by Caitlin Smith
The home of your cell culture and tissue culture work can be nearly everything that you need it to be, if you know what to look for. An incubator will protect your cells from wide-ranging changes in temperature, humidity, CO2 levels and O2 levels (also known as hypoxia, or hypoxic conditions). Incubators are also safe havens for your next big idea. Here you can find some important qualities and what to look for in incubators, your cells’ home away from home.
Temperature control
Every time someone opens the door of an incubator, they disrupt the temperature of the interior. Currents of warm and cool air swirl around inside, and the incubator’s temperature control system must work to return the interior’s temperature to the set level. Water jackets are one type of mechanism that makes incubators less susceptible to fluctuating temperatures; water jackets can be particularly valuable in the event of a power failure.
Although water jackets offer many advantages, they also can incur disadvantages: maintenance includes draining and filling with water and adding algaecide; also, the water is heavy enough to make it difficult to move the incubator without first draining the jacket. Hoping to lighten the load, Caron recently introduced its new GelJacket™ technology. The 10-cubic foot GelJacket benchtop CO2 incubator (offered in non-refrigerated or refrigerated models) retains uniform heating and also includes other vital features, such as a decontamination cycle, infrared (IR) sensor for CO2 control (see below) and humidity control without a water pan (see below). “Caron’s new GelJacket incubator easily exceeds the thermal protection of water jacket incubators by retaining a far greater amount of heat than any other insulation technology available in CO2 incubators,” says Leah Harris, director of marketing communications at Caron. “In addition, the GelJacket incubator includes a 90˚C moist heat decontamination cycle, not possible in water jackets. GelJacket’s sealed gel requires no maintenance, is lightweight and has virtually no risk of leaking.”
Another jacket-type temperature regulator is offered by BINDER and is included in the recent CB model addition to its line of CO2 incubators. The CB is compact and space-efficient while retaining all the advanced features of its larger cousins. For example, the VENTAIR™ air-jacket system maintains accurate temperatures and a homogeneous temperature distribution, and it speeds the recovery of the set temperature after door openings. Within the chamber, the “APT.line™ preheating chamber technology [ensures] accurate and homogeneous growth conditions over all racks,” says Andreas Baeurer, product manager at BINDER. And being smaller, like the CB incubator, has advantages that are attractive to users: “It is 45% more cost-effective than the CB 150 model in terms of energy use and CO2 consumption,” says Baeurer.
SANYO’s MCO-19M multi-gas incubator also uses an air jacket system for temperature regulation. “Temperature is controlled using SANYO’s patented Direct Heat and Air Jacket conditioning system,” says Paul Freeland, senior product manager in Europe for the biomedical division at SANYO. “This combination of direct heating with a surrounding air jacket provides rapid response after door opening and excellent temperature uniformity to maintain cultured samples at the optimum temperature.”
New Brunswick Scientific's Galaxy® CO2 Incubators have replaced the need for a traditional jacket design and instead use a fanless system with six-sided direct-heating profile. “The unique system provides gentle convection circulation of the atmosphere within the chamber for exceptional uniformity of temperature and gasses,” says Thomas Uschkureit, senior product manager at New Brunswick Scientific. “The Galaxy system guards against the wide fluctuations in temperature and CO2 that can shock cells, often observed in traditional forced-air culture systems.” Uschkureit adds, “By removing conventional fans, Galaxy incubators have eliminated a classic source of repeat contamination, while also maximizing interior space by enabling the entire incubator — including the upper shelf — to be utilized. In addition, since there is no fan, there is no need for an expensive internal HEPA filter that needs to be replaced frequently.”
Humidity control
Maintaining the ideal humidification is necessary because it prevents the cell cultures from drying out. At the same time, it supports the important function of maintaining uniform osmotic cell pressure. BINDER’s CO2 incubators, including its newest compact CB model, control the humidity within the inner chamber using BINDER’s Permadry® system. “The Permadry® humidification system ensures dry, condensation-free walls while operating at a relative humidity of more than 95%,” says Baeurer. “The water level can be visually inspected, and water replacement is easy,” using simple water exchange via a water pan. Permadry® also shortens the time it takes to recover the set humidity level after a disruption such as a door opening. Thermo Fisher Scientific offers a pan-less, rapid-response humidity system that uses a humidity water trough built into the incubator chamber. “This system also features an optical water level sensor which will tell the user when the humidity water needs to be refilled, by audible alarm and a visual indication on our iCAN touch-screen user interface,” says Douglas Wernerspach, global product manager for CO2 incubation at Thermo Fisher Scientific. “This feature is found in our Heracell 150i and 240i models.”
The airflow within the incubator’s interior can lead to evaporation of media (possibly changing the relative composition of the media). Preventing sample desiccation is not only a function of humidification controls—it also is affected by the amount and type of airflow that occurs within the chamber. “The CO2/air mixture is injected into the inner chamber through a jet; the mixture is distributed homogenously because of the generated Venturi effect,” says Baeurer. “This obviates the need for a fan, which creates turbulence and complicates cleaning.” For similar reasons, NuAire reduces the airflow within its incubators to avoid drying out cell cultures. “The Closed Loop HEPA Filtration System,” says Buck Richerson, vice president of international sales at NuAire, “which is standard on all NuAire Water Jacket Models and will become standard on all Direct Heat Models shortly, slows airflow to one air exchange per 30 minutes within the inner chamber, which minimizes evaporation or desiccation of the cell samples.” New Brunswick tackles sample desiccation from another angle – offering an active humidification option that rapidly humidifies the chamber, with user-definable settings to 95% humidity and an external UV disinfection of humidified atmosphere.
CO2 control
Maintaining a healthy CO2 level within the incubator is important, because the CO2 interacts with the buffering system of the cell culture media to determine the media’s pH. One of the most important choices to make for CO2 control is what type of CO2 sensor you would like your incubator to have. Many incubators use either the more traditional thermal conductivity (TC) sensor or the newer type of IR sensor.
Uschkureit says that “the traditional thermal conductivity sensor is highly sensitive to changes in chamber humidity and temperature fluctuations making it unsuitable for use in CO2 incubators. Uniquely, the Galaxy IR Sensor can remain in the chamber during the entire high-temperature disinfection cycle, ensuring that all chamber components are disinfected.”
At Caron, Harris says that many customers need CO2 incubators that also feature IR sensors. “All Caron CO2 incubators were designed within the last four years and incorporate new energy-efficient components and technologies,” she says. “Caron reach-in CO2 incubators [include] IR sensors, controlled humidity without water pans and high-heat decontamination.” For the MCO-19 incubator series, SANYO includes an infrared CO2 control sensor with a new, continuous auto-zeroing function. “SANYO’s proprietary CO2 sensor is not affected by humidity or temperature changes normally associated with frequent door opening,” says Freeland. “Together with sophisticated PID microprocessor control of CO2 and O2 levels, the MCO-19M provides precise control and rapid recovery.”
O2 control
In recent years, researchers have shown more interest in experiments that test the effects of different levels of O2 on cells in culture. “In their efforts to provide an optimized environment for important cultures, scientists are becoming more interested in controlling the oxygen concentration that their cells are exposed to,” says Wernerspach. “This has created an enhanced demand for incubators with variable oxygen control to provide a reduced O2 atmosphere more closely simulating physiological oxygen conditions.”
HypOxygen’s HypOxystation is a hypoxic incubator built specifically for cell culture, with individual controls for oxygen, carbon dioxide, temperature and humidity levels. “Researchers can control O2 in 0.1% increments from 0.1% [to] 20%, and CO2 in 0.1% increments from 0.1% [to] 15%,” says Julie Magruder, marketing and sales associate at Microbiology International, which sells the HypOxystation. Among other applications, the HypOxystation is being used for examining the role of hypoxia in tumor microenvironments. “With such accurate control and the ability to manipulate cells in situ without having to alter the incubation environment, research into cell biology can be performed over a comprehensive range of oxygen tensions with precision.”
The HypOxystation’s real-time feedback system lets you monitor and adjust O2 and CO2 levels; for example, oxygen profiling lets you set a range of oxygen tensions and have them adjust automatically over user-defined time periods. For a stable temperature and gas mixture, the HypOxystation uses constant air circulation created by two tangential fans that run the length of the chamber. “Our most recent innovation is a blackout cover for the HypOxystation, which allows cells to be cultured in the dark,” says Magruder.
The New Brunswick Galaxy incubators also offer oxygen control in three ranges: 1 - 19% for most common hypoxic applications, 0.1 – 19% for more stringent O2 requirements, and 1 - 95% for hyperoxic and hypoxic incubation. “Stem cell and primary cell research is advancing at a rapid pace, and other recent studies have shown that for many cell types, an environment closer to the physiological oxygen concentrations [2 to 5%] is needed for maintaining cell functions,” says Uschkureit. “We offer highly regulated CO2 and O2 environments with the ability to closely mimic physiological normoxic conditions and to provide the optimal environment for stem cell and primary cell work.”
Contamination prevention
In addition to maintaining the right culture conditions for your cells, the other main challenge is preventing contamination of your cultures. Contaminants may be introduced when a person’s contaminated hand or glove touches the inside of the incubator, or when an open door makes it vulnerable to airborne contaminants. A filtration system such as NuAire’s Closed Loop HEPA Filtration System can help to reduce contamination by airborne species. “[It] provides continuous Class 100 Clean Air conditions inside the inner incubator chamber,” says Richerson. “This system guarantees the elimination of any airborne contaminant before it has a chance to contaminate cell culture work.” Thermo Fisher Scientific’s incubators also use HEPA air filtration in their interiors, “assuring the quality of the air surrounding samples by rapidly providing Class 100 [ISO Class 5] conditions, similar to those encountered in the biological safety cabinet,” says Wernerspach.
For cleaning the interior surfaces of the incubator, most models use sterilization cycles (run at high heat, so the incubator must be emptied first). NuAire’s model NU-5510(E) has two types of sterilization cycle (+95°C wet and +145°C dry). It also uses air pressure, another incubator defensive device, to protect cultures during door openings. “The inner chamber is kept under slight positive pressure so the incubator can ‘breathe,’” says Richerson. “The absence of negative pressure guards against entry of any potential contaminants into the inner chamber from outside, dirty ambient air.” In addition, it helps if the interior of the incubator has rounded corners—this makes it easier to clean by chemical disinfection, as there are no tight crevices in which contaminants could grow. Similarly, Uschkureit says that for all New Brunswick models, “the chambers are pressed from a single sheet of stainless steel, with no welds or seams, eliminating a traditional area where contaminants can collect, as well as making wipe-down easy. Of course we also offer a high-temperature (120°C) disinfection option and copper interior option too.” Baeurer also says that along with hot air sterilization at 356°F, BINDER uses “a seamless deep-drawn stainless steel inner chamber [that] reduces the risk of contamination to an absolute minimum.”
Another way to obtain low-maintenance, continuous protection is to choose an incubator whose inside surfaces are 100% copper, which has anti-microbial properties. This can protect against contaminants introduced by a user or perhaps by the bottoms of culture plates. “The natural antimicrobial properties of copper have been known for quite some time,” says Wernerspach. “However, as a result of a number of recent high-profile research studies demonstrating the powerful capability of 100% pure copper to eradicate high levels of MRSA and numerous other bacterial strains quickly, incubators with full solid-copper interiors are in very high demand today.”
SANYO’s multi-level approach to contamination conundrums also emphasize prevention. “SANYO’s InCu saFe® copper-enriched stainless steel interior continuously inhibits growth of moulds, fungi, mycoplasma, and bacteria, and the optional patented SafeCell™ UV system eliminates airborne and water contaminants without harming cell cultures,” says Freeland. SANYO also incorporates a low-temperature H2O2 sterilization technology. “When total chamber decontamination with verification is required, SANYO’s new rapid hydrogen peroxide decontamination option limits down time to less than 3 hours, making it suitable for use in all laboratories, and especially those operating to GMP/GLP.”
According to Wernerspach, cell culture samples are becoming increasingly valuable (for applications such as in vitro fertilization, stem cell research and cell-based vaccine production), and therefore we should take more precautions to protect them. “Contamination prevention technology should be considered essential, providing a safety net for culturing activities and protecting against the potential loss of valuable work,” he says. “Much in the way you would routinely protect your computer files with antivirus software, so too should contamination-control features that protect you every time you open the incubator door be viewed as a necessity.”
The image at the top of this article shows the Galaxy 48 R and 14 S incubators from New Brunswick.