The culture of mammalian cells is without a doubt one of the most important model systems in biomedical research. Despite their widespread use, mammalian cells are relatively delicate and require a specialized environment in which to grow. This environment is supplied almost exclusively by CO2 incubators. Keeping your CO2 incubator functioning properly and free of contamination are of paramount importance in the successful culture of mammalian cells. The following tips are a starting point for developing your own maintenance campaign.
Avoid contamination
Mammalian cells are cultured in nutrient-rich media at a temperature that is conducive to the growth of almost any microorganism, wanted or unwanted. Although many researchers use antibiotics and antimycotics in their culture medium as a precaution against contamination, this won’t solve persistent methodological errors and often hides the fact that there is a problem. Avoid the use of these agents, if possible, and work on improving your sterile technique and developing a system to keep your incubator clean.
The main source of incubator contamination is the entry of microorganisms through the access door. This may be obvious, but keeping it top of mind will help you develop habits that will go a long way in preventing a full-blown contamination. Always wear gloves when placing something in the incubator and minimize the time the inner glass door is open. Occasionally wipe down the door handle with 70% ethanol. Keep the water in the humidity pan clean by changing it weekly. Periodically scrub the pan with soap and water and then wipe it down with ethanol; or, autoclave the pan. If culture medium spills inside the incubator, remove the shelf and clean it thoroughly.
The frequency of these cleaning rituals will depend on how much traffic your incubator gets. Incubators in larger labs may require more frequent cleaning. If you start with a clean incubator and are careful about what goes in and out, it should remain clean for weeks or months.
Multiple inner doors increases temperature and CO2 recovery time following door openings.
Make sure the CO2 level is what it should be
The majority of mammalian-cell culture media use a bicarbonate buffer system. CO2 is a vital part of this buffer system and must be kept at 5% to maintain the medium at physiological pH. Although essentially all modern incubators are equipped with a CO2 sensor to maintain the desired CO2 level, with time, the calibration may drift from the set point, necessitating an off-set recalibration with a direct determination of the CO2 level by the operator (i.e., you).
The easiest and most accurate method to measure the CO2 level in an incubator is with a gas analyzer such as a Fyrite® device. These can be purchased from many scientific supply houses and are available in models designed to measure CO2 or O2 levels. The operation of the gas analyzer is based on the volume expansion of a fluid following the chemical absorption of the gas to be measured. The device is ‘zeroed’ using room air (0.04% CO2), and the air in the incubator is sampled through a port in the door or side of the incubator.
Older model incubators may require more frequent recalibration. The best new models perform an automatic re-zero of the CO2 sensor every month. Although these models rarely require manual recalibration, users should still check the chamber CO2 level with a Fyrite device according to the provisions of their Standard Operating Procedure (SOP).
Maintain 37°C temperature
Given than our bodies strive to maintain a core temperature of 37°C, it’s no surprise that mammalian cells in culture are happiest at this temperature. Most CO2 incubators are equipped with a display that reports the temperature of the inner chamber, but it’s a good idea to double-check it periodically, especially if you notice suboptimal or uncharacteristic cell growth. This is best accomplished with a high-quality, calibrated thermometer. Open the outer and inner incubator doors and tape the thermometer to the inside of the glass door so the temperature can be read from the outside when the glass door is closed. Do this before you leave the lab for the day. The next morning, open the outer door of the incubator and read the thermometer. Once again, if the reading is different from 37°C (and the thermometer is accurate), it will be necessary to recalibrate the onboard temperature sensor.
Incubator display: Precise temperature and CO2 control for high cell yields and viability.
Keep it humid
The inside of your incubator is maintained at a relatively high temperature, so evaporation of the culture medium would be a real problem without high humidity. Sustaining a high relative humidity (90% to 95%) inside your incubator is achieved by keeping water in the tray located on the bottom level of the incubator. The downside of this is that the water-filled tray is a potential source of contamination (see above).
Adopting a regular schedule of maintenance and cleaning is the most effective way to keep your CO2 incubator functioning properly and your cells free of contamination.
Charlie Villano, Product Manager - Eppendorf CO2 Incubators
Related Products from: Eppendorf North America