CO2 Incubator Buyer’s Guide

CO₂ or gassed incubators are fixtures in mammalian cell culture laboratories. Although incubator technology has not changed much in recent decades, deciding on the right options could save hundreds of hours per year.

Considerations when purchasing a CO₂ incubator include:

• Size—how much room do you need for your experiments? How much equipment do you need to squeeze inside the main incubator chamber, and how many experiments or cultures do you need to run simultaneously?
• Atmospheric control—how much do you have, and for which gases?
• Potential to work inside the incubator with minimal door openings
• Humidity and its flip side, evaporation
• Heating
• Contamination risk mitigation
• Decontamination protocol
• Parameter recovery after door opening
• Temperature and environmental uniformity
• Monitoring and data capture capability, particularly for regulated processes
• Familiarity with the process or operator

Incubators use CO₂ to control pH within culture media and to maintain an atmosphere optimal for cell growth. Typical conditions inside the chamber are a temperature of 37°C, 95% relative humidity, and a CO₂ concentration of about 5%. Microbiological incubators, which will not be discussed at length in this article, feature wider temperature ranges (typically between 5ºC and 70ºC).

Due to a surge in interest in exotic mammalian cell cultures, cell-based assays, and cell therapy, CO₂ incubators are a growth business.

Due to a surge in interest in exotic mammalian cell cultures, cell-based assays, and cell therapy, CO₂ incubators are a growth business. Business intelligence firm Research and Markets estimates demand for CO2 incubators will grow at a rate of nearly 8% per year for the next four years. Considering the purchase price (from about $3,000 to just south of $20,000), and an average usable life of about ten years, the projected growth rate is robust for a laboratory asset.

“Our customers increasingly work with fragile cell types like stem and primary cells,” says Molly Love, senior global product manager for CO₂ incubators at Thermo Fisher Scientific. Thermo Fisher recently launched a new product, the Cell Locker System, which segregates the incubator’s larger working chamber into removable, protected areas for improved culturing efficiency and security for sensitive cultures. “Cell culturists are now able to quarantine cell types or different projects in one incubator, saving space and gas in an undisturbed environment while protecting against circulating cross-contamination including mycoplasma.”

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While some labs obsess over incubator size and features, bigger and more complex is not always better. Needs sometimes dictate a small, personal-sized incubator that users can tuck into one corner of their lab and forget about when it’s not in use. For these users the 18-pound and eminently movable Mini Microbiology and Hematology Incubator from Labnet might be a good choice. Labnet also offers the significantly larger 211DS model with an internal electrical outlet, stackable shelves, large observation panel, and the SmartChek™ temperature control system.

Binder carries a line of compact incubators as well, in their Model C 170, which are stackable and use 180°C air to sterilize the units.

Features that matter

“Before we built our first incubator we asked microbiologists what they were most concerned about, and they answered ‘dessication’ and ‘contamination,’” says Buckner Richerson, vice president of international sales at Nuaire.

Nuaire reduces contamination risk in its incubators (and biosafety cabinets as well) by judicious application of HEPA filtration, to maintain the inside of the incubator at the ISO Class 5 cleanliness standard. The main capsule filter gently and continuously scrubs the chamber atmosphere by filtering and conditioning it; filter, pump, and temperature sensor all reside outside the working area.

“This design exchanges the incubator’s atmosphere every twenty minutes while minimizing evaporation, and returns the unit to optimal operation within twenty minutes of closing the door,” Richerson adds. Nuaire sells about 2,000 incubators per year and, according to Richerson, has not heard of a contamination incident in several years. “When customers do experience contamination we’re the first ones to hear about it.”

As a general contamination-mitigation and evaporation-minimization strategy Richerson recommends slow air exchange, with air flow as vertically laminar as possible. Horizontally flowing air creates a wind tunnel effect that promotes contamination and evaporation by creating air flow eddys around samples.

To eliminate pathogens that enter and are not filtered out, Nuaire uses heat sterilization in all of its direct heat incubators, except for its most basic model. Decontamination occurs at 145ºC for an extended period, usually overnight. Nuaire also offers a copper-lined interior as an option, which Richerson says some users prefer as an additional step to avoid contamination, but is not generally necessary.

Richerson also notes that some heat sterilization protocols are so complex that users eventually stop using them. The cycles may require human intervention, the removal of internal structures such as shelving, or replacement of the temperature sensor with a dummy unit. “Everything inside our incubator survives temperatures of 145ºC, so you never have to take anything out. The incubator essentially becomes an autoclave.”

Manual cleaning is possible in all incubator styles, but in Nuaire incubators all internal corners are rounded, which reduces areas where microbes could take hold and facilitates cleaning with chemical agents.

NU-5710 Direct Heat Incubator. Image courtesy of NuAire.

Heating & sterilization

Water-jacketing is an old strategy for incubator heating. These units are still available, particularly in used markets, but water-jacketing has given way to air-jacketing, direct heating, or a combination of the two techniques.

Incubator manufacturers introduce their own improvements within those three heating alternatives. For example the Galaxy® 170S Direct Heat CO₂ Incubator from Eppendorf is an example of a directly heated unit with a proprietary twist—a six-sided, fan-less, filter-less heating system which, according to the company, protects against wide fluctuations in temperature and CO2 ,which stress cells.

“Customers like the temperature security of water-jacketed incubators, but have been driven to direct-heat models by the need of water-jacketed models for constant maintenance, and the risk of leaks,” says Alan Campbell, vice president of marketing at Caron Products & Services. Late in 2017 Caron introduced its Wally® line of CO₂ incubators, which, according to Campbell, provides the advantages of water-jacketed and direct-heat designs, “with none of their disadvantages.” Caron uses a phase-change material surrounding the culture space instead of water.

Wally also sports a slim form factor that allows placing the unit in smaller labs, hallways, aisles, and other tight areas in which incubator doors swinging open might cause issues. “Wally saves up to nineteen inches of depth versus conventional cube incubators,” Campbell explains.

Space utilization extends to the incubator itself. Most lab workers use only the front areas of incubators because the back is difficult to reach. “Up to half the capacity they've paid for is wasted space. Wally’s slim chamber depth makes all of chamber volume accessible for use, while providing enough space for most commonly used cell culture flask/plate sizes.”

Caron has also upgraded the sterilization cycle, which in standard incubators is time-consuming (heat-based) or labor-intensive (wet H₂O₂). Wally uses (another patent-pending) dry H₂O₂ cycle, which Campbell says provides validatable 12-log sterilization in just two hours, “with none of the time-consuming chamber configuration and cleanup required for a wet H₂O₂ process.”

Peroxide sterilization units are most often added on to incubators as third-party upgrades. Panasonic manufactures an H₂O₂ sterilization kit that it sells through distributors, for example VWR, and which the company claims is eight times faster than heat sterilization. It also sells a full line of incubators ranging in size from just a few cubic feet to 30 cubic feet, with standard and optional features like heat sterilization, infrared carbon dioxide and oxygen sensors, gentle airflow, microprocessor control, and graphical monitoring and data collection.

That disinfection alternatives exist is not surprising given the diverse applications and operating parameter ranges for a “typical” incubator. Analytic Jena offers one incubator model, the UVP, which employs ultraviolet disinfection instead of heat, chemical cleaning, or peroxide.

Fan-less, microbe-less

According to Rick Ellison, business development manager at BMT USA, the leading issues affecting CO₂ incubators are cross contamination, vibration, slow recovery and readings after door openings, and condensation on the door and walls that creates opportunities for microbiologic contamination.

BMT uses a fan-less design in its CO₂ incubators, which significantly reduces the impacts of the first two problems. “Zero vibration practically eradicates any possibility of cell edging effects,” Ellison says. “The fan-less design also induces less evaporation, thus minimizing water loss in samples.”

Fan-less incubators also increase usable space since no ducting or built-in HEPA components are required. “Maintenance and replacement of those components are eliminated, as are the costs of replacing fan HEPA filter elements.” Turbulent airflow are a major source of potential contamination in CO₂ incubators. “Our incubator is designed specifically to provide the lowest risk of contamination,” Ellison adds.

CO2 Cell 190 Comfort Incubator with 200C Sterilization. Image courtesy of BMT.

Contamination enters incubators from any source inside the lab, including individuals’ hair and clothing, open windows, and even the lab’s ventilation system. Once inside the incubator microorganisms latch onto the fan, where they spread throughout the unit.

“Fans remain the single most difficult component in an incubator to clean,” Ellison explains. “For this reason, incubators with fans require the addition of an expensive HEPA filter in front of the fan to protect it. This adds another dimension of risk and cost.”

That is not to say all air movement will hurt cultures. Amerex, for example, uses forced air circulation in its INCUMAX™ incubators to provide temperature uniformity and rapid recovery after door opening. It should be possible to have robust air flow until equlibrium is reached, then maintain much less robust air flow in maintenance mode, but no incubators on the market seem to have this feature.

But when airborne organisms do enter an incubator, condensation becomes their growth medium. BMT minimizes this risk by wrapping the incubation chamber in a direct-heat cable, which brings all surfaces to specified temperatures rapidly. The three-circuit direct heat design allows for the programing of four critical heating zones: the chamber bottom, chamber door, chamber walls, and chamber top. BMT programs these to heat at slightly different rates to create soft, gentle convection.

“Most importantly for condensation control, a cold spot (relative to the other heating zones) is created to draw moisture to the bottom of the chamber and water pan,” Ellison adds. “The glass door is heated by the external insulated door to stop condensation forming, thus eliminating the environment for bacterial growth in ways that water-jacketed chambers cannot.