Cell culture is an important process used in cellular and molecular biology, providing model systems for studying the physiology and biochemistry of cells, the effects of drugs and toxic compounds, and mutagenesis and carcinogenesis. Contamination is one of the most common challenges encountered in cell culture laboratories, and contaminants can be divided into two main categories: chemical (impurities in media, sera and water, endotoxins, plasticizers, and detergents), and biological (bacteria, molds, yeasts, viruses, and mycoplasma). Cross-contamination among cell lines can also occur.
The consequences and implications of contamination on the viability and reliability of results can be costly in terms of sample or cell line loss and time invested. While it is impossible to entirely eliminate the risk of contamination, it is possible to reduce its frequency and significance, and increase the odds of protecting the viability of cell cultures, by following best practices. As such, all cell culture laboratories share the requirement for equipment and consumables that help to reduce contamination, protecting sample integrity for downstream use.
Setting up an aseptic work area
A typical cell culture workflow involves a number of steps, streamlined to ensure the highest yields possible are obtained. The first step involves setting up an aseptic work area within a biological safety cabinet (BSC), which is protected from dust and other airborne contaminants by maintaining a constant, unidirectional flow of air filtered using high-efficiency particulate air (HEPA) filtration technology. As a result, the potential for contamination from outside air within the internal working environment of the BSC is low, and involves risk only from the user and the materials inside the workspace.
Classification is an important consideration when searching for a BSC. The U.S. Centers for Disease Control and Prevention (CDC) classifies BSCs into three classes, with each class representing different capabilities and performance attributes. Classification is awarded based on two key criteria: the level of personnel and environmental protection provided, and the level of product protection provided. Class II cabinets are most widely used in cell culture laboratories as they meet requirements for the protection of product, personnel, and the environment.
Following selection of the most appropriate BSC to meet the specific application needs, proper maintenance will ensure that the cabinet will achieve the best possible performance. The internal workspace of the BSC must be kept clean and uncluttered. Each item placed within the cabinet should be disinfected by spraying it with 70% ethanol and allowing it to air dry in the BSC work area. Overall, a properly maintained and certified instrument can protect cultured cells, as well as users, by containing infectious splashes or dangerous aerosols, as long as proper aseptic technique is observed.
Maintaining a safe growth environment
CO2 incubators provide a strictly controlled environment for the growth and propagation of mammalian cells. At the same time, they provide an excellent environment for the growth of microorganisms. A well-designed CO2 incubator should offer a set of capabilities targeting the air quality, surfaces, and water reservoirs to prevent and/or eliminate the growth of microorganisms in the chamber.
Optimal contamination protection could be achieved by utilizing HEPA filtration, combined with active airflow technology, to constantly filter the entire chamber air volume. Ideally, this technology should be able to rapidly provide International Standards Organization (ISO) Class 5 cleanroom air quality following door openings. Furthermore, it is necessary to implement automated high-temperature sterilization protocols, which have been tested and proven effective following the standards set in the U.S. and EU Pharmacopeias. This, in combination with monthly or semimonthly manual cleaning and disinfection best practices, can help keep the chamber free from contamination.
Water used to humidify the growth chamber can be an additional place for microorganisms to reside. To eliminate any microorganisms that may have colonized the water, the reservoir should be emptied every week and refilled with sterile distilled water. No items should be stored on top of the incubator, since dust and dirt among these items can be swept inside the chamber with air currents created during door openings.
Even when all of these measures have been applied, it is still recommended to place cell cultures under a microscope for examination before beginning work with them, to ensure they are healthy (i.e., there is no contamination and only a few dead cells) and growing as expected.
Best practices for nonstationary cultures
Orbital shakers are used when cell cultures need to be aerated, for blending purposes, or for agitating substances in tubes or flasks. Traditional CO2 orbital shakers are built in CO2 incubators, limiting the space available for stationary cell cultures. Recent innovations have led to the development of portable orbital shakers, which offer the flexibility to be placed within or outside a CO2 incubator. As a result, space is maximized and many different cell types can be cultured simultaneously.
Inside a CO2 incubator, the warm, moist, and slightly acidic environment can prove challenging for electronics. It is therefore important that the shaker’s electronic elements are sealed to provide a robust, long life. Furthermore, a magnetic drive will offer long service life, minimal heat output, and vibration-free agitation. When it comes to using large incubated/refrigerated floor models, it is necessary to ensure that the air within the chamber is automatically purified using proper airflow driven by a HEPA filtration system. Ideally, the operation of the shaker should be controlled and adjusted using an external controller to minimize door openings and avoid disturbances to culturing conditions.
In addition to attending to sudden spills, regular cleaning and disinfection of the shaker at least once every month is key. Spills can seep under the platform, so a removable platform is important to ease cleaning and disinfection.
Obtaining high-purity cells
Centrifuges separate and purify cells from the supernatant/suspension liquid used for cell growth. The high pressure exerted on sample tubes, bottles, and plates during centrifugation can result in their failure and sample leakage. To prevent subsequent release of pathogens to the laboratory environment, it is necessary to use biocontainment lids and rotors with proven efficacy. The centrifuge lid should be kept closed when the system is not in operation to prevent condensation and liquid in the bowl of the centrifuge. This approach will eliminate the need to use drainage catch basins, which can be a source of contamination by aerosolizing drained material.
It is also important to implement routine cleaning procedures since any contaminants present in the centrifuge can be aerosolized into the laboratory environment and not only cross-contaminate, but also present a safety concern for the operator. Cleaning under the unit is also essential since the high-speed rotor of the centrifuge produces an air current that can move contamination from beneath the system to the air around it.
Establishing optimal cold storage conditions
A cell culture laboratory requires cold storage equipment to safely house media and reagents, as well as substances such as drugs and antibodies. Proper storage temperature plays a vital role in keeping these materials safe from contaminants and preserving their structural integrity throughout storage for future use.
Laboratory-grade refrigerators and freezers are often located inconveniently, since the constant noise generated can compromise communication and working conditions. It is, however, much more convenient and productive to be able to have all components required for cell culture together, to avoid exposure to additional sources of contamination during transportation. As such, laboratories would benefit from cold storage equipment that operates quietly and has a smaller footprint, enabling them to be located at the point of use in the laboratory.
As with all cell culture laboratory equipment, maintenance and best practices are key to ensuring optimal performance. Refrigerators and freezers must be cleaned regularly to avoid contamination, while ensuring no cardboard is stored in or around the systems, since it can get wet and breed fungi.
Cell culture is an essential technique that enables studies in drug discovery and scientific research. It is important to avoid contamination in the everyday workflow of a cell culture laboratory to ensure that cellular integrity is maintained in order to yield high numbers of quality cells. To achieve this, laboratories must adhere to strict best practices and use advanced technologies designed to minimize contaminants. Maintaining an aseptic work area that is dedicated to cell culture work is a major requirement. Once established, laboratories must ensure appropriate equipment is in place to facilitate the sterile handling and containment, incubation, centrifugation, and cold storage of the cells in culture. Such an all-inclusive approach to maximize cultivation is critical to ensuring a streamlined and productive approach to success.
Mary Kay Bates, M.S., is senior global cell culture specialist, Thermo Fisher Scientific, 275 Aiken Rd., Asheville, NC 28804, U.S.A.; tel.: 218-525-2293; fax: 218-525-1156; e-mail: [email protected]; www.thermofisher.com