Over the past three decades, the continuous culture of eukaryotic cells has become a mainstay technique in many different forms of biological, biochemical, and biomedical experimentation. While at first, to the uninitiated, the techniques, methodology, and equipment can appear daunting, clear specification of the experimental requirements can help make choices straightforward. This article does not purport to be an exhaustive guide but rather aims to prompt the researcher to plan and make choices appropriate to their experimental, environmental, and financial resources.
Cell culture needs a commitment of energy and resources to be undertaken in a professional manner for any continuous period. Therefore, the biggest decision to be made before going down this experimental road is whether there will be an ongoing need for culture facilities or whether for short periods of work it might be more economical to collaborate with an established laboratory or sub contract work. Assuming that there is an agreed need to set up a cell culture facility, there are several fundamental considerations.
A. EnvironmentPurpose-built facilities are optimal for ergonomic and experimental reasons but often this choice is not available. Small cell culture facilities can be engineered (with inherent limitations) without making significant modifications to laboratory rooms. Before going into the detailed choices available, it is perhaps timely to go through some of the basics.
There are two fundamental considerations that govern most choices available to the would-be cell culture researcher: contamination and safety.
The fundamentals of cell culture owe much to the basic methodologies developed by microbiologists over the last two centuries. However, microorganisms reproduce several orders of magnitude more rapidly than eukaryotic cells and, in direct competition, bacteria and fungi will rapidly reproduce more biomass than eukaryotic cells. Eukaryotic cells are also very sensitive to the primary and secondary metabolic products of microbes. Bacteria and fungi are therefore the biggest problem, for those growing eukaryotic cells. As mentioned in the article by Meleady and O'Connor, there is also potential for cross contamination of one cell line by another if proper procedures are not observed (a major problem with the first cultured human cell lines).
Eukaryotic cells can potentially harbour subcellular microbes that could cause disease to human beings or animals. More specifically, most human cell cultures are derived from human cancers. Being derived from human beings who could be harbouring several known (and potentially, as yet, unknown) pathogens, appropriate steps must be taken to ensure that cells do not pose a risk of passing disease on to human beings, including the researcher or others including visitors or cleaning staff. In practice, the majority of pathogenic organisms are quite fragile and do not survive well under general culture conditions. The risk of disease is therefore greatest when working with primary biological material, i.e., material recently removed from another (human) being. However, one should assume that any eukaryotic cells could potentially harbour microbes and/or viruses (including oncogenic viruses) and/or prion-contaminated material. As a general rule, taking the maximum amount of reasonable precautions (in procedures and equipment) gives the best margin of protection for staff, provides peace of mind for operators, and limits the culpability of supervisors/ managers. The Centers for Disease Control (CDC) in the United States stipulate that general cell cultures should be undertaken in biosafety level 2 containment facilities (CDC, 1999).
B. LocationHaving briefly outlined these fundamentals, it should be clear that correctly locating a cell culture facilities is of paramount importance. The working environment needs to be clean, free from dust, and easy to disinfect. The immediate area should have limited/restricted access, with no passing traffic. Consideration should be given to proper ergonomics in the area, e.g., correct heights of equipment, nothing requiring bending down, suitable chairs, and so on, to reduce the chance of chronic or acute injuries to staff (see the excellent laboratory website by the NIH for further details, specifications, and illustrations). Thought should also be given to how large and often heavy equipment, particularly laminar flow cabinets, can be brought in and out without major disruptions. If a building is being planned, this may include provisions for large lifts with fully opening doors or direct door access to higher floors with high loading equipment. Door openings need to be extra wide, typically at least l m full clearance, and corners designed that large equipment can get by. Provision must also be made for the movement and storage of consumables and bulk items. In practice, it is usually better to pool certain sets of equipment together in individual rooms, e.g., several biosafety cabinets being located with an incubator and other ancillary equipment. This reduces the overall equipment/cost necessary and can also make for a more "communal" working environment. Quarantine areas are an obvious exception to this suggestion.
C. GasesMany cell cultures can be maintained in HEPESbuffered medium, which utilises carefully controlled incubators that do not need a separate carbon dioxide gas supply. However, some cells do not grow optimally in HEPES and need the buffering provided by the equilibrium of 5% CO2 with bicarbonate in medium. This can be important for some specific cell lines, primary culture, and hybridoma work (Freshney, 2000). At a minimum, such incubators should have two separate CO2 sources supplying them. Direct connection of a single cylinder means that the incubators are vulnerable every time a cylinder is changed and cylinders may fail to provide an adequate pressure of gas as they near an empty state. Cylinder changeover units permit, for example, one main line of gas and one backup cylinder, which means that incubators can be left for weeks or months (depending on use) without needing gas replacement and, when cylinders are replaced, there is no interruption in supply to the incubator (see NTC services website). Cylinders in a laboratory environment must always be fully fixed to an immovable object to lessen the risk of them toppling and doing serious injury and damage. Appropriate automatic changeover units can also be incorporated into external supplies of CO2. It is always better not to have large cylinders of gas in a culture room for safety, practical, and aesthetic reasons. Building regulations in some areas may also legislate against the use of large cylinders in enclosed rooms.
D. VentilationVentilation and airflow in the cell culture environment are critical to operation. At its simplest, there must be no disruption to the laminar airflow pattern in the biological safety cabinet and no undue circulation of dust and dirt that could occur with, for example, significant staff movement around the unit or location near drafts or vents. Ideally, there should be no openable windows; if so, they should be sealed to prevent drafts, insects, and dust entering (Freshney, 2000). However, in a purpose-built facility, if possible, it is ergonomically and aesthetically desirable that there be a source of natural light. When planning, one should also make provisions in case there is a need to fumigate the room or equipment (Doyle, 1998). Cabinets will usually need to be fumigated in advance of any filter changes, although this can now usually be performed on single cabinets (using a cabinet bag system) rather than whole rooms.
Clearly, whatever the room design, there must be some replenishment of air in a room and such ventilation must not interfere with the operation of the cabinet. If liquid nitrogen is being utilised in the same location as the cabinet, the ventilation must be adequate to remove the continuous evaporation of liquid and the ventilation/air space sufficient to ensure that if there is an acute spillage of liquid or a rupture of the vacuum vessel, there will still be sufficient oxygen concentration to support life and permit evacuation, i.e., that instant evaporation of all the liquid nitrogen will not reduce the oxygen concentration below 14%. If this cannot be guaranteed at all times, oxygen monitoring and alarm equipment will be required (Angerman, 1999).
High efficiency particulate air (HEPA) filtration within the safety cabinet will ensure a good quality working environment. However, if the air around the unit is dusty, the cabinet filters will age and clog quite rapidly. HEPA filtration is a statistical process (99.999%-99.97% efficient depending on the manufacturer) and unduly contaminated air may also reduce the air quality inside the cabinet. HEPA filtration of air into a cell culture room should improve operation and cabinet filter longevity and is also a requirement of biocontainment-classified rooms. However, if the room itself is not maintained properly, expensively ducted HEPA-filtered air may merely be clean air being utilised to circulate dust and microbes. Depending on the biocontainment level required for operation, HEPA filtration may also be required on exhaust vents for a cell culture room (class 2+ biocontainment and above). In larger cell culture facilities, careful balancing of air pressures in culture and anterooms may be useful or may be required for operation to appropriate biocontainment levels, specifically the positive and negative air pressures required for class 2 and greater biocontainment (CDC, 1999). Balancing and filtration require careful installation and validation from the outset and continued regular maintenance and monitoring to ensure appropriate function. Ventilation systems will also need to be tied into building fire management systems with, for example, automatic smoke dampeners to prevent the ventilation system from fanning or spreading smoke and fire.
E. Basic Cell Culture RequirementsTo perform a basic range of cell culture procedures for any prolonged period the following list of equipment is required (Freshney, 2000).
- Laminar flow cabinet
- Incubator
- Centrifuge
- Refrigerator
- Freezer
- Microscope
- Haemocytometer
- Pipette boy
- Micropipette
- Autoclave
References: EPlantScience
