Selecting the Right CO2 Incubator for Your Lab

The current interest in cell-based assays, therapies, and biomanufacturing has transformed the CO₂ incubator from a laboratory curiosity to an essential component of lab-based operations.

Selecting a mammalian cell culture incubator involves several steps. The process begins with having an established, well-characterized process or application, understanding the cells involved and their environmental requirements, and familiarity with applicable regulations. From there, lab managers assess specific needs for optimal growth, cleaning and disinfection, the unit’s potential to introduce contamination into the cleanroom (for GMP facilities), materials of construction, validation, physical size, and proximity to utilities and other equipment.

“Materials of construction affect cleanability,” says Kris Wronski, Cell Culture Applications Scientist at Thermo Fisher Scientific. Several obvious differences exist between academic and biopharma labs and these affect incubator selection. “GMP cleaning protocols are much more stringent and impactful, especially in facilities producing cell therapies for which the cells are the end product.”

Where merely specifying cGMP compliance simplifies selection in manufacturing environments, no single workflow classification exists for universities.

“What complicates the purchase decision at schools are the myriad lab types, ranging from “biology 101” to facilities that treat patients, all with varying needs and expectations in terms of performance and value,” Wronski explains.

According to Wronski, stainless steel construction covers the most bases since its strength, durability, and chemical inertness are outstanding, as is the material’s most critical characteristic for cell culture: cleanability.

“Stainless steel has high compatibility with many cleaning agents you would use in a laboratory,” Wronski says, and the closer one gets to a cGMP cleanroom the more critical cleanability becomes. “Cleaning is not as ritualized in academic labs as under GMP, where companies need to develop detailed Standard Operating Procedures (SOP) and often use high concentrations of such agents as hydrogen peroxide to disinfect both incubator and cleanroom. Cleaning protocols are much simpler and relaxed in academic labs.” For example it is common practice for academic research labs to use equipment with stainless steel outer housing that has been painted or coated. “The extremely stringent cleaning protocols in GMP facilities can cause these coatings to peel over time.”

Over many years in the incubator business, Thermo Fisher Scientific has developed proprietary cleaning protocols and incorporated them into specific products. But as Wronski notes many service companies have sprung up solely to provide cleaning and fumigation of both cell culture appliances and cleanrooms.

The next level: cGMP

The selection challenge takes on new meaning for cGMP-level facilities, where researchers investigate cell-based therapies and the extremely high level of quality and consistency these processes demand. Here, Wronski tells Biocompare, is where customers start to look for advanced contamination control features and increased compatibility with cleaning procedures.

“Therapeutic cells can be challenging, so all incubator parameters must be accurately controlled to sustain cell health and viability,” Wronski says. This is where users look for rapid recovery of operating parameters such as temperature, CO₂ level, and humidity after door openings, especially for cells grown under hypoxic conditions. The cells are expensive to begin with but often require some genetic manipulations, then several days of culturing and meticulous care. “Throwing these cultures away costs money and resources and causes delays.” That is why, even when such labs do not operate strictly under GMP, specifying rapid recovery and uniformity is a good idea. “If your culture parameters are not exactly the same every time your work will not be reproducible.”

Looking out for the cleanroom

At the GMP-level, incubators are often housed inside cleanrooms, so lab managers must consider the impact of introducing an incubator on the cleanroom environment.

“The process of installing an incubator into a cleanroom must itself be controlled, documented, and validated,” Wronski says. Validation requires formal regulatory filings, can be time-consuming, and requires availability of the manufacturer documentation to support regulatory audits. “You need to identify all the elements required for timely validation, like availability of product-specific factory documentation, regulatory certificates, or even a detailed list of all components and structural materials like silicones, plastics, type of metals, etc. Everything must be checked for compatibility with the cleanroom, and all this must occur in compliance with not just GMP regulations but company, environmental, OSHA, etc.”

Another huge difference between academic labs and industrial cleanroom environments is the emphasis placed by the latter on contamination control. We focus here on particulate contamination since microbial contamination in GMP facilities is a topic of its own and chemical contamination is rare.

“Particulate contamination is not a significant issue in academia, but GMP facilities are constantly on guard for particles generated from all physical objects in the room, including humans and equipment,” Wronski says.

Ironically CO₂ incubators, which protect cells and processes from the outside world (and vice versa), can contribute to the particulate emission into the outside cleanroom air, especially when a CO₂ incubator is equipped with high temperature sterilization.

According to Wronski, any piece of equipment placed into a cleanroom may contribute to particle emissions. “During normal operation, CO₂ incubators can release particles to the cleanroom environment. The levels released are usually quite low during normal operation but increase during the high temperature sterilization cycles. If equipment is not designed to emit as few particles as possible, it can affect operation and regulatory compliance for the entire cleanroom.”

Thermo Fisher Scientific has addressed this problem directly through a line of cleanroom-compatible incubators announced in April 2021. The Heracell Vios CR CO₂ Incubator employs many standard and add-on features, but is unique in its fully encased construction and brushed 304 stainless steel exterior, which the company claims minimizes particle release into cleanrooms. Heracell Vios CR is third-party validated to be appropriate for use in ISO Class 5 and GMP grade A/B environments in accordance with ISO 14644-1. This next level contamination control CO₂ incubator is also compatible with STERIS dry, non-condensing vaporized hydrogen peroxide, a popular cleanroom decontamination procedure.

Incubator selection depends on cells’ nutritional and environmental requirements, the particular lab’s workflows or processes, and where they sit on the continuum of “teaching lab” to full GMP. This article touched on the most critical features of disinfection and parameter control. Among the less popular but (for some labs) essential features is modularity or movability, which can be critical in versatile production environments or cleanrooms in which space is extremely limited.

“Especially under GMP, operators must be able to move equipment around both to adapt to changing workflows and to access hard-to-reach parts during cleaning, maintenance, and decontamination,” Wronski says.

Dry H₂O₂: The Expert's Choice

After years of agonizing over the best way to sterilize CO₂ incubators, many (if not most) experts now back dry hydrogen peroxide (H₂O₂) gas as the method of choice. H₂O₂ gas reaches every nook and cranny in the device and, when combined with high temperature, leaves no condensed water behind.

Many manufacturers sell incubators that can use H₂O₂ out of the box, and most provide technical guidance and background. Caron, for example, has published a very nice primer on the subject to promote its own H₂O₂-ready line of incubators. Numerous other manufacturers have followed suit as well, including PHC, and of course Thermo Fisher Scientific, whose selection guide is well worth examining.

Still other companies specialize in decontamination but do not sell incubators. Among these are STERIS, Vaisala, and MesaLabs. Steris manufactures sterilization systems for biotech and other industries, while Vaisala and Mesa focus on tools for validating dry H₂O₂ sterilization.