Cell culture mycoplasma

Various antibiotics have been investigated for their ability to eradicate mycoplasma from cell lines. Historically the antibiotics of choice were either minocycline or tiamulin. However, many cell culture mycoplasmas are now resistant to these antibiotics. Today the most effective are the quinalone antibiotics ciprofloxacin (Mowles, 1988) and MRA (see Table 1.9.3). 

In 1961 it was reported that human leukocytes were capable of producing IFN in response to viral infections [8,9]. This viral stimulation of white blood cells was initially used to produce leukocyte IFN for clinical applications. Identification of a number of varied IFN inducers such as mycoplasma or other microorganisms in cell cultures, lipopolysaccharides (LPS, derived from bacteria membranes), tumor-derived or virus-transformed cells, and synthetic chemical compounds such as polyanions and poly I C (poly inosine-cytosine) suggested that different IFN mixtures could be derived from interaction of various inducing agents and appropriate target cells [10-16]. Another pH-labile, nonvirus-induced IFN termed immune-IFN (induced by immune effector cells) was discovered in 1965. It was produced by... 

There are various methods for detecting Mycoplasma contamination of cell culture. A sensitive polymerase chain reaction test with broad specificity for Mycoplasma species is our method of choice (8). There are several products available for the eradication of Mycoplasma species from cell lines. The effectiveness of the treatment will depend on the cells and involves trail and error. This is because some cell lines are very sensitive to the chemicals used to eradicate Mycoplasma and may become static or die during treatment. 

Pruckler, J. M., Pruckler, J. M., and Ades, E. W (1995) Detection by polymerase chain reaction of all common Mycoplasma in a cell culture facility. Pathobiology 63,9-11. 

Such a catabolic reaction is indeed excluded in 6 alkyl purine derivatives. The parent compound of this group, 6-methylpurine, is known for its cytotoxicity its libera tion from the 2 -deoxyribonucleoside by purine nucleo side phosphorylases is used for detection of mycoplasma in cell cultures.19 It is highly potent and toxic to nonproliferating and proliferating tumor cells. Recently, the use of cytotoxic bases liberated by purine nucleoside phosphorylases such as 6-methylpurine was proposed as a novel principle in the gene therapy of cancer.20... 

Another factor to be taken into account when using animal serum is the potential risk for human health, due to the possible presence of adventitious agents, such as virus and proteins as prions. In addition, serum can contain contaminants such as bacteria, fungi, and mycoplasmas (small bacteria without cellular walls), which can negatively affect cell culture. 

The production of heterologous proteins for therapeutic use requires selection of the producer cell line, based on yield, monoclonality (for proteins), product quality, stability, and absence of contaminants like bacteria, molds, mycoplasmas, and viruses. Progress in the production of biopharmaceuticals by cell culture is due mainly to the use of diploid cells and continuous cell lines, together with the maintenance of cells by cryo-preservation. It is important to guarantee that the expression system chosen is able to generate the product in a consistent and economically feasible way (Levine and Castillo, 1999). 

Although primary cell cultures are usually free from contamination by mycoplasmas, many cell strains and lines in use are contaminated. This probably arose from the use of animal sera contaminated with A. laidlawii and M. arginini or pig trypsin contaminated with M. hyorhinis. These are probably no longer a significant source of contamination and, although M. hominis, M. pharyngis and M. salivarium are readily isolated from human mouths and throats, the most frequent source of contamination today is from working with contaminated cells. It has been estimated that 50-95% of cells in use today are contaminated with mycoplasma. 

As mycoplasmal contamination of cell cultures is not always so obvious as bacterial contamination, it is important to 1) be aware of the effects of mycoplasmas on cell cultures, and 2) carry out routine tests for their presence. This is especially important as a contaminated culture may have 108 mycoplasma per ml, i.e. there may be 100 mycoplasma per cell. The mycoplasma often grow attached to the surface of the cell providing it with a prokaryotic coat. 

The lethal effect of mycoplasmas on cell cultures can be accentuated by culture in the presence of 6-methylpurine deoxyribonucleoside (6-MPDR) and this forms the basis of the MycoTect kit available from Gibco. All mycoplasmas possess high levels of adenosine phosphorylase which converts the non-toxic 6-MPDR into 6-methylpurine and 6-methylpurine ribonucleoside, both of which are toxic to mammalian cells (McGarrity and Carson, 1982). 

Mycoplasma can be eliminated from cell culture by treatment with immune serum (Pollock and Kenny, 1963) and passage through an animal is often effective in removing mycoplasma from tumour producing cell lines. 

As detailed in Section III, initial raw materials for the production of antibodies are very different. Animal sera, transgenic milk, bovine colostrum, cell culture (hybridomas or other recombinant cells), ascites fluid, and egg yolk are the best known. Each raw material represents a potential carrier for adventitious agents. All of them can contain viruses. More specifically, animal sera used as source of antibodies, or as a cell culture medium supplement, can additionally carry mycoplasma and pyrogens depending on the method of collection. 

Of particular importance in the routine handling of cell cultures is the mycoplasma contamination status of cell lines. If present, the concentration of mycoplas-mas in the culture supernatant can be in the region of 10 -10 mycoplasmas mH. Unlike bacterial and fungal contaminants, they do not necessarily manifest themselves in terms of pH change and/or turbidity and they can be present in low... 

Mycoplasma is a generic term given to organisms of the order Mycoplasmatales that can infect cell cultures. Those that belong to the families Mycoplasmataceae Mycoplasma) and Acholeplasmataceae (Acholeplasma) are of particular interest. 

The first observation of mycoplasma infection of cell cultures was by Robinson et ah (1956). The incidence of such infection has since been found to vary from laboratory to laboratory. At present 12% of cell hnes received by the ECACC are infected but this may be an uncharacteristically low figure because many lines are screened for mycoplasma prior to deposition. 

The importance of mycoplasma detection in cell cultures should not be underestimated. The concentration of mycoplasmas in the supernatant can be typically in the region of 10 -10 mycoplasmas mH. Additionally, mycoplasmas will cytadsorb to the host cells. They do not necessarily manifest themselves in the manner of most bacterial or fungal contaminants, e.g. pH change or culture turbidity. It is important therefore to adopt an active routine detection procedure. Mycoplasmas have been shown to elicit various effects, including the following ... 

Note Both methods used for the detection of mycoplasma have the following general principles in common. The cells to be tested should, before testing, complete at least two passages in antibiotic-free media. Infection may be hidden by the presence of antibiotics. Cell cultures tested from frozen ampoules should undergo at least two passages because cryoprotectants may also mask infection. 

Each new batch of media ingredients should be subject to quality control before agar or broth preparation. It is especially important to show that new batches of pig and horse serum can support the growth of a representative sample of species found infecting cell cultures, e.g. any two of M. orale, M. hominis, M. fermentans, M. arginini, M, hyorhinis or A laidlawii. The National Collection of Type Cultures (Colindale, London, UK) or the American Type Culture Collection (Manassas, Virginia, USA) may supply type strains, or wild-type strains may be used. Stock positive control cultures may be kept frozen at -70°C in mycoplasma broth. 

The fluorochrome dye Hoechst 33258 binds specifically to DNA. Cultures infected with mycoplasma are seen under fluorescence microscopy as fluorescing nuclei with extranuclear mycoplasmal DNA (Plate 1.6.1a, b) whereas uninfected cell cultures contain fluorescing nuclei against a negative background (Plate 1.6.1b, c). 

Levine EM, Thomas L. McGregor D, Hayflick L Eagle M (1968) Altered nucleic acid metabolism in human cell cultures infected with mycoplasma. Proceedings of the National Academy of Sciences of the USA 60 583-589. 

Stanbridge EJ, Hayflick L Perkins FT (1971) Modification of amino acid concentrations induced by mycoplasmas in cell culture medium. Nature (London) New Biology 232 242-244. 

Many different techniques, such as bacteriological culture, DNA staining using fluorochrome and immunological or biochemical methods, are available to detect mycoplasma contamination (see section 1.6). However, none seem to be fully efficient, so a combination of different methods is often necessary. Molecular tools such as hybridization using rDNA gene probes or polymerase chain reaction (PCR) have been developed over the past few years. Several studies using 16S rDNA-based PCR concluded that PCR seems to be a very convenient method for routine detection of cell culture contaminations (Spaepen, 1992 Teyssou, 1993 van Kuppeveld, 1994). 

The PCR method that we have described is able to detect cell cultures containing contaminating mycoplasma species with great sensitivity (1.10 cfu/10 p.1) and seems to be more sensitive than the other techniques available. Also, PCR analysis can be performed from lyophilized cell cultures, which facilitates the transport of samples. Moreover, sample preparation is very simple and does not require any DNA extraction, so the results are obtained in 1 day. 

However, several disadvantages can be reported. This PCR method requires two different PCR systems mollil + molli2a to detect all the Mycoplasma and Spiroplasma spp. and mollil + molli2b to detect Acholeplasma spp. The set of primers mollil + molli2a amplifies two phylogeneticaUy closely related species -Clostridium ramosum and C. inocuum - but these species are rarely found as cell culture contaminants. Moreover, this method does not allow bacterial identification of the detected species or antibiotic susceptibility testing. 

Is there a better PCR technique Over the past few years different authors have described other 16S rDNA-based PCR methods. Spaepen et ah (1992) used a nested PCR system with great sensitivity, but the use of a second amplified cycle dramatically increased the risk of DNA carryover contaminations, van Kuppeveld et al. (1994) reported a single PCR system that seems to be very suitable to detect cell culture contamination but it requires a DNA extraction stage, which is very time consuming. Moreover, a new marked PCR method is available (Stratagene, CA). The primers used make it possible rapidly to (4-5 h) test eukaryotic cells for mycoplasma infection but this method seems to be less sensitive than our PCR technique. 

Spaepen M, Angulo AF, Marynen P Cassiman JJ (1992) Detection of bacterial and mycoplasma contamination in cell cultures by polymerase chain reaction. FEES Microbiology Letters 99 89-94. 

This incident emphasizes the critical importance of diligent testing of cell cultures for contaminant microorganisms. By combining procedures such as those described here with procedures included elsewhere in this volume (e.g. fluorescent or nucleic acid probes for mycoplasma and viruses) one can be more certain that clean cell cultures are available for experimentation. 

In the event of cell cultures becoming contaminated with bacteria, fungi or mycoplasmas, the best course of action is to discard the culture, check cell culture reagents for contamination, thoroughly disinfect all safety cabinets and work surfaces and resuscitate a fresh culture from previously frozen stock. In the case of contamination with a spore-forming organism, and where such facilities exist, room fumigation may also be advisable. 

Unlike bacterial or fungal contamination, mycoplasma infection is not always detectable in a cell culture by the usual microscopic methods. 

Marcus M, Lavi U, Nattenberg A, Rottem S Markowitz D (1980) Selective killing of mycoplasma from contaminated mammalian cells in cell cultures. Nature London) 285 659-699. 

A more general example from virus vaccine production is the rigorous examination of tissue cultures to exclude contamination with infectious agents from the source animal or, in the cases of human diploid cells or cells from continuous cell lines, to detect cells with abnormal characteristics. Monkey kidney cell cultures are tested for simian herpes B virus, simian virus 40, mycoplasma and tubercle bacilli. Cultures of human diploid cells and continuous line cells are subjected to detailed kary-ological examination (examination of chromosomes by microscopy) to ensure that the cells have not undergone any changes likely to impair the quality of a vaccine or lead to undesirable side-effects. 

Testing of cell cultures for the presence of key adventitious agents should be routine in any tissue culture facility. Altough bacterial and fungal contaminations can be detected by microscopic and sometimes by macroscopic examination, the detection of mycoplasma and virus contaminants require the use of specific test procedures, including isolation by culture, PCR methods, electron microscopy, and analysis of cytopathic effects. 

Microbial contamination can exert numerous effects, and bacterial and yeast contamination will cause nutrient depletion resulting in the death of the cell culture. Although mycoplasma are very fastidious in their growth requirements, contamination with these organisms is known to exert more insidious effects, such as alteration of the growth rate of cells (3), induction of chromosomal aberrations (4), changes in amino acid and nucleic acid metabolism (5,6), and membrane aberrations (7). 

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