Cell culture contamination testing

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. 

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. 

The need for cell cultures for tests with viruses requires the incorporation of controls over and above those necessary for working with bacteria. In addition to cell culture controls to demonstrate lack of contamination and vims controls to demonstrate a functioning assay system, the level of input vims and the loss in virus infectivity upon the drying of the inoculum on the carrier need to be measured. In some cases, these measurements are only made once and the data used with a series of tests. However, it is recommended that, for proper accuracy, such measurements should be included with every test due to inherent variations in cell cultures and viruses. When there is a need to separate virus kill from simple mechanical removal of the test vims during the test, it is recommended that a control also be included to determine the mechanical removal of the test virus standard hard water should be used for this purpose in place of the test topical product. For any claim of topical antisepsis, reduction in the vims titer on treatment with the test product must be substantially higher than that obtained with the standard hard water treatment alone. 

Biological methods This is the oldest group of test methods. In the past, cats were administered food filtrates intraperitoneally, and if the samples being tested were contaminated the cats would vomit. Animal tests are no longer used due to ethical and financial considerations. Cell cultures are now recommended. They are sensitive, cheap, and rapid tests - the cytotoxicity effect may be observed after only two hours (Normanno et al., 2001) - however such tests are not widely applied. 

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. 

Bioassays appeared to fit the bill to perform this service to monitor chemical contamination. They have been around for a while. Until relatively recently, however, they remained in the realm of the laboratory. Only over the last two decades have they found a niche in testing for toxic chemicals in water and sediment, but not yet specifically as a tool for routine water quality monitoring. As Small-scale Freshwater Toxicity Investigations, Volumes 1 and 2 amply demonstrates, the science has now come of age. Assays based on bacteria, microscopic or multi-cellular algae, protozoa, invertebrates and vertebrates (freshwater fish cell cultures) are discussed in... 

Affinity chromatography techniques have shown less utility in analytical testing than in preparative separations for a variety of reasons, including cost and the difficulty of validating consistent operation as the column changes over time. Protein A affinity has been commonly used to quantitate the total antibody content of either ascites or cell culture fluids. To provide guidance in the development of a purification process, specific immunoaffinity resins are either available or can be readily prepared to quantitate the levels of unrelated protein contaminants. To rapidly determine what the active species in a mixture is, a monoclonal antibody that... 

The norms for medicinal production are particularly stringent. Biological products are composed of complex molecules, produced by cell lines with a relatively recent history, and difficult to characterize. Tests performed only on the final product do not guarantee consistency of production. The purification procedures should be planned and validated for the removal of potential contaminants from diverse sources cells, culture media, equipment, and reagents used in the purification or even degradation products derived from the protein itself. There are examples of products with unexpected risks that have caused serious problems such as blood contamination by HIV-1 virus between 1980 and 1985 (Bloom, 1984) or the presence of residual infectious viruses in the poliomyelitis vaccine due to inefficient inactivation (Lubiniecki et al., 1990). 

Sterility checks should be made on all batches of media and trypsin before these are used. As some tests take several weeks to complete, it is essential that adequate stocks are maintained. The use of untested medium is a sure way to introduce contamination into all cell lines in use in a laboratory. Similarly, cell cultures should be routinely checked for contamination. Such checks will involve growth tests on the medium in which the cells have been growing as well as tests on the cells themselves. 

No single test is sufficient to detect all possible contaminants and hence multiple procedures must be adopted. Organisms that grow rapidly in cell culture medium are readily apparent when contaminated medium is incubated at 37°C for a few days. Such contamination is not a serious problem as the experiment can quickly be terminated and the contaminated culture eliminated. It is the slow-growing contaminant which produces no obvious change in the medium and which exerts no marked cytotoxic effect, which may be overlooked and yet may dramatically interfere with a biochemical investigation, e.g. satellite DNA bands in CsCl density gradient analyses or unusual forms of enzyme may reflect the presence of a contaminant. 

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. 

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. 

Cour I, Maxwell G Hay RJ (1979) Tests for bacterial and fungal contaminants in cell cultures as applied at the ATCC. Tissue Culture Association Manual 5 1157-1160. 

Quality assurance by means of strict quality control of all aspects of a cell culture process has always been of prime importance, given the sensitivity of cells to sub-optimal medium and environmental factors and the ease with which cells can become contaminated with viruses and other microorganisms. The potential for biological changes in scale-up of cell culture processes demands even greater standardization and testing of the system. 

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. 

The final product must be free of adventitious agents that primarily include bacterial or viral pathogens and other biologic contaminates contributed during cell culture, such as DNA from prokaryotic or eukaryotic host cells and endotoxin. Purity testing for these factors must be performed throughout the production procedure and meet defined criteria before administration into humans. 

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