The CO2 incubator is an essential piece of lab equipment for anyone doing cell-culture work. It creates a contamination-free environment for cells while maintaining temperature, humidity and O2/CO2 levels. The common cause of fluctuations in cultured cells’ environment is a door opening—even briefly—bringing an influx of ambient air that disrupts temperature and O2/CO2 levels and introduces potential contaminants. Vendors are constantly improving incubators to help researchers protect their hard work, even as newer and more complex types of cell cultures are creating growth and maintenance challenges. Here are some recent advances in cell-culture incubators.
Kinda humid in here
In addition to keeping O2/CO2, temperature and humidity levels steady, incubators mimic the cells’ natural environment—at least to the extent possible. “Increasingly, we are seeing cultured cells used in medical applications or for production of biopharmaceuticals, including monoclonal antibodies, vaccines, cell therapies and more,” says Molly Love Parrucci, global product manager for CO2 incubators at Thermo Fisher Scientific. “Uniformity throughout the chamber [and] rapid recovery after a door opening … are important for ideally modeling native conditions in the body.”
After the major disruption of a door opening, the interior conditions return to set point faster if there is airflow in the incubator. Thermo Scientific THRIVE™ Active Airflow, in the Thermo Scientific™ Heracell™ VIOS and Forma™ Steri-Cycle™ i160 Tri-Gas CO2 incubators, uses a circulating fan to bring temperature, CO2 and humidity back to their set levels within 10 minutes, following a 30-second door opening. Different gases can be slower to mix without airflow, leading to localized areas of nonhomogeneity. “Those variations could affect cellular reactions that are reflected in experimental or production results,” says Parrucci. She notes that sensors within the incubator must be positioned directly within the chamber, rather than in a separate electronics compartment, so that “they experience the same conditions as cultured cells and to react quickly to any minute changes.”
Maintaining high humidity is important for preventing desiccation, as even moderate evaporation can change the concentrations of important components in growth media. “Evaporation is four times faster at 80% humidity than at 93% or greater,” says Parrucci. “So fast humidity recovery and high relative humidity are important for cells that react to changing nutrient concentrations or drug cocktails.” Different types of cell-culture vessels can also present different types of humidification challenges, according to Jens Thielmann, product manager for growth and preservation at BINDER. “For high-throughput screening with multiwell plates, high humidity is essential to avoid media evaporation of the outer rows and columns,” he notes.
Get enough oxygen
Increasing interest in O2-control functions is emerging with observations that “oxygen control is found to be increasingly important for studying solid tumors, for growing neurons or immune cells, even for studying gastrointestinal pathogens,” says Parrucci. Also some cell types, such as primary or stem cells, grow better with lower O2 levels, which more closely approximate their in vivo conditions.
According to Buckner Richerson, vice president of international sales at NuAire, traditional sensors control O2 levels down to about 3%. However, NuAire has introduced a zirconia ceramic oxygen sensor to control O2 levels down to 0.5% (for example, in its In-VitroCell ES NU-5831 Direct Heat Hypoxic CO2 Incubator) to meet customer demand for maintaining O2 levels at physiological levels. Parrucci recommends that researchers consider variable O2 control even if they don’t need it, because they may in the future. “Get an oxygen-control incubator now and turn off the O2 control. [Use it] as a standard CO2 incubator until [you] need physiological oxygen capability,” she says.
Biospherix's Xvivo System X3 incubator lets researchers program changes in O2 and CO2 levels during the course of a culture’s development. Biospherix’s CEO, Randy Yerden, is enthused about the advantage of O2/CO2 dynamics. “Bigger and better cell expansions can be achieved by raising the O2 over the course of the culture to accommodate higher cell densities, and then lowering the CO2 near the end of the culture to counter the lactic acidosis,” he says. Yerden says dynamic O2 also can be used to physiologically induce or influence cell fate and phenotype or to simulate “pathophysiologic conditions like ischemia, tumor formation or wound healing.”
Maintain a natural environment
Recent developments in temperature control tend to be more important outside the United States. In some parts of the world, labs don’t have air conditioning (AC)—or even electricity—around the clock. But scientists still need to protect their cell cultures from temperature fluctuations. NuAire works with scientists worldwide to help them find incubators that can solve these conundrums. Currently, the company is working on software controls for temperature and pH that respond faster to shutoffs. “In Taiwan in the summer, labs can get really hot because they turn everything off at night,” says Richerson. “In Hokkaido, Japan, on the other hand, it can be near freezing in wintertime.”
Labs with temperature-regulation problems also turn to incubators with older, water-jacketed technology. “In India, the water barrier helps with daily temperature fluctuations in a non-AC lab, which helps to avoid problems holding the set point,” says Richerson. “This is also true in places that have trouble with reliable electricity, such as Mexico.” In contrast, most labs in the United Kingdom and Europe use direct-heat (or air-jacketed) incubators. The United States is still a mix, he says, but he sees direct-heat incubators gradually predominating.
Keeping it clean
Another crucial role of incubators is protecting cells from contamination. The most common method is heat sterilization, in which the empty incubator is put through a high-heat cycle to kill microorganisms. Some now have sensors that can withstand the high-heat cycle. These include BINDER’s new Series CB incubators, which “are equipped with a heat-sterilizable CO2 sensor, which remains in the chamber during the 180°C sterilization cycle,” says Thielmann.
Some incubators include both heat sterilization and air filters. For example, some of Thermo Fisher Scientific’s incubators include both the Thermo Scientific Steri-Run™ high-temperature sterilization cycle, for a 12-log sterility assurance level, and an in-chamber HEPA filter for decontaminating the interior air. NuAire also offers heat sterilization and a HEPA filtration system, which constantly withdraws air and passes it through a class 100, sterile HEPA filter to make a complete exchange every 20 minutes. Each NuAire incubator contains four separate HEPA filters: one for interior air, one for entering CO2 gas, one for fresh air and one for air surrounding the electronics. “We’ve found that this means [fewer] electronic failures,” says Richerson.
Biospherix offers the option to integrate its new Xvivo System X3 incubator with closed hoods or aseptic, gloved work spaces. Not only do these extend the aseptic work space, they are also kept at the same O2/CO2 and temperature conditions as the incubator interior—so it’s as if the cells never left their cozy haven. “Since the incubator opens only into this aseptic, cell-optimized space, it eliminates the major source of contamination in most incubators—room air—and eliminates the major source of variation in most cell cultures,” says Yerden. “Because now cultures are never exposed to suboptimal conditions when the incubator door opens, or when cells are removed from the incubator for handling and processing.”
Special features for special cells
As researchers use more complex cell types with different needs—such as primary cells, stem cells, tissue explants and 3D cultures—incubator manufacturers are responding with creative solutions. “Researchers tracking cells from many different patients wanted to have a way to keep them all straight, yet expose them to the same incubator conditions,” says Richerson. NuAire partnered with a company that sells a structure that divides the interior of NuAire’s incubators into individual compartments.
For greater flexibility, Biospherix’s X3 incubator can be configured into individual chambers that in effect become multiple smaller incubators, “each with independent controls to optimize different conditions in each chamber, simultaneously, for different culture protocols,” says Yerden. Biospherix also offers retrofitting accessories to compartmentalize traditional incubators.
If you’re in the market for a new cell-culture incubator, keep your eyes open for new developments in technology. Incubators themselves have been around for a long time, but it’s the small yet powerful changes—for example in O2 control or decontamination—that can make a big difference to your cells.
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