Cell Culture Contamination

Cell Culture Contamination

An Overview

Simon P. Langdon

1. Introduction

For the cell culturist, two types of contamination require careful monitoring and constant vigilance: the contamination of cell cultures with microbiological organ-isms and the contamination of one cell line with another. Both forms of contamina-tion are extremely prevalent and cannot be underestimated. Neither type can be eliminated, only controlled and managed to minimize the possibility of occurrence. Contamination consequences can range from minor inconvenience (a flask of cells becoming contaminated with bacteria) to a major disaster (published results that may be invalid owing to cross-contamination of one cell line with another). Other types of contaminants, such as chemical contamination, may also cause problems (e.g., deposits of disinfectants or detergents on glassware; residues, impurities, and toxins in water, media or sera), but the common recurring problems are likely to be biologi-cal in origin.

2. Microbial Contamination

The major microbial contaminants in cell culture are mycoplasma, bacteria, fungi, yeasts, and viruses (see Table 1). Sources of contamination include the cells them-selves, (e.g., for viruses and mycoplasma), the media or serum, poor aseptic tech-nique, and airborne contamination. Even when excellent aseptic technique is in place, it is essential to monitor and test for contamination. In general, bacteria, yeasts, and fungi are easy to detect while mycoplasma and viruses are more difficult (Table 2). In the absence of antibiotics, they will grow rapidly; however, if antibiotics are routinely used, low level infections may develop that may be more difficult to observe.

2.1. Mycoplasma

Mycoplasmas are the smallest prokaryotes (approx 0.3–0.8 μm diameter) and their presence is generally not obvious in cultures either macroscopically or microscopi-From:Methods in Molecular Medicine, vol. 88: Cancer Cell Culture: Methods and Protocols

Edited by: S. P. Langdon © Humana Press Inc., Totowa, NJ

309Table 1

Tissue Culture Contaminants

Contaminant General indications Microscopic appearance Common source Bacteria pH change Fine granules Cell culturist Turbidity/cloudiness Water bath

Precipitation

Yeast Cloudiness Oval organisms Airborne

PH change Budding/chains

Fungus Spores Thin spores Airborne

Furry growths

PH change

Mycoplasma Often covert—Cell culturist Poor cell adherence Other cell lines

Reduced growth

Virus Sometimes cytopathic—Serum

Cell lines cally. Screening in the United States by the Food and Drug Administration for over 30 yr indicated that of 20,000 cell cultures examined, over 3000 (15%) were contami-nated with mycoplasma (1). In studies conducted in Japan and Argentina, incidence rates of mycoplasma contamination of 80% and 65% respectively have been reported (1). It is likely that laboratories that do not routinely screen and eliminate sources of mycoplasma will be rife with the organism and may have all cell lines contaminated. Contaminating organisms include members of both the Mycoplasmataceae (Myco-plasma) and Acholeplasmataceae (Acholeplasma) families, and although at least 20 distinct species have been isolated from continuous cell lines, the most common spe-cies include M. fermentans(human),M. orale(human),M. arginini(bovine), and A. laidlawii(1). The major sources of mycoplasma contamination are incoming cell cul-tures or cell lines, sera, or human contact (poor aseptic technique).The level of con-tamination can reach densities of 106to 108organisms/mL. The consequences of mycoplasma contamination on any individual cell line or culture are often difficult to predict but can include effects on growth rates (2), chromosome aberrations (1,3), nucleic acid and amino acid synthesis, and metabolism (4–6), and membrane alter-ations(7). Their presence can also modulate the effects of viruses (1)and influence results of techniques such as the MTT assay (8).

Many methods of detecting mycoplasma contamination have been described, and each has advantages and disadvantages with respect to cost, time, reliability, specific-ity, and sensitivity. These include culture methods, DNA staining techniques, immu-nological methods, transmission electron microscopy, nucleic acid hybridization, and polymerase chain reaction (PCR) identification. Traditionally, the two most widely used methods of detection involve staining with Hoescht 33258 (6)and microbiologi-cal culture. The Hoeschst 33258 dye binds to DNA producing fluorescence that canTable 2

Methods to Identify Contaminants

Mycoplasma Bacteria/fungi Viruses

PCR Microbiological culture PCR

DNA fluorescence Transmission electron microscopy Microbiological culture Immunodetection

Transmission electron microscopy Hemadsorption

Nucleic acid hybridization

3H-Thymidine

be observed by fluorescence microscopy. Mycoplasma-infected cultures will demon-strate extranuclear staining. An indicator cell line is often used, which allows for a level of standardization. Culture techniques are more sensitive but can take several weeks and require more expertise. Furthermore, certain mycoplasmas do not grow readily and may be overlooked. With the widespread use of PCR technology in most cell and molecular biology laboratories, a very sensitive, specific, and rapid option has now become available and an example of this method is described in Chapter 31. The target choice for primer design is the 16S rRNA. Use of size determination of PCR fragments, together with restriction enzyme analysis, can allow detection of spe-cies-specific sequences.

The elimination of mycoplasma is not straightforward and several approaches using antibiotics are described in detail in Chapter 32. This may be the only option for rescuing unique samples that have become infected. For cultures that cannot be returned to previous or frozen stocks, this is the normal course of action. In addition to applying appropriate aseptic technique, a number of simple precautions will help minimize mycoplasma contamination. Cells should be tested for mycoplasma at fre-quent intervals if they are to be maintained in long-term culture. Ideally cell lines should only be cultured for a limited number of passages before returning to frozen stocks. Cell lines imported into a laboratory should be quarantined until verified mycoplasma-negative.

2.2. Bacteria

Bacteria are another widespread cell culture contaminant. These are usually intro-duced through poor aseptic technique and are often first recognized in culture medium.

A downward shift in pH (yellow with phenol red as indicator) with aerobic bacteria and an increased turbidity or cloudiness will be evident. Microscopic inspection indi-cates many fine granules if there is gross contamination (Fig. 1A). This should be distinguished from cell debris, media, or sera precipitates. Under high magnification, several principal forms of bacteria can be distinguished including cocci (round-shaped), bacilli (rod-shaped), and spirilla (spiral-shaped). Movement may also be observed. To detect lesser levels of contamination, media from the suspect cells is cultured and observed for the growth of colonies in antibiotic-free media for 2–3 wk.

Fig. 1. Microscopic appearance of bacteria and fungus, (A) bacteria, (B) fungus. Good aseptic technique should avoid contamination with bacteria. One of the major sources of bacterial contamination is the water bath used to warm media and sera before use.

Once recognized, contaminated flasks should be discarded immediately and new stocks used to regenerate the cell culture. If the culture is unique and irreplaceable, then antibiotics may be able to eliminate the bacteria. It may be necessary to try sev-eral antibiotics to find an effective one, and when one has been identified, the culture should be maintained in the presence of antibiotic for a number of subcultures. Eradi-cation of the contaminant should be confirmed by testing cell-conditioned media anti-biotic-free media. There is divergence of opinion as to whether antibiotics should be used routinely in cell culture. If they are added, they will reduce the incidence of bacterial infection but resistant organisms may appear. Penicillin and streptomycin are widely used in combination.2.3. Fungi

Fungi (molds) routinely appear as a contaminant either as thin filamentous mycelia or as clumps of spores (Fig. 1B). In their advanced stages, they can take over a culture as furry growths that can vary in color from white to black. Fungi are ubiquitous in the environment and generally enter cultures via the air. Seasonal changes, air-condition-ing, and heating can all have a major effect on levels in the atmosphere. Antimycotics such as amphotericin B (Fungizone) and mycostatin (Nystatin) can be used to treat essential cultures if the fungus is not too advanced.

2.4. Yeast

Yeasts characteristically appear as small oval shaped organisms that are substan-tially smaller than mammalian cells. They can be seen “budding” from other yeast particles in short chains. Eventually these chains will form multibranches. Yeasts gen-erally infect cell culture materials by airborne routes and are transferred easily by contact. Mycostatin (Nystatin) can be used to treat essential cultures in early stages of infection.

2.5. Viruses

Viruses are the most difficult contaminants to detect, but unless they are cytopathic may have little effects on their host cells. Viral contaminants have been detected in bovine serum intended for use in tissue culture and in cell lines including bovine viral diarrhea virus (BVDV), bovine herpes virus (BHV), bovine parainfluenza-3, and epi-zootic hemorrhagic disease (9). Because BVDV was widely present in serum, it may be present in many cell lines unless the cultures have been grown in rigidly tested sera or sera of nonbovine origin. A variety of methods have been used to identify viruses, including identification of cytopathic effects in susceptible cells, hemadsorption, elec-tron microscopy, and immunofluorescence, but more recently PCR-based techniques have been developed to detect these contaminants (9). Some ATCC human cell lines are known to contain Epstein-Barr virus, human T cell leukemia virus, and hepatitis virus, and this information is available in their catalogue and data sheets. Their pub-lished recommendation is to use caution when handling any human cell line and to regard it with the same level of biosafety caution as a line known to carry human immunodeficiency virus.

The possible hazard from bovine spongiform encephalopathy (BSE) has become a concern over the last few years and although the risk for tissue culture workers from fetal calf serum (FCS) contamination may be trivial, some regulatory authorities now insist that FCS used for culture should be obtained from countries where BSE has not been diagnosed. Therefore, countries such as the USA will only allow import of cells cultured in serum from BSE-free areas.

2.6. Sources of Contamination

There are many different entry points for contaminants into the culture system. The first is via tissue culture materials. Sterilize all plastics and glassware prior to use and although many plastics are sterilized commercially, care should be taken when pack-ages are unsealed. Similarly, tissue culture liquids, such as media, sera, and washingsolutions should be filter sterilized. When bottles are opened, frequent observation and monitoring is required to check that the liquid is not infected. All equipment entering the tissue culture cabinet should be sprayed with 70% ethanol or an alterna-tive disinfectant (e.g., dilute hypochlorite or 1% benzalconium chloride). One major source of contamination are the still damp external surfaces of bottles taken from water. These should be sprayed well with ethanol, as should tissue culture flasks that have been used for some period of time, because they are effective at attracting fungi to their external surfaces. A dirty lab coat is another excellent source of contamina-tion. Working within the sterile environment of the cabinet, care should be taken to avoid constant or unnecessary moving in and out of the hood. Aerosols should be minimized as far as possible. Airborne contamination may also increase seasonally, for example in spring and summer when pollen counts can be high and during con-struction work when there may be an increased amount of dirt and dust. When spill-ages occur, they should be cleaned immediately and incubators and cabinets should be cleaned thoroughly at regular intervals. Minimizing personnel traffic in cell culture rooms and reducing the opening and closing of incubators reduces the probability of spreading microorganisms. Finally, one of the common means of transferring infec-tions is via the transfer of cell lines into a laboratory from another location. Cell lines new to a laboratory should be quarantined and treated with caution until proven free of microorganisms, unless they have been confirmed free prior to transfer (for exam-ple, if obtained from a cell bank).

2.6. Prevention of Microbial Contamination and the Use of Antibiotics

The cornerstone to preventing microbial contamination is the practice of aseptic technique. The use of antibiotics in routine culture varies but is generally discouraged for a number of reasons. First, good practice should make their addition unnecessary. Although antibiotics may suppress infection, they may not eliminate the infection and may permit resistant organisms to develop. Poor technique should not rely on assis-tance from antibiotic use, and it is important to detect infection as early as possible. Evidence to support this view was obtained in one analysis where 72% of cultures grown continuously in antibiotics were shown to be mycoplasma-positive while only 7% grown in the absence of antibiotics were infected (1). Secondly, the antibiotics may influence the biochemistry of the cultured cells, which may influence the experi-mental endpoint. However, the short-term use of antibiotics may have useful strategic value in a number of different situations. These include initial culture of primary samples and also curing unique and essential contaminated samples. The latter will be dependent on the extent of contamination and is only feasible if still at a relatively early stage. In most instances, contaminated culture should be autoclaved.

2.7. General Procedure for Decontaminating Infected Cultures

For cultures that are considered unique and become infected, it may be feasible to disinfect and eliminate the infection. Having established the type of infection (bacte-rial, fungal, yeast, or mycoplasmal), a range of antibiotics are available to treat the problem (see Table 3). As a higher concentration of antibiotic is likely to be more effective, it can be worthwhile evaluating the antibiotic over a range of concentrations

Cell Culture Contamination315 Table 3

Commonly Used Antibiotic and Antimycotic Treatments

Responsive microorganisms Reagent Working Conc Bacteria Yeast Fungus Mycoplasma

Amphotericin B 2.5 mg/L+–+–Ampicillin100 mg/L+–––Dihydrostreptomycin100 mg/L+–––Erythromycin100 mg/L+–––Gentamycin sulfate 50 mg/L+––+ Kanamycin sulfate100 mg/L+––+ Neomycin sulfate50 mg/L+– ––Nystatin100,000 U/L–++–Penicillin G100,000 U/L+–––Polymyxin B sulfate50 mg/L+–––Streptomycin sulfate100 mg/L+–––Tetracycline hydrochloride10 mg/L+–––Tylosin tartrate10 mg/L+––+

to establish a maximum tolerated concentration, i.e., a concentration that is just subtoxic. Having established this concentration, culture infected cells for several (3–4) passages in antibiotic to eliminate infection.

Cells should be cultured in antibiotic-free conditions for several passages and then tested for the presence of infection. Repeat this process if the infection is not fully eliminated.

3. Cell Line Cross-Contamination

The use of multiple cell lines in any laboratory leads to the possibility of contami-nation of one cell line with another. The cross-contamination of cultures has plagued many researchers, often leading to mistaken results, retractions of results, cover-ups, and some out-and-out falsification of data and results following inadvertent use of the wrong cells (10). The realization that this was not a trivial problem came initially in the 1960s and 1970s with the observation that many cell lines had become cross-contaminated with HeLa cells, the first established human cell line. This had occurred to such a degree that a number of cell lines, such as KB, Hep-2, and INT407 were so cross-contaminated that it was unclear if the original cell line existed any longer or had been replaced completely by HeLa. In 1967, Gartler, using isoenzyme analysis reported that 20 human cell lines had been contaminated by HeLa cells (11). Nelson-Rees and colleagues systematically investigated large numbers of cell lines and demonstrated that widespread contamination of cell lines with HeLa had316Langdon occurred(12–14). In 1977, this group demonstrated that of a series of 253 cultures examined, 21 were of the wrong species and 15 were contaminated with HeLa (15). Another contemporary study investigating 246 cultures showed that 14% of cultures were contaminated with another species while 25% were of HeLa origin (16). In 1984, a study by Hukku showed that the situation had not improved and testing of 275 cultures received by that laboratory for analysis indicated that 36% of cul-tures were cross-contaminated: 25% by cells of another species and 11% by another human line (17). In a study published in 1999 by investigators from the German Collection of Microorganisms and Cell Cultures (DSMZ), analysis of 252 human cell lines indicated cross-contamination in 45 (18%) of cell lines (18). This recent report indicates that complacency about this issue still exists despite the ongoing warnings of the dangers (19,20).

Most cross-contamination of this nature is the result of poor tissue culture tech-nique and the culture of multiple cell lines at one time. In addition to accidental mixing of cell lines, the use of pipets or media bottles for multiple cell lines and the creation of aerosols can allow contamination to occur. As with microbial contamina-tion, microscopic monitoring of cultures is likely to be the first indication of cross-contamination, and the observation of mixed morphologies or changed growth rates should alert the culturist to potential problems. A number of recommendations to minimize the possibility of cross-contamination (19,21) include:

1.When new cell lines are derived, archive representative samples of the original tissue,

cells, or DNA for later authentication of stocks.

2.Disseminate cell lines from authenticated sources, such as cell collections if possible,

rather than transfer them frequently through multiple laboratories.

3.Cell lines transferred between laboratories should be confirmed free of mycoplasma, and

full information should accompany the cell line attesting to its authenticity.

4.Rapidly expand new cell lines to produce frozen stocks.

5.Treat changes in cell morphology and characteristics with suspicion.

6.Only one cell line should be used at one time when working in a microbiological safety

cabinet. After removal of the line, disinfect the cabinet and run for several minutes before the introduction of another line.

7.Dedicate bottles of media for use with individual cell lines.

8.The formation of aerosols should be kept to a minimum.

9.After 3 months or 10 passages, discontinue the working cell line and obtain a new stock

of cells from liquid nitrogen storage.

10.All culture vessels should be carefully and correctly labelled.

Following these recommendations should minimize the risk of contamination.

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