Incubators: Providing a Stable Home for Precious Cellular Samples

The CO₂ incubator, along with the laminar flow tissue-culture hood, is a mainstay of any cell-culture laboratory—so much so that it’s generally part of the package supplied to incoming investigators at the University of North Carolina’s Lineberger Comprehensive Cancer Center.

Because there are so many things to consider when deciding which incubator might be right for your lab—from size, cost and its ability to prevent and deal with contamination, to the user interface, to how temperature, humidity and gases are regulated—other important considerations may be overlooked initially. Here we examine some obvious, and some not-quite-so-obvious, decision points for a new CO₂ incubator.

Size matters

Defining a “standard-size” CO₂ incubator can be a challenging task.

That being said, a typical CO₂ incubator is in the roughly five- to seven-cubic foot, or 140- to 200-liter, range, allowing it to be placed on a bench or on the floor.

“Generally, all the incubators available today will be separate but stackable—most people will get a stack of two,” says Steve Oglesbee, longtime director of the Lineberger tissue-culture facility.

It's good to know ahead of time where the incubator will be placed, so that it can be ordered with the doors opening in the correct direction, he notes. “It depends on where the air is coming from, because you don’t want room heating or air-conditioning air to be blowing into a chamber when you open it up—so right or left hinging can make a big difference for blocking that.” Although doors may be reversible, it may require a service call to do so.

Castors or roller platforms can typically be ordered for dry-jacketed, floor-mounted models. Not only does this make relocating incubators easier, “it’s really great,because it allows you to get to the back of the instrument much more quickly,” says David Killilea, staff scientist at the Children’s Hospital Oakland Research Institute. Killilea created and manages an academic core facility.

Jackets and fans

Once upon a time, the chamber of almost all cell-culture incubators was enclosed with a water jacket. The thermal mass properties of water create an interior temperature that remains fairly consistent and constant (as long as the door is kept closed), even in the event of a power failure. As an added benefit, the water helps dampen any vibrations. But such incubators are very heavy, requiring them to be drained before being moved. This style of incubator can take a while to get up to, or return to, operating temperature. They are prone to leakage. And they require maintenance, lest contamination set in.

The majority of incubators still in operation are water-jacketed. But the majority of incubators being purchased by new, expanding and upgrading labs incorporate dry technologies (air jacket, direct heat or a hybrid system).

Improvements over the past decades have enabled these incubators to equal the performance of water-jacketed models (with the exception of maintaining temperature without power), and in addition, to provide high-heat sterilization cycles.

Vendors use different technologies to assure that the air inside the chamber is uniform in composition and temperature. The majority of incubators on the market have circulating fans that “prevent stratification within your incubator … and also ensure fast recovery of all conditions,” notes Mary Kay Bates, senior global cell-culture specialist at Thermo Fisher Scientific. “We also put a HEPA filter on our airflow, to capture contaminants that are circulating in the air.” 

In the case of the Thermo Scientific Heracell™ VIOS CO₂ incubator, the fan is turned off when the door is opened, to help reduce air exchange. Other vendors offer incubators with pumps or fans that blow “like a Class II hood, with vertical direction of the air. So when you open the door of the incubator, you have this vertical laminar flow, which helps keep things inside the incubator from getting out and things outside the incubator from getting in,” remarks Oglesbee.

A number of manufacturers worldwide—including Eppendorf and NuAire—offer fanless designs that eliminate a potential source of contamination and vibration. “It’s not just gravity—not entirely passive,” notes Rick Ellison, BMT’s business unit manager, Scientific Division. In BMT’s case, three programmed heating circuits are used to differentially control heat and thus create natural convection. 

Keep it simple, keep it clean

With temperature, CO₂ and humidity well controlled in the modern incubator, perhaps the most pressing general concern is the prevention and elimination of contamination: bacterial or fungal growth within the incubator, cross-contamination from other cultures or something introduced from outside.

Many incubators are designed with smooth, seamless interior surfaces to minimize growth and make the unit easy to clean. “The entire chamber can be disassembled, wiped down and reassembled in less than three minutes,” says Rebecca Guarino, Eppendorf North America’s product manager for CO₂ incubators, of the company’s Galaxy line.

Robert Hunter, program manager of the University of Washington’s Transgenic Resources Program, whose core had experienced a couple of fungal outbreaks, had chosen copper interior in place of other costly add-ons. But Killilea didn’t like the “ugly green/brown patina” that forms when pure copper oxidizes, nor the dust that eventually comes off, so he opted for a stainless-steel alloy that “had enough copper to be antimicrobial.”

There are even options to have the air cleaned by UV light hidden behind the ductwork. 

Routine maintenance, like a weekly wipe-down, changing the water and water pan and changing the HEPA filters (where applicable), can go a long way.

Oglesbee cautions before you buy to make sure those filters are accessible and can be easily changed by lab personnel.

And when contamination does occur—there’s an adage that “it’s not if, but when”—vendors have a variety of ways of dealing with it. Most common in dry-jacketed incubators are either wet or dry heat cycles; these are typically overnight, but some are faster. In the past, some incubators required preparations such as removing sensors that could not tolerate the heat—be sure to find out what the procedure is for the instrument you’re looking to purchase. Other less common options, such as H₂O₂ or other chemical sterilization, may be available, as well.

Breathe deeply and keep it cool

With accumulating data supporting the idea that cells do better under more physiological hypoxic conditions—especially cells such as stem cells, 3D cultures and primary cells that have not been adapted to culture—many researchers are looking for incubators that can reduce oxygen content. This is most commonly achieved by allowing nitrogen gas to offset the oxygen, and it requires an oxygen sensor as well as nitrogen regulation. “That’s not something you can add on. You have to order the incubator that way,” says Bates. “But you can order a hypoxic incubator and turn off the option until you need it, investing in the future.” Many hypoxic incubators offer divided inner glass doors, allowing the researcher to access a single shelf at a time, lessening overall gas exchange.

Vendors also make hyperoxic incubators. “They’re less popular. It’s a bit more of a niche application,” Bates notes.

If a researcher will be putting equipment—a shaker or a microscope, perhaps—into an incubator, they’ll likely need power and perhaps other cables that go to the exterior. Oglesbee advises to make sure there are ports designed for the purpose and to plan placement of the incubator accordingly. If that equipment generates enough heat—especially if the room itself is very warm—a unit with refrigeration capabilities may be necessary.

CO₂ incubators are designed to keep precious cell cultures safe and happy. To keep the users happy, a host of bells and whistles—from automated decontamination to password protection, data logging and onboard help screens—can be had, for a price. 

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