[Skip to Content](https://www.sigmaaldrich.com#main-content) [](https://www.sigmaaldrich.com/US/en) Products Cart0 USEN Products [Login / Register](https://www.sigmaaldrich.com/oidc-sign-in) [Order Lookup](https://www.sigmaaldrich.com/US/en/order-lookup) [Quick Order](https://www.sigmaaldrich.com/US/en/quick-order) Cart0 [Home](https://www.sigmaaldrich.com/US/en)[Applications](https://www.sigmaaldrich.com/US/en/applications)[Cell Culture & Cell Culture Analysis](https://www.sigmaaldrich.com/US/en/applications/cell-culture-and-cell-culture-analysis)Cell Culture Troubleshooting # Cell Culture Troubleshooting   Cell culture has become one of the most fundamental techniques for modeling biological systems, and is of increasing importance in the biotechnology and pharmaceutical sectors as well as an essential process in life science research labs. Though this technique is highly accessible, successful propagation of cells for stock expansion or modeling experiments can be plagued by contamination or other conditions that negatively impact cell viability. The common practice of sharing cells has led to well-published evidence of cross-contamination of cell stocks whose identity was previously unquestioned. Identifying both common and infrequent reasons why cells may fail to thrive can increase lab efficiency, enhance yield of cellular products, and ensure meaningful, reliable downstream data from *in vitro* models.       * * * ## RELATED PRODUCTS Slide 1 of 19 1 of 5 [ \ Sigma-Aldrich \ SLM-241 \ DMEM Complete Medium, with 2mM L-Glut, Sodium Pyruvate, and 10% FBS](https://www.sigmaaldrich.com/US/en/product/mm/slm241) Quick View [ \ HeLa Cell Line human](https://www.sigmaaldrich.com/US/en/product/sigma/cb_93021013) Quick View [ \ Millipore \ SEGPU0538 \ Stericup® E-GP Funnel-less Sterile Vacuum Filtration System](https://www.sigmaaldrich.com/US/en/product/mm/segpu0538) Quick View [ \ Millipore \ S2VPU11RE \ Millipore® Stericup® Quick Release Vacuum Filtration System](https://www.sigmaaldrich.com/US/en/product/mm/s2vpu11re) Quick View [ \ Sigma-Aldrich \ MP0040A \ LookOut® Mycoplasma qPCR Detection Kit](https://www.sigmaaldrich.com/US/en/product/sigma/mp0040a) Quick View [ \ Millipore \ PFHYS1008 \ Millicell® HY Multilayer Culture Flask](https://www.sigmaaldrich.com/US/en/product/mm/pfhys1008) Quick View [ \ Millipore \ PSET010 \ Millicell® 24 well Plates](https://www.sigmaaldrich.com/US/en/product/mm/pset010) Quick View [ \ Millipore \ PHCC20060 \ Scepter 2.0 Handheld Automated Cell Counter](https://www.sigmaaldrich.com/US/en/product/mm/phcc20060) Quick View [ \ Sigma-Aldrich \ C6164 \ Cell Freezing Medium-DMSO 1×](https://www.sigmaaldrich.com/US/en/product/sigma/c6164) Quick View [ \ Sigma-Aldrich \ C1562 \ Freezing container, Nalgene® Mr. Frosty](https://www.sigmaaldrich.com/US/en/product/sigma/c1562) Quick View [ \ Sigma-Aldrich \ E1270 \ ECM Gel from Engelbreth-Holm-Swarm murine sarcoma](https://www.sigmaaldrich.com/US/en/product/sigma/e1270) Quick View [ \ Sigma-Aldrich \ A9418 \ Bovine Serum Albumin](https://www.sigmaaldrich.com/US/en/product/sigma/a9418) Quick View [ \ Sigma-Aldrich \ C2674 \ Collagenase from *Clostridium histolyticum*](https://www.sigmaaldrich.com/US/en/product/sigma/c2674) Quick View [ \ Sigma-Aldrich \ D5652 \ Dulbecco′s Phosphate Buffered Saline](https://www.sigmaaldrich.com/US/en/product/sigma/d5652) Quick View [ \ Sigma-Aldrich \ E8008 \ Ethylenediaminetetraacetic acid solution](https://www.sigmaaldrich.com/US/en/product/sigma/e8008) Quick View [ \ Supelco \ 04511 \ Live/Dead Cell Double Staining Kit](https://www.sigmaaldrich.com/US/en/product/sigma/04511) Quick View [ \ Sigma-Aldrich \ T8154 \ Trypan Blue solution](https://www.sigmaaldrich.com/US/en/product/sigma/t8154) Quick View [ \ Sigma-Aldrich \ SCT149 \ ATP Cell Viability Luciferase Assay](https://www.sigmaaldrich.com/US/en/product/mm/sct149) Quick View [ \ CLS4487 \ Corning® Costar® Stripette® serological pipettes, individually paper/plastic wrapped](https://www.sigmaaldrich.com/US/en/product/sigma/cls4487) Quick View * * * ## Featured Categories [](https://www.sigmaaldrich.com/US/en/products/cell-culture-and-analysis/cell-culture-media-and-buffers/classical-media-and-buffers) [Classic Media & Buffers](https://www.sigmaaldrich.com/US/en/products/cell-culture-and-analysis/cell-culture-media-and-buffers/classical-media-and-buffers) Choose from a range of optimized cell culture media formulations, including DMEM, RPMI-1640, and serum-free options for diverse cellular needs. 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Factors leading to cell line misidentification/cross-contamination include: - Contamination with aggressive cell lines: Isoenzyme analysis has demonstrated that many cell lines share a rare enzyme isoform with HeLa cells. HeLa has since been shown to be the most common invasive cell line in culture stocks. - Documentation and labeling practices: Cell culture flasks, plates, cryovials, and freezer containment must be clearly labeled, and liquid nitrogen storage maps strictly maintained to reflect the addition, withdrawal, and relocation of cell stocks. - Cell line borrowing: Sharing of cells amongst neighboring labs is a common practice that leads to errors in cell line identity. Cell lines should only be obtained from reputable cell repositories such as ECACC, the European Collection of Authenticated Cell Cultures at Public Health England. [Be informed about common causes of cell line cross-contamination](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/mammalian-cell-culture/cell-culture-troubleshooting-cell-line-misidentification) and how to protect your work from its consequences.  ## Microbial Contamination of Cell Cultures Cell culture media and incubation conditions provide the ideal environment for cells, as well as bacterial, fungal, and viral contaminants. To augment diligent adherence to aseptic culture technique, cultures must be regularly examined microscopically for evidence of bacterial and fungal intruders. Some ubiquitous microbial contaminants evade visual detection, as up to 30% of all cultures are estimated to be contaminated with mycoplasma. Reagents and kits designed for detection of invisible mycoplasma are often based on PCR amplification, which can also be used to detect viral contaminants. [Learn how to keep your cultures free of biological and chemical contaminants](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/mammalian-cell-culture/cell-culture-troubleshooting-contamination).  ## Poor Cell Growth It's frustrating when cells in culture that otherwise appear healthy fail to attain confluency. When contaminants are ruled out, some possible barriers to healthy cell doubling include: - Culture/freezing media condition and quality - Quality and application-suitability of supplements - Inaccurate cell enumeration during freezedown or passage When cells seem viable but fail to expand, [learn what you can do to improve culture conditions for optimal growth](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/mammalian-cell-culture/poor-cell-growth) before heading back to the liquid nitrogen.  ## Detachment of Adherent Cell Phenotypes Adherent cells may spontaneously detach in culture, singly or in sheets. When adherent cultures don't stay that way, [know what to check and how to restore cohesion in culture](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/mammalian-cell-culture/poor-adherent-cell-attachment-troubleshooting).  ## Cell Clumping Cells in suspension mimic *in vivo* conditions as singlets. When cells start to clump, this can be due to sticky nucleic acids, present in media when cultures are stressed. What's hiding in media that can cause cells to clump? [Read more here](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/mammalian-cell-culture/cell-clumping-troubleshooting) about how to banish clumping from cultures.  ## Cell Death in Culture When cells die in culture, contamination with microorganisms must first be ruled out. Other factors that may contribute to poor cell health or behavior inconsistent with phenotype include: - Frozen stock conditions - Cell passage number/overconfluency - Environmental stress - Overdigestion with dissociation enzymes Appropriate equipment, qualified reagents, and expert protocols are key to [resolving unexpected cell culture outcomes](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/mammalian-cell-culture/cell-culture-troubleshooting-cell-death).  ## Precipitates in Culture Media If it's not contamination, particulates in culture often have an inorganic explanation. Understand balanced buffer and media constituents, and [learn more about keeping media constituents in suspension](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/mammalian-cell-culture/troubleshooting-precipitates). 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[Find Documents](https://www.sigmaaldrich.com/documents-search) ## Related Articles - [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) Know when to use antibiotics to prevent bacterial or fungal, mycoplasma, or viral contamination in cell culture and find suitable antibiotics or other biological agents. - [Antibiotic Kill Curve](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/mammalian-cell-culture/antibiotic-kill-curve) Antibiotic kill curve is a dose response experiment in which mammalian cells are subjected to increasing amounts of selection antibiotic - [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 antibiotic selection guide, including antibiotics for mammalian cell culture, plant cell culture, and antibiotic selection agents for cell culture. - [Cell Culture FAQs: Bacterial Endotoxin Contamination](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/microbiological-testing/pyrogen-testing/what-is-endotoxin) FAQs on bacterial endotoxin contamination, details on endotoxin testing using the LAL assay, and tips to avoid contamination in cell cultures. - [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) Extracellular matrix proteins such as laminin, collagen, and fibronectin can be used as cell attachment substrates in cell culture. - [See All (22)](https://www.sigmaaldrich.com/US/en/search/facet-search?focus=sitecontent&term=facet-search) ## Related Protocols - [Thawing of Frozen Cell Lines](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/cell-culture-and-cell-culture-analysis/mammalian-cell-culture/thawing-of-frozen-cell-lines) Cell culture protocol for proper thawing of cryopreserved cell lines. - [Subculture of Adherent Cell Lines](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/cell-culture-and-cell-culture-analysis/mammalian-cell-culture/subculture-of-adherent) Cell culture protocol for passaging and splitting adherent cell lines using trypsin EDTA. Free ECACC handbook download. - [Cryopreservation of Cell Lines](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/cell-culture-and-cell-culture-analysis/mammalian-cell-culture/cryopreservation-of-cell-lines) Cell culture protocol for high cell viability freezing using cryopreservation reagents like DMSO. 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