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Order](https://www.sigmaaldrich.com/US/en/quick-order) Cart0 [Home](https://www.sigmaaldrich.com/US/en)[Mammalian Cell Culture](https://www.sigmaaldrich.com/US/en/applications/cell-culture-and-cell-culture-analysis/cell-culture-by-cell-type/mammalian-cell-culture)Quality Control Considerations in Cell Culture # Quality Control Considerations in Cell Culture *ECACC Laboratory Handbook 4th Edition* - [Reagents And Materials](https://www.sigmaaldrich.com#reagents) - [Cell Line Integrity and Identification](https://www.sigmaaldrich.com#cell) - [Avoidance of Microbial Contamination](https://www.sigmaaldrich.com#avoidance) - [What to Do in the Event of Cell Culture Contamination](https://www.sigmaaldrich.com#what) - [Environmental Monitoring](https://www.sigmaaldrich.com#environmental) - [Aseptic Technique and Contamination Control](https://www.sigmaaldrich.com#aseptic) Quality is important in all aspects of tissue culture. The quality of materials used (cell lines, media and other reagents) will affect the quality of the cultures and the subsequent scientific data and products derived from them. The main areas of quality control that are of concern for tissue culture are: - The quality of the reagents and materials - The cell line integrity and identification - The avoidance of microbial contamination [Monitor Cell Health](https://www.sigmaaldrich.com/US/en/products/cell-culture-and-analysis/cell-analysis/cell-health-analysis) ## [](https://www.sigmaaldrich.com)Reagents and Materials Potential sources of contamination are reagents and materials, in particular bovine serum which has been identified as a source of bovine viral diarrhea virus (BVDV). Porcine trypsin is also a potential source of Mycoplasma. Good quality reagents and materials are available from numerous manufacturers of tissue culture media and supplements. In addition, reputable manufacturers will carry out a range of quality control tests including screening for mycoplasma and BVDV and supply a Certificate of Analysis with their products. These state the product and lot numbers and form a vital part of record keeping and tracking of reagents used in the production of cell stocks. It is advisable to further test key reagents such as fetal bovine serum to ensure that they are ‘fit for purpose’ due to batch-to-batch variation. Manufacturers of sterile plasticware (flasks, centrifuge tubes, pipettes) designed for tissue culture use also supply Certificates of Analysis for each batch produced, which should be kept for future reference. ## [](https://www.sigmaaldrich.com)Cell Line Integrity and Identification The sourcing of cell lines can have an important effect on quality; freshly imported cell lines are a major source of contamination. The advantages of obtaining cell lines from a recognized source such as a ECACC culture collection are that the cultures will be: - Contaminant free - Authenticated by use of short tandem repeat profiling for human cell lines, and determination of the species of origin by DNA barcoding for other species - Supplied with a detailed data sheet Once cell lines have been obtained from a reputable source it is important to implement master and working cell banking procedures and the associated quality control steps such as routine testing for microbial contaminants and confirming the identity of cultures. ## [](https://www.sigmaaldrich.com)Avoidance of Microbial Contamination Potential sources of contamination include other cell lines, laboratory conditions and staff poorly trained in core areas such as aseptic techniques and good laboratory practice. Thus, the use of cells and reagents of known origin and quality alone is not sufficient to guarantee quality of product (cell stock or culture products); it is necessary to demonstrate quality throughout the production process and also in the final product. Routine screening aids the early detection of contamination since all manipulations are a potential source of contamination. The three main types of microbial contaminants in tissue culture are: - Bacteria and Fungi - Mycoplasma - Viruses ### Bacterial and Fungal Contamination Bacterial contamination is generally visible to the naked eye and detected by a sudden increase in turbidity and colour change of the culture medium as the result of a change in pH. The cell culture may survive for a short time but the cells will eventually die. Daily microscopic observation of cultures will ensure early detection of contamination and enable appropriate action to be taken as soon as the first signs of contamination become apparent. In addition, specific tests for the detection of bacteria and fungi should be used as part of a routine and regular quality control screening procedure. ### Mycoplasma Contamination Mycoplasmas are the smallest free-living self-replicating prokaryotes. They lack a cell wall and the ability to synthesize one. They are 0.3μm in diameter and can be observed as filamentous or coccal forms. There are 6 major species that are tissue culture contaminants, namely M. hyorhinis, M. arginini, M. orale, M. fermentans, M.hominis and Acholeplasma laidlawii. The effects of mycoplasma infection are more insidious than those of bacteria and fungi, inducing several long-term effects in cell cultures. These can include: - Altered growth rate - Morphological changes - Chromosome aberrations - Alterations in amino acid and nucleic acid metabolism However, despite these well documented effects the presence of mycoplasma is often not tested for with the consequence that in such laboratories the majority of cell lines are positive for mycoplasma. Mycoplasma contamination is difficult to detect requiring the use of specialist techniques. In the past only specialist laboratories, such as culture collections, have performed these tests. However, a variety of commercial mycoplasma detection kits are now available although the performance characteristics of these kits can be extremely variable. A combination of these should be used as part of a routine and regular quality control screening procedure. ECACC tests cultures for the presence of mycoplasma on a routine basis and offers a mycoplasma testing service. ### Viral Contamination Some cell lines contain endogenous viruses and secrete virus particles or express viral antigens on their surface, e.g. Epstein-Barr Virus (EBV) transformed lines. These cell lines are not considered contaminated. However, bovine serum is a potential source of bovine viral diarrhoea virus (BVDV) contamination and use of infected serum will lead to contamination of cell lines with the virus. Contamination of cell lines with BVDV may cause slight changes in growth rate but since this virus is non-cytopathic, macroscopic and microscopic changes in the culture will not be detected. Suppliers of bovine serum are aware of this and screen sera accordingly; generally, serum is sold as BVDV tested. Having said this you should be always check carefully to ensure that you understand the results of any testing that is performed on serum. Is the serum indicated as BVDV tested and none detected? What is the sensitivity of detection? BVDV tested serum is not necessarily BVDV free. ## [](https://www.sigmaaldrich.com)What to do in the Event of Cell Culture Contamination One hugely underestimated problem in tissue culture is the routine use of antibiotics. Continuous use of antibiotics is unnecessary and can lead to the development of resistant strains that are difficult to eradicate and may require the use of more exotic antibiotics that may be toxic to the cell cultures. In addition, the use of antibiotics may mask a low level of contamination. Once a contamination has been detected, whether it is due to bacteria, fungi or mycoplasma, the recommended course of action is to discard the culture and continue the work with earlier stocks that are known to be free of contaminants or obtain fresh stocks from a recognized source. Viral infections are virtually impossible to remove from cultures since they do not respond to antibiotic treatment. Also, as they are intra-cellular parasites it is not possible to remove them by centrifugation or other separation techniques. If virus free stocks or a virus free alternative is not available, then a thorough risk assessment should be undertaken prior to continuing work with the infected cell line. ## [](https://www.sigmaaldrich.com)Environmental Monitoring It is good practice to monitor the laboratory environment where cell cultures and their products are prepared. Class 2 microbiology safety cabinets, fitted with HEPA filters, should be tested every 6 months to ensure that they are working efficiently, i.e. the level of airflow across the filter should be tested. However, it is also advisable to monitor the level of ingress of contaminants into the cabinet by periodically placing open settle plates (Tryptone Soya Bean Agar bacteriological culture plates) on the cabinet work surfaces. In addition, settle plates should be used to assess airborne microbial burden at selected points around the laboratory. Plates should be left open for a period of 4 hours. After this time, they should be covered, placed in sealed boxes and incubated at 32 °C and 22 °C for up to 7 days. At the end of this period the plates should be examined for the presence of microbial growth. The position of each plate in the cabinet should be recorded and results stored for trend analysis. Acceptable limits should be defined in terms of “alert” levels and “action” levels, the actual values being dependent on the containment classification of the work area, the criticality of the work and the levels of cleanliness that can be achieved under normal operating conditions. ## [](https://www.sigmaaldrich.com)Aseptic Technique and Contamination Control ### Personal Hygiene When entering the laboratory, it is important to wash hands since this will remove dry skin and loosely adherent microorganisms which could potentially contaminate cell cultures. Gowns and disposable gloves must be worn. Gloves should be frequently swabbed with 70% (v/v) sterile alcohol. Other personal protective equipment includes head caps and face masks, but these are not always necessary, particularly when a class 2 microbiological safety cabinet is being used. Long hair should be tied back to remove obstruction and reduce the risk of contamination. ### Working within the Microbiological Safety Cabinet When working within the cabinet the operator should remember that the air-flow does not make the environment sterile but keeps it clean. Before any practical procedure is conducted the cabinet should be stocked with all the materials required for the experiment. In doing so the operator restricts the number of times that their hand/arm is removed from the cabinet into a non-clean environment. When stocking up the cabinet it is essential that a clutter free condition is maintained. Each item within the cabinet should be positioned to minimize movement and traffic over the area where cell culture operations are performed. Both the rear and front of the cabinet should be cleared to achieve maximum airflow. Flasks and dishes should be the last items to enter the cabinet. All items that enter the cabinet must be sprayed with 70% (v/v) sterile alcohol to prevent dust and particulates from entering the cabinet. ### Pipetting and Prevention of Aerosols Disposable plastic pipettes (1mL, 2mL, 5mL, 10mL and 25mL) are the easiest forms to use for cell culture. Microbial and cellular contamination can arise through pipetting errors such as spillage of material. Adherence to the following guidance can minimize contamination and safety risks associated with pipetting: - Never mouth pipette - Use automatic pipette aids, with one pipette aid designated to each cabinet. Ensure the pipette fits into the pipette aid without any force. To avoid contamination, disinfect the pipette aid regularly and ensure that filters are changed regularly - Use plugged pipettes when transferring medium - Avoid drawing liquid into the pipette plug. Use an individual pipette once only - To avoid generating aerosols do not create bubbles in the medium or pipette. Aerosols can spread contaminating microorganisms and by introducing cells into the air increases the potential risk of cross-contamination - Clean spills that arise immediately with 70% (v/v) sterile alcohol ## Products Sorry, an unexpected error has occurred Response not successful: Received status code 500 __Related Articles__ - [Mycoplasma Detection and Elimination in Cell Culture](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/cell-counting-and-health-analysis/mycoplasma-detection-and-elimination) - [Why Use Antibiotics in Cell Culture?](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/mammalian-cell-culture/antibiotics-in-cell-culture) - [Cell Culture Antibiotic Selection Guide](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/cell-culture-troubleshooting/antibiotic-selector) - [Cell Culture Protocol 10: Testing Cells for Mycoplasma Contamination by Hoechst DNA Staining](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/microbiological-testing/mycoplasma-testing/testing-for-mycoplasma) - [Hepatotoxicity/Cytotoxicity Assays](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/cell-counting-and-health-analysis/hepatotoxicity-cytotoxicity-assays) - [Optimized PCR-based Detection of Mycoplasma](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/cell-culture-and-cell-culture-analysis/cell-counting-and-health-analysis/optimized-pcr-based-mycoplasma-detection) - [Protocol Guide: WST-1 Assay for Cell Proliferation and Viability](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/cell-culture-and-cell-culture-analysis/cell-counting-and-health-analysis/cell-proliferation-reagent-wst-1) - [Extracellular Matrix Proteins and Tools for Cell Culture Optimization](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/3d-cell-culture/attachment-factors-for-cell-culture) - [View More](https://www.sigmaaldrich.com/US/en/search/facet-search?focus=sitecontent&term=facet-search) __Related Product Categories__ - [Cell Health Analysis](https://www.sigmaaldrich.com/US/en/products/cell-culture-and-analysis/cell-analysis/cell-health-analysis) - [Cell Counting Equipment](https://www.sigmaaldrich.com/US/en/products/cell-culture-and-analysis/cell-analysis/cell-counting-equipment) - [Antibiotics](https://www.sigmaaldrich.com/US/en/products/chemistry-and-biochemicals/biochemicals/antibiotics) - [Mammalian Cell Lines](https://www.sigmaaldrich.com/US/en/products/cell-culture-and-analysis/cell-lines-and-specialty-cell-culture/mammalian-cell-lines) - [Animal Component Free Cell Culture Reagents](https://www.sigmaaldrich.com/US/en/products/cell-culture-and-analysis/animal-component-free-cell-culture-reagents) - [Cell Culture Supplies](https://www.sigmaaldrich.com/US/en/products/labware/cell-culture-and-cryogenics/cell-culture-supplies) - [Cell Imaging Instruments](https://www.sigmaaldrich.com/US/en/products/cell-culture-and-analysis/cell-analysis/cell-imaging-instruments) - [Fetal Bovine Serum (FBS)](https://www.sigmaaldrich.com/US/en/products/cell-culture-and-analysis/cell-culture-supplements-and-reagents/fbs) Top __Sign In To Continue__ To continue reading please sign in or create an account. 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[Duolink^® PLA - Protein Interaction Technology](https://www.sigmaaldrich.com/US/en/products/protein-biology/duolink-proximity-ligation-assay) - [Immunoassay Platform Solutions](https://www.sigmaaldrich.com/US/en/products/protein-biology/immunoassay-platform-solutions) - [ELISA Kits](https://www.sigmaaldrich.com/US/en/products/protein-biology/immunoassay-platform-solutions/elisa-kits) - [MILLIPLEX^® Kits](https://www.sigmaaldrich.com/US/en/products/protein-biology/immunoassay-platform-solutions/milliplex-multiplex-assays-for-luminex-technology) - [SMC^™ Technology](https://www.sigmaaldrich.com/US/en/products/protein-biology/immunoassay-platform-solutions/single-molecule-counting-technology) - [Protein Electrophoresis & Western Blotting](https://www.sigmaaldrich.com/US/en/products/protein-biology/protein-electrophoresis-and-western-blotting) - [Electrophoresis & Transfer Equipment](https://www.sigmaaldrich.com/US/en/products/protein-biology/protein-electrophoresis-and-western-blotting/electrophoresis-and-transfer-equipment) - [Transfer Membranes & Kits](https://www.sigmaaldrich.com/US/en/products/protein-biology/protein-electrophoresis-and-western-blotting/transfer-membranes-and-kits) - [Immunodetection Reagents & Substrates](https://www.sigmaaldrich.com/US/en/products/protein-biology/protein-electrophoresis-and-western-blotting/immunodetection-reagents-and-substrates) - [Membrane Immunodetection Devices](https://www.sigmaaldrich.com/US/en/products/protein-biology/protein-electrophoresis-and-western-blotting/membrane-immunodetection-devices) - [Protein Electrophoresis Gels & Buffers](https://www.sigmaaldrich.com/US/en/products/protein-biology/protein-electrophoresis-and-western-blotting/protein-electrophoresis-gels-and-buffers) - [Protein Electrophoresis Gel Stains](https://www.sigmaaldrich.com/US/en/products/protein-biology/protein-electrophoresis-and-western-blotting/protein-electrophoresis-gel-stains) - [Protein Markers](https://www.sigmaaldrich.com/US/en/products/protein-biology/protein-electrophoresis-and-western-blotting/protein-markers) - 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[Recombinant/Fusion Tag Protein Purification](https://www.sigmaaldrich.com/US/en/products/protein-biology/protein-sample-prep/recombinant-fusion-tag-protein-purification) - [Subcellular Fractionation Reagents](https://www.sigmaaldrich.com/US/en/products/protein-biology/protein-sample-prep/subcellular-fractionation-reagents) - [Whole Cell Lysis Reagents & Enhancers](https://www.sigmaaldrich.com/US/en/products/protein-biology/protein-sample-prep/whole-cell-lysis-reagents-and-enhancers) - [Water Purification](https://www.sigmaaldrich.com/US/en/products/water-purification) - [Milli-Q^® Benchtop Water Purification Systems](https://www.sigmaaldrich.com/US/en/products/water-purification/milli-q-benchtop-water-purification-systems) - [Milli-Q^® High-Flow Water Purification Systems](https://www.sigmaaldrich.com/US/en/products/water-purification/milli-q-high-flow-water-purification-systems) - [Milli-Q^® Clinical Water Purification Systems](https://www.sigmaaldrich.com/US/en/products/water-purification/milli-q-clinical-water-purification-systems) - 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[Primary Cell Culture](https://www.sigmaaldrich.com/US/en/applications/cell-culture-and-cell-culture-analysis/cell-culture-by-cell-type/primary-cell-culture) - [Stem Cell Culture](https://www.sigmaaldrich.com/US/en/applications/cell-culture-and-cell-culture-analysis/cell-culture-by-cell-type/stem-cell-culture) - [Cell Culture by Technique](https://www.sigmaaldrich.com/US/en/applications/cell-culture-and-cell-culture-analysis/cell-culture-by-technique) - [3D Cell Culture](https://www.sigmaaldrich.com/US/en/applications/cell-culture-and-cell-culture-analysis/cell-culture-by-technique/3d-cell-culture) - [Cell Culture Media Preparation](https://www.sigmaaldrich.com/US/en/applications/cell-culture-and-cell-culture-analysis/cell-culture-by-technique/cell-culture-media-preparation) - [Cell Growth & Maintenance](https://www.sigmaaldrich.com/US/en/applications/cell-culture-and-cell-culture-analysis/cell-culture-by-technique/cell-growth-and-maintenance) - [Transfection & Gene Editing](https://www.sigmaaldrich.com/US/en/applications/cell-culture-and-cell-culture-analysis/cell-culture-by-technique/transfection-and-gene-editing) - [Cell Culture for Manufacturing](https://www.sigmaaldrich.com/US/en/applications/cell-culture-and-cell-culture-analysis/cell-culture-for-manufacturing) - [Cell Culture Troubleshooting](https://www.sigmaaldrich.com/US/en/applications/cell-culture-and-cell-culture-analysis/cell-culture-troubleshooting) - [Chemistry & Synthesis](https://www.sigmaaldrich.com/US/en/applications/chemistry-and-synthesis) - [ADC & Bioconjugation](https://www.sigmaaldrich.com/US/en/applications/chemistry-and-synthesis/adc-and-bioconjugation) - [Lead Discovery](https://www.sigmaaldrich.com/US/en/applications/chemistry-and-synthesis/lead-discovery) - [Peptide Synthesis](https://www.sigmaaldrich.com/US/en/applications/chemistry-and-synthesis/peptide-synthesis) - [Protein Degradation](https://www.sigmaaldrich.com/US/en/applications/chemistry-and-synthesis/protein-degradation) - [Reaction Design & Optimization](https://www.sigmaaldrich.com/US/en/applications/chemistry-and-synthesis/reaction-design-and-optimization) - 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[Interrogation of Protein Pathways](https://www.sigmaaldrich.com/US/en/applications/protein-biology/interrogation-protein-pathways) - [Lysis & Protein Extraction](https://www.sigmaaldrich.com/US/en/applications/protein-biology/lysis-and-protein-extraction) - [Protein & Nucleic Acid Interactions](https://www.sigmaaldrich.com/US/en/applications/protein-biology/protein-and-nucleic-acid-interactions) - [Protein Concentration & Buffer Exchange](https://www.sigmaaldrich.com/US/en/applications/protein-biology/protein-concentration-and-buffer-exchange) - [Protein Expression](https://www.sigmaaldrich.com/US/en/applications/protein-biology/protein-expression) - [Protein Labeling & Modification](https://www.sigmaaldrich.com/US/en/applications/protein-biology/protein-labeling-and-modification) - [Protein Pulldown](https://www.sigmaaldrich.com/US/en/applications/protein-biology/protein-pulldown) - [Protein Purification](https://www.sigmaaldrich.com/US/en/applications/protein-biology/protein-purification) - 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Services - [Contract Manufacturing](https://www.sigmaaldrich.com/US/en/services/contract-manufacturing) - [ADC & Bioconjugation](https://www.sigmaaldrich.com/US/en/services/contract-manufacturing/adc-bioconjugation) - [Diagnostic OEM & CMO Services](https://www.sigmaaldrich.com/US/en/services/ivd-development-manufacturing/diagnostic-oem-cmo) - [mRNA & LNP](https://www.sigmaaldrich.com/US/en/services/contract-manufacturing/mrna-and-lnp-formulation-ctdmo-services) - [Lipids CDMO Services](https://www.sigmaaldrich.com/US/en/services/contract-manufacturing/mrna-and-lnp-formulation-ctdmo-services/lipids-cdmo-services) - [Lipid Nanoparticle CDMO Service](https://www.sigmaaldrich.com/US/en/services/contract-manufacturing/mrna-and-lnp-formulation-ctdmo-services/lipid-nanoparticle-cdmo-services) - [Small Molecule](https://www.sigmaaldrich.com/US/en/services/contract-manufacturing/small-molecule) - [Activated Polyethylene Glycol (PEGs)](https://www.sigmaaldrich.com/US/en/services/contract-manufacturing/peg) - 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