Josh P. Roberts has an M.A. in the history and philosophy of science, and he also went through the Ph.D. program in molecular, cellular, developmental biology, and genetics at the University of Minnesota, with dissertation research in ocular immunology.
Cell culture typically takes place at atmospheric oxygen concentrations, about 20% to 21%, despite the fact that virtually every tissue in the body (except the cornea) grows under lower oxygen tension. Lungs, for example, see about 14% O2 in vivo, while the brain experiences 3% to 6%. Since at least the 1960s, researchers have reported differential effects of culturing cells in reduced oxygen (hypoxia), and many researchers now routinely grow tumors, stem cells and embryos for in vitro fertilization under hypoxic conditions.
There are various tools researchers can use to control how much oxygen cells are exposed to in the lab, from relatively inexpensive sealed boxes to all-encompassing, modular workstations costing more than $250,000. Here are some of your options.
Hypoxic chambers
Perhaps the simplest, least expensive way to create a hypoxic environment is to seal the cells into a container with the desired atmosphere and stick the container in a 37oC incubator. The Billups-Rothenberg Modular Incubator Chamber, for example, is a round, airtight polycarbonate box with shelves that can hold up to nine 96-well plates or 18 T-25 flasks. Humidity is provided by a dish of water placed on the bottom.
The company recommends flushing the system with 100 liters of gas using the chamber’s built-in ports. A (preferably certified) mixture of gas can be used, or researchers can mix their own gases using an optional dual-flow meter to assure the desired concentrations. A battery-powered O2 sensor also can be placed inside the chamber.
To get started, a user can simply buy one of the company’s incubators and use premixed gas, says founder and chief scientific officer Barry Rothenberg. “So for about $500, you can do a low-oxygen experiment.”
“If you’ve got a couple of these, you can do a couple of different conditions side by side,” adds Mary Kay Bates, global cell-culture specialist at Thermo Fisher Scientific, which is among the distributors of the Billups-Rothenberg chamber.
Tri-gas
Most major CO2 incubator manufacturers offer incubators designed to control both CO2 and O2 concentrations in a heated, humidified environment. “Just punch in the O2 concentration just like you do for CO2, and that triggers influx of N2 gas,” says Bates, explaining that these so-called “tri-gas incubators” are a bit of a misnomer, as they typically use only two gases. “A lot of people don’t understand that lowering the O2 requires N2. That’s how it works.”
Some researchers require an oxygen concentration of virtually zero, termed anoxia. Incubators that have this capability typically achieve it by introducing hydrogen gas and allowing it to interact with residual oxygen in the presence of a palladium catalyst to form water.
More rare are customers interested in hyperoxic conditions (oxygen concentrations up to about 90% with the balance being water vapor and CO2), says Bates, and few incubators are designed to handle such conditions. Those that are often use different oxygen sensors that measure a wider range of concentrations but are not as accurate at the lower end of the scale.
Humidity generally is provided via evaporation from a pan of water placed on the bottom of the incubator. Thermo Scientific’s Heracell™ incubators feature a large-surface-area integrated water reservoir, allowing for up to five times faster humidity recovery after the doors have been opened, according to Bates. Other manufacturers, including NuAire, offer an active humidification system—misting sterile water into the incubator from an external reservoir—which eliminates a potential source of contamination.
Tri-gas incubators often come standard or can be ordered with split inner glass doors to minimize atmospheric exchange.
Glove boxes
The traditional hypoxia workflow involves taking cells from a tri-gas incubator to a tissue-culture hood for feeding and to the benchtop for viewing or reading, and then returning them to the incubator. As a result, says Betty Cosgrove, national sales manager at Ruskinn Technology (a subsidiary of Baker Company), researchers may well be studying the shock effects of those oxygen-concentration changes as much as what is occurring in vivo. She suggests users instead use an in vivo chamber “so that you are controlling your cells’ level of oxygen the whole time.”
Such systems are like a cross between a tri-gas incubator and a lab bench. There typically are two (or more) arm holes fitted with sleeves—gassed with nitrogen to remove oxygen—or gloves, as well as an air lock allowing items to enter and leave without changing the interior atmosphere. Ruskinn’s offerings range from shoulder width to about 4 feet wide. Depending on the size of the box, instruments such as a pipettor or plate reader can be placed and used inside. They also can be customized with extra sockets, vacuum support lines and ocular cutouts for a microscope.
Glove boxes range from about $20,000 to more than $250,000 for units with multiple modules. For about $22,000, Plas-Labs offers a clear-top acrylic box with touch-screen-controlled oxygen (from 0% to 25%), CO2 and temperature. “It offers better visibility, with no shadows, and improved ergonomics,” says Mike Regan, vice president of product development.
HypOxygen’s offerings look more like tissue-culture hoods with a removable front panel. The lower-cost and smaller H20A HypOxystation uses a premixed gas, and the $50,000-plus H35 HypOxystation can precisely regulate the gases and make changes on the fly using a touch keypad, says Bill Richman, regional sales manager.
Companies such as Biospherix offer modular workstations consisting of interconnected chambers. These let a researcher do everything from sacrificing an animal to running assays under controlled oxygen conditions, notes Kevin Murray, director of sales and marketing.
Achieving equilibrium
Whether cell cultures temporarily exposed to 21% O2 will immediate become shocked is open to debate. It takes 90 minutes for a tissue-culture plate with the lid off to equilibrate to its surroundings, and three hours for it to do so with the lid on, Rothenberg says.
That being said, he does recommend preconditioning the medium either by bubbling it through with hypoxic gas (don’t add serum until afterward) or letting it equilibrate overnight at 4oC under hypoxic conditions.
However you do it, if you culture cells you should give hypoxia a try. Your cells—and your data—will likely be happy you did.