PerspectivesAre you interested in submitting a Perspective Article? Be sure to read The Science Advisory Board's Editorial Guides for Perspective Articles. Click here. The Significance of Chemicals as Contaminants of Cultured Cell Lines by Janice A. Schwartz, Ph.D. Assistant Professor of Physiology Wayne State University School of Medicine Detroit, MI Contaminants can pose a significant threat to cell lines. Their presence has been associated with alterations in growth, long term survival, and even artefactual results in experiments (1). Contaminants come in all varieties, including infectious agents, such as mycoplasma, bacteria, viruses, yeast, and fungi; unwanted chemicals, like phenolics, xenoestrogens, hydrogen peroxides, free radicals, and trace metals; as well as other cell lines, like the HeLa cell line in a culture of COS-1 cells. Although infectious agents may be the more familiar form of contamination, cell cultures that are contaminated with unwanted chemicals are, by far, the more widespread of the two. However, surprisingly little is known about what actually constitutes chemical contamination and how it can be recognized when it does occur. Any chemical, whose presence is unwanted, has effects that are deleterious or can somehow alter the performance or survival of a cell line, is considered a contaminant. The impact that a chemical contaminant has on a cell line depends on the cell line itself, the type of chemical and its concentration. Some chemicals are directly cytotoxic, abruptly curtailing cell growth and survival within hours of exposure. At the other extreme, there are chemicals whose presence, although chronic, has an impact so slight that it usually goes unnoticed. Most of the chemicals that contaminate cell cultures are subtle, sometimes requiring weeks or even months of exposure before their presence can be detected. The effects of chemicals such as these are likely to be manifest as gradual changes in the growth, morphology, or bioactivity of the cells they contaminate. Chemical contaminants may make their way into the media used to culture cell lines from a number of different sources. They may be leached into the culture medium from other sources (2), produced by the cell culture medium itself (3), or result from interactions between constituents in the medium and agents that have been added to it (4,Table 1). Chemical Contaminants Have a Variety of Sources Unwanted chemicals that can be leached into the cell culture medium have several potential sources (Table 1). They may come from: (a) the residues of other solutions that were previously kept in the bottles used to make media; (b) traces of detergents or disinfectants that remain after incomplete rinsing, (c) deposits left after autoclaving (2); or (d) the phenolics, plasticizers or other coatings that were used to manufacture the particular cell cultureware product. Residues from these and many other sources can be leached into the media during repeated cycles of warming to 37 degrees C followed by prolonged refrigeration. Measures That Can Be Taken to Avoid Chemical Contamination Certain measures can be taken to avoid contamination with chemicals. Approaches taken to avoid chemical contamination include using new glassware for media preparation and storage, enforcing scrupulous rinsing procedures before media containers are re-used, and selecting low resin plastics for cell cultureware products. Phenolics, plasticizers/hardeners, and coatings used by the manufacturers of flasks, culture dishes, multi-well plates, and media bottles, for example, can accumulate in the culture medium and influence the biologic activity of a cell line in ways which are rarely anticipated. We provide an example of the impact that plastic-borne chemicals can have on the results of an experiment. In the figure shown here (Figure 1A and 1B), two identical experiments were conducted in parallel using cell culture dishes from different manufacturers. In these experiments estrogen receptor positive breast cancer cells were transiently transfected with a reporter gene that is highly sensitive to estrogen stimulation. Reporter activity levels for the estrogen-treated cells are calculated relative to the untreated control cells that have been deprived of estrogen exposure. All of the experimental parameters used, the reporter construct, the transfection reagent, the cell line and the culture medium, were kept strictly identical in both sets of experiments; the only difference being the cell culture dishes that were used. When the reporter activities of estrogen- (or estradiol)-treated cells were compared to those of the untreated control samples measurable increases were observed in both experiments. However, Figure 1A shows that the level of estrogen-stimulated reporter activity was 4.5-fold greater than the corresponding control levels whereas, in Figure 1B the effect of estrogen stimulation on reporter activity was much weaker, being only 1.2-fold relative to the untreated control. The cell culture dishes that were used in Figure 1B contained labile plasticizers that were able to accrue in the media. Once in the media these chemicals behaved as exogenous estrogens (or xenoestrogens) causing reporter activity levels to increase whether or not bone fide estrogens were added therein. This caused the level of reporter activity in control cells to increase to levels that were nearly equal to those of the estrogen-treated samples and masked any differences that would have otherwise been detected.  Exposing Culture Media to Ordinary Laboratory Lighting Can Cause Free Radical Production Can Chemical contaminants can also be produced by the cell culture medium itself or result from interactions between constituents in the medium and agents that have been added to it. Some components of commonly used biological media may be responsible for the production of free radicals and other by-products that are damaging to cell lines grown in culture (see Table 1). In particular, sublethal or even toxic products may form when a certain combination of constituents in the cell culture media, such as HEPES or other Good�s buffers, riboflavin, tyrosine, and/or tryptophan, are exposed to ordinary laboratory lighting (3-4). This is because light exposure causes nitric oxide consumption to increase, and photo-oxidation products, free radicals, reactive oxygen species, and other cell-damaging by-products to form in these media (5). The situation is made worse by prolonged light exposure, exposure to UV, near UV or fluorescent room lighting, and by the presence of trace metals (5-8). Shielding cell culture media and the components that are to be added to it, from laboratory lighting should be routine practice.  Other Chemicals Other chemicals can inadvertently enter cell cultures as gases or particulates during storage in liquid N2, as CO2 contaminants in cell culture incubators, or as aerosolized particles in laminar flow hoods. The knowledge that chemical contaminants may be present in the media, which inflict damage upon the health and well-being of cells grown in culture and threaten to alter the results of experiments, is important in understanding and preventing their widespread occurrence. References 1. Keynes RG, Griffiths C, Garthwaite J. Superoxide-Dependent Consumption of Nitric Oxide In Biological Media May Confound In Vitro Experiments. Biochem. J. (2003) 369, 399-406 2. Grady, J. K., Chasteen, N. D., Harris, D. C. Radicals from �Good's� buffers. Anal. Biochem. (1988)173, 111-115 3. Lepe-Zuniga JL, Zigler JS Jr, Gery I. Toxcity of Light-Exposed HEPES Media. J Immunol Methods (1987) 103(1) 145 4. Zigler, Jr, J. S., Lepe-Zuniga, J. L., Vistica, B., Gery, I. Analysis of the Cytotoxic Effects of Light-Exposed HEPES-Containing Culture Medium. In Vitro Cell Dev. Biol. (1985)21, 282-287 5. Wang, R. J., Nixon, B. R. Identification of Hydrogen Peroxide As A Photoproduct Toxic To Human Cells In Tissue-Culture Medium Irradiated With �Daylight� Fluorescent Light. In Vitro (1978)14, 715-722 6. Wang RJ. Effect of Room Fluorescent Light on the Deterioration of Tissue Culture Medium. In Vitro (1976) 12(1) 19-22. 7. Stoien, J. D., Wang, R. J. Effect of Near-Ultraviolet and Visible Light on Mammalian Cells In Culture II. Formation of Toxic Photoproducts in Tissue Culture Medium by Blacklight. Proc. Natl. Acad. Sci. U.S.A. (1974) 71, 3961-3965 8. Grzelak, A., Rychlik, B., Bartosz, G. Light-Dependent Generation of Reactive Oxygen Species in Cell Culture Media. Free Radicals Biol. Med. (2001)30, 1418-1425 ### << Previous Next >> [ View All Perspectives ] |
|