analyser and the requirement for addition of whole blood to the medium to improve the accuracy of readings. In order to characterise oxygen levels within the oxygen controlled cell culture environments used in our laboratory in a way that best compensated for the factors discussed above, we chose an oxygen electrode with automatic temperature compensation and measured dissolved oxygen levels in test culture medium maintained within the same environments as our cultured cells, rather than directly in cell cultures themselves. Dissolved oxygen levels in culture medium incubated in a hypoxic environment of 2% oxygen following
355
prior exposure to atmospheric oxygen or standard culture conditions decreased gradually to 2% over an incubation period of 24 h. During this time cultured cells would be exposed to varying oxygen concentrations and degrees of hypoxia at the cell/culture medium interface depending on the length of time in culture and before the desired oxygen concentration, in this case 2%, is reached. These experiments were carried out using a large volume of culture medium due to the requirements of the oxygen electrode in use. Presumably, with the much smaller volumes of culture medium used in our actual cell culture experiments the rate of gaseous exchange between the culture medium and the environment would be greater and hypoxic conditions would be established more rapidly. However, problems associated with culturing cells in a static environment, which may limit the rate of gaseous exchange, still remain. A rocking platform placed inside the incubator may be useful in instances where cells are firmly attached to substrate to allow diffusion. However, the authors should ensure that effects observed are not due to flow/shear stress per se. One way of circumventing the delay in establishing the appropriate culture conditions may be to pre-gas the culture medium, either with nitrogen to eliminate oxygen from the medium or with a gas of the desired mix and oxygen level. As we have shown, after pre-gassing with nitrogen to 0% oxygen, dissolved oxygen levels will equilibrate to 2% and remain at that level when maintained within a 2% oxygen environment. However, manipulation and microscopic monitoring of cultured cells is often carried out in ambient air, thus exposing the culture medium and cells to atmospheric oxygen. We have demonstrated that culture medium that has been pre-gassed with nitrogen to eliminate oxygen quickly absorbs atmospheric oxygen on exposure to ambient air. Further incubation in a 2% oxygen environment for 16 h was required for dissolved oxygen levels in the culture medium to equilibrate back to 2%, thus limiting the advantage of pregassing the culture medium. Gassing cultures directly to avoid uptake of atmospheric oxygen also has inherent difficulties particularly when millicell dishes are used. Such difficulties include dislodging the cells or explants during gassing, the cooling effect of the gas on the culture medium and the time involved in gassing individual cultures in an experiment. Furthermore, cultures maintained in a low oxygen environment would be exposed to increased oxygen levels during handling and during the associated time delay before the cell culture environment equilibrates to appropriate levels on return to the incubator, thus disrupting the exposure of the cells to the required oxygen concentration. The only way to avoid uptake of atmospheric oxygen would be to use a chamber flushed with the appropriate gas mix to allow manipulation of cultures in the same oxygen environment as the incubator atmosphere [3] and then to be able to transfer the dishes to the incubator without exposure to any atmospheric air. This work highlights the importance of, and difficulties associated with, the validation of oxygen levels in vitro cell
Placenta (2005), Vol. 26
356
culture studies investigating the effects of oxygen on placental development and function. Our studies focused on plastic dishes since this is what most workers use. However, studies with glass dishes may, because of differences in oxygen retention, reveal other issues. The issues highlighted are particularly important with regard to reproducibility and the comparison of data between different research laboratories. Based on our experiences we propose a number of recommendations when investigators perform culture experiments using varying oxygen conditions. Clearly, different groups will be asking different questions in their model systems and therefore the recommendations can only be used as a guideline. These have been sub-divided into two categories; Group 1: what this paper’s results have provided evidence to support and Group 2: what the authors’ ‘‘expert opinion’’ adds to the recommendations.
4. If the culture dishes are to be removed for examination under the microscope, authors should state how this aected the oxygen concentration.
Group 2 recommendations
Group 1 recommendations 1. The oxygen concentration in the incubator or chamber should be validated using a suitable device such as the Fyrite Gas Analyser used in the present study. 2. Authors should state the volume of medium per area of culture dish and how long it took to reach the required oxygen concentration before the experiment in question started. 3. Authors should specify if the culture medium was changed during the course of the experiment and how this aected the oxygen concentration. Ideally medium should be changed in a chamber flushed with the desired oxygen concentration and with medium that has been pre-bubbled to the required oxygen concentration. We do not recommend bubbling directly into culture dishes where cells are growing. A gloved box cabinet would be required with the pipette and pre-gassed medium placed inside the box before the box was flushed to obtain the desired oxygen concentration. Pipettes or tips should be flushed once with medium to remove air trapped inside the pipette. At this point the lid of the culture dish can be removed. However, authors should bare in mind that even with the lid on air exchange will still be taking place, albeit more slowly.
5. Measurement of the oxygen concentration in the culture medium should be made using an oxygen electrode with a tip size which can be fully immersed in the culture medium. A number of companies including Jencons (http:// www.jenway.com) and Diamond General (http://www. diamondgeneral.com) sell a range of electrodes with differing tip sizes. The smaller tip electrodes are more expensive. These must be calibrated before use according to the manufacturer’s instructions. The electrodes must have an integral temperature compensation function as temperature affects the dissolved oxygen in the culture medium. 6. We do not recommend shaking the culture dish containing cells to measure oxygen concentration when villous explants are cultured on matrigel since this may dislodge them slightly or completely thus altering their invasive potential. However, gentle shaking of a control dish which contains medium but no explant will give the researcher an idea of how the oxygen concentration has changed as a result of the electrode being in a static environment. 7. Authors should specify the oxygen concentration of the culture medium that the placental villous tissue was collected into after delivery of the placenta. The culture medium should be gassed to the appropriate level in the laboratory in a universal container and then the sample could be quickly dropped into it at collection. They should state how long it took from collection to preparation and the conditions used to dissect the piece of tissue. Ideally all procedures should be performed in the starting oxygen concentration of the experiment. We feel it would be unrealistic and technically very dicult to isolate single trophoblast cells in a low oxygen environment because of the many steps involved. 8. After consideration of the above manipulations in their experiment it would be helpful to other readers if the authors then defined whether, on balance, their culture system represents a model of hypoxia or a model or re-perfusion.
ACKNOWLEDGEMENT We are grateful to Action Research and the British Heart Foundation for funding.
REFERENCES [1] Kingdom JCP, Kaufmann P. Oxygen and placental villous development: origins of fetal hypoxia. Placenta 1997;18:623e4. [2] Zamudio S, Palmer SK, Regensteiner JG, Moore LG. High altitude and hypertension during pregnancy. Am J Human Biol 1995;7:182e93. [3] Genbacev O, Joslin R, Damsky CH, Polliotti BM, Fisher SJ. Hypoxia alters early gestation human cytotrophoblast differentiation/invasion in
vitro and models the placental defects that occur in preeclampsia. J Clin Invest 1996;97:540e50. [4] Genbacev O, Zhou Y, Ludlow JW, Fisher SJ. Regulation of human placental development by oxygen tension. Science 1997;277:1669e72. [5] Watson AL, Skepper JN, Janiaux E, Burton GJ. Susceptibility of human placenta syncytiotrophoblastic mitochondria to oxygen-mediated damage in relation to gestational age. J Clin Endocrinol Metab 1998;83: 1697e705.
Newby et al.: Dissolved Oxygen Concentration in Culture Medium [6] Caniggia I, Homa M, Winter J, Gassmann M, Lye SJ, Kuliszewski M, et al. Hypoxia-inducible factor-1 mediates the biological effects of oxygen on human trophoblast differentiation through TGFb3. J Clin Invest 2000;105:577e87. [7] Huppertz B, Kingdom J, Caniggia I, Desoye G, Black S, Korr H, et al. Hypoxia favours necrotic versus apoptotic shedding of placental syncytiotrophoblast into the maternal circulation. Placenta 2003;24: 181e90. [8] Fitzpatrick TE, Graham CH. Stimulation of plasminogen activator inhibitor-1 expression in immortalized human trophoblast cells cultured under low levels of oxygen. Exp Cell Res 1998;245:155e62.
357 [9] Kudo Y, Boyd CAR, Sargent IL, Redman CWG. Hypoxia alters expression and function of syncytin and its receptor during trophoblast cell fusion of human placental BeWo cells: implications for impaired trophoblast syncytialisation in pre-eclampsia. Biochim Biophys Acta 2003;1638: 63e71. [10] Nelson DM, Smith SD, Furesz TC, Sadovsky Y, Ganapathy V, Parvin CA, et al. Hypoxia reduces expression and function of system amino acid transporters in cultured term human trophoblasts. Am J Physiol Cell Physiol 2003;284:C310e5. [11] Kilani RT, MacKova M, Davidge ST, Guilbert LJ. Placenta 2003;24: 826e34.