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CO2 Incubators: Mainstays of Cell, Tissue Culture, and Microbial Testing

Carbon dioxide (CO2) incubators are mainstays in traditional biology labs engaging in cell or tissue culture. Incubators provide a stable environment designed to mimic a cell’s natural environment: pH of 7.2 to 7.5, temperature of 37°C, and a relative humidity of about 95 percent.

Angelo DePalma, PhD

Angelo DePalma is a freelance writer living in Newton, New Jersey. You can reach him at

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Carbon dioxide (CO2) incubators are mainstays in traditional biology labs engaging in cell or tissue culture. Incubators provide a stable environment designed to mimic a cell’s natural environment: pH of 7.2 to 7.5, temperature of 37°C, and a relative humidity of about 95 percent. The CO2 concentration, about 5 percent, is controlled to match physiologic conditions and to maintain a constant pH.

Incubators are used wherever cells must be maintained, expanded, or cultured over periods ranging from a few hours to many weeks. Common applications include expansion of manufacturing cells for cell banking, virology, microbiological testing (both environmental and medical/ diagnostic), small-scale production of cell products (proteins, genes, toxins, viruses), in vitro fertilization, and drug testing. Very high-volume applications such as clinical microbiology often use large reach-in incubators.

Deepak M. Mistry, manager for strategic development and marketing at Sanyo (Wood Dale, IL), notes that an upswing in biological therapeutics and stem cell research has created additional demand for high-performance CO2 incubators. “Applications that fall under pharmaceutical Good Manufacturing Practices (GMPs), which employ highly regulated protocols, use incubators extensively.”

The need to validate incubator operations and cleaning for regulated work is a driving force behind the tight control over conditions inside incubators. Conditions inside an incubator change rapidly when someone opens the door to introduce or remove samples. In some cases, the health or viability of cells may be compromised. “Uniformity of conditions is a problem in incubators just as with refrigerators,” Mistry tells Lab Manager. Sanyo and other companies use infrared sensors that automatically measure the CO2 concentration and initiate re-equilibration of gas concentration.

Linda CO2 Video

Keeping it clean

Maintaining cleanliness inside an incubator is of prime importance, particularly for units that house several cell lines at once or that change over samples rapidly. Incubators, after all, provide ideal growth conditions not just for cells of interest but for invasive fungi, yeast, and bacteria.

Traditionally, disinfection is achieved via heat or ultraviolet (UV) light. Heat is effective, but is energy-intensive and stresses materials of construction. UV also works quite well, but only on line of sight: nooks and crannies that are not directly irradiated may remain contaminated.

Sanyo has pioneered the use of hydrogen peroxide gas for incubator disinfection. Gas permeates every surface area within the box and provides close to 100 percent kill of pathogens. The company claims that cycle time between removal of cells, cleaning, and introduction of new cells is about two hours after peroxide disinfection.

Several vendors make peroxide sterilization equipment that is catching on in cell culture, pharmaceutical, and food safety work. Mistry mentioned two: Steris (Mentor, OH) and Bioquell (Hampshire, UK).

Maintaining the health of cultured cells used in research or biomanufacturing is of utmost importance—not just while they’re in the incubator, but during use and storage. “Since cell lines can take weeks or months to make, they’re very expensive,” Mistry says. “Users expect them to be viable for many months after they leave the incubator.

David Craig, product manager at BINDER (Bohemia, NY), sees increasing demand for tri-gas incubators, which are traditional CO2 devices that incorporate oxygen control. Oxygen normally ranges between 15 percent and 20 percent by volume; here O2 is as low as 0.2 percent.

The term “tri-gas” refers to separate hookups for CO2, nitrogen and oxygen. “Low levels are achieved by injecting nitrogen to drive out the oxygen,” Craig says.

Tri-gas units are popular in biology and oncology labs handling tissues that thrive under specific atmospheric conditions. For example, they are used for cancer cells grown under low oxygen and stem cells under higher oxygen concentration. The explosion in tissue regeneration studies has been a boon for incubator manufacturers. “The idea is to replicate physiologic growth conditions.”

A lot of hot air

BINDER specializes in hot air sterilization, which occurs at 180°C. According to Craig, sterilization provides the most robust and reliable cleaning. “The next closest, decontamination at 140 degrees, doesn’t remove everything, while disinfection at 90 degrees just knocks down some organisms. You need at least 170 degrees to eliminate not just microorganisms and cells but DNA.”

Hot air sterilization practically eliminates condensation—a breeding ground for microorganisms— and does not require the use of HEPA filtration. HEPA units are expensive and are another potential source of contamination.

Perhaps the most attractive feature of sterilization is that it occurs without user intervention. “You complete one experiment, press a button, and the next morning you’re ready to go,” Craig observes. The sterilization cycle takes just under ten hours. Cleaning incubators normally involves disassembly, autoclaving parts, and hand-scrubbing.

“It’s important to remember that CO2 incubators don’t just grow things you want; they also grow things you don’t want,” Craig says.