( ...)The QIAGEN Guide to Animal Cell Culture

Part II: Safety and handling considerations for animal cell culture

Welcome to the second of a new series of articles aimed at providing useful hints for culturing animal cells. This article describes considerations for working with animal cell cultures as well as information on cell culture contamination. The series will continue in future issues of QIAGEN News with information on cell culture growth conditions, followed by cell culture protocols.

Legislation and regulatory guidelines

Before undertaking any work with human or animal tissue (e.g., to establish a primary cell culture), it is necessary to ensure that the nature of the work conforms to the appropriate medical-ethical and animal-experiment legislation and guidelines. It may be necessary to seek approval from the relevant regulatory authorities and/or individuals.

Safety considerations and biohazards

When working with potentially hazardous material, it is important to be aware of the possible risks associated with both the material and the experimental protocol. All cell cultures are considered a biohazard because of their potential to harbor an infectious agent (e.g., a virus). The degree of hazard depends on the cells being used and the experimental protocol. Primary cell cultures in particular should be handled carefully as these cultures have a high risk of containing undetected viruses. Although commonly used cell lines are generally assumed to be free of infectious agents, care should still be exercised when working with these cell lines as it is possible that they contain infectious agents, such as latent viruses. Cell cultures used to study specific viruses should be assumed to have the same degree of hazard as the virus under study. We recommend handling all material as potentially infectious to ensure the safest possible working environment. Work should be performed in an approved laminar flow hood using aseptic technique, and the creation of aerosols should be avoided (see below). After the work is complete, all waste media and equipment (i.e., used flasks, pipets, etc.) should be disinfected by autoclaving or immersion in a suitable disinfectant according to institutional and regional guidelines.

Handling cell cultures

Adherence to good laboratory practice when working with cell cultures is essential for two reasons: first, to reduce the risk of exposure of the worker to any potentially infectious agent(s) in the cell culture, and second, to prevent contamination of the cell culture with microbial or other animal cells (see below).

Aseptic technique and minimization of aerosols

Aseptic technique and the proper use of laboratory equipment are essential when working with cell cultures. Always use sterile equipment and reagents, and wash hands, reagent bottles, and work surfaces with a biocide or 70% ethanol before beginning work.

Creation of aerosols should be avoided aerosols represent an inhalation hazard, and can potentially lead to cross-contamination between cultures. To avoid aerosols, use TD (to deliver) pipets, and not TC (to contain) pipets; use pipets plugged with cotton; do not mix liquids by rapidly pipetting up and down; do not use excessive force to expel material from pipets; and do not bubble air through liquids with a pipet. Avoid releasing the contents of a pipet from a height into the receiving vessel. Expel liquids as close as possible to the level of liquid of the receiving vessel, or allow the liquid to run down the sides of the vessel.

Proper use of equipment can also help minimize the risk of aerosols. For example, when using a centrifuge, ensure the vessel to be centrifuged is properly sealed, avoid drops of liquid near the top of the vessel, and use centrifuge buckets with caps and sealed centrifuge heads to prevent contamination by aerosols.

Laminar flow hoods

For the most efficient operation, laminar flow hoods should be located in an area of the laboratory where there is minimal disturbance to air currents. Avoid placing laminar flow hoods near doorways, air vents, or locations where there is high activity. Hoods are often placed in dedicated cell culture rooms.

Tip - Keep laminar flow hoods clean, and avoid storing equipment inside the hood.

Tip - Before starting work, disinfect the work surface of the hood as well as the outside of any bottles (e.g., by wiping with 70% ethanol), and then place everything needed for the cell culture procedure in the hood.

Tip - Arrange equipment, pipets, waste containers, and reagent bottles so that used items are not placed near clean items, and avoid passing used items over clean items.

Tip - Place used items (e.g., pipets) in a container inside the hood, and disinfect or seal before removing from the hood.

Contamination

The presence of microorganisms can inhibit cell growth, kill cells, and lead to inconsistent results. Contamination of cell cultures can occur with both cell culture novices and experts. Potential contamination routes are numerous. For example, cultures can be infected through poor handling, from contaminated media, reagents, and equipment (e.g., pipets), and from microorganisms present in incubators, refrigerators, and laminar flow hoods, as well as on the skin of the worker and in cultures coming from other laboratories.

Bacteria, yeasts, fungi, molds, mycoplasmas, and other cell cultures are common contaminants in animal cell culture. To safeguard against accidental cell culture loss by contamination, we recommend freezing aliquots of cultured cells to re-establish the culture if necessary. A protocol for freezing cell cultures will be provided in a future issue of QIAGEN News.

Microbial contamination

The characteristic features of microbial contamination are presented in Table 1. The presence of an infectious agent sometimes can be detected by turbidity and a sharp change in the pH of the medium (usually indicated by a change in the color of the medium), and/or cell culture death. However, for some infections, no turbidity is observed and adverse affects on the cells are not easily observed.

Table 1. Characteristic features of microbial contamination
Cell cultures should be routinely evaluated for contamination. Mycoplasmal infections are one of the more common and difficult-to-detect infections; their detection and eradication are described in further detail below.


Mycoplasmal infection detection

Mycoplasmas are small, slow-growing prokaryotes that lack a cell wall and commonly infect cell cultures. They are generally unaffected by the antibiotics commonly used against bacteria and fungi. Furthermore, as mycoplasma do not overgrow cell cultures and typically do not cause turbidity, they can go undetected for long periods of time and can easily spread to other cell cultures. The negative effects of mycoplasmal contamination include inhibition of metabolism and growth, as well as interference with nucleic acid synthesis and cell antigenicity. Acute infection causes total deterioration of the cell culture, sometimes with a few apparently resistant colonies that may, in fact, also be chronically infected. There are two main approaches to detect mycoplasma Hoechst 33258 staining (1, 2) and mycoplasma-specific DNA probes (Fisher Scientific). Alternatively, a PCR-based, mycoplasma-testing service is offered by the ATCC (www.atcc.org) or BioReliance (www.biomeva.com) on a fee-for-service basis.

Mycoplasmal infection - eradication

The best action to take with a culture containing chronic mycoplasmal infection is to discard it by either autoclaving or incineration. Only if the cell culture is absolutely irreplaceable should eradication be attempted. This process should be performed by experienced personnel in an isolated hood that is not used for cell culture, preferably in a separate room. Elimination of mycoplasma is commonly achieved by treatment with various commercially available antibiotics such as a quinolone derivative (Mycoplasma Removal Agent), ciprofolxacin (Ciprobay), enrofloxacin (Baytril), and a combination of tiamulin and minocycline (BM-Cyclin). Treatment procedures and appropriate antibiotic concentrations can be found in the suppliers' instructions and in references 1 and 3.

Cross-contamination of cell lines

Cross-contamination of one cell culture with fast-growing cells from another culture (such as HeLa) presents a serious risk. To avoid cross-contamination, only use cell lines from a reputable cell bank; only work with one cell line at a time in the hood; use different pipets, bottles of reagents, and bottles of media for different cell lines; and check cells regularly for the correct morphological and growth characteristics. The QIAGEN Guide to Animal Cell Culture will continue in future issues of QIAGEN News with information on cell culture growth conditions and cell culture protocols. If there is any other information you would like to see on these pages of QIAGEN News, please let us know by calling QIAGEN Technical Services or your local distributor.

References
1. Freshney, R.I. (1993) Culture of Animal Cells, a Manual of Basic Technique. 3rd ed. New York: Wiley-Liss.

2. Spector, D., Goldman, R.R., and Leinwand, L.A., eds. (1998) Cells: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

3. Drexler, H.G. et al., eds. (1997) DSMZ Catalog of Human and Animal Cell Lines. 6th ed.



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