The air is warm and humid, there is an abundance of food, and your friends come and go with their shiny toys. What sounds like a dreamy summer holiday is also the reality of in vitro cell culture experiments, and a golden opportunity for contaminants to intrude. Every person, reagent, and piece of equipment in the laboratory is a potential vehicle for invasive microbes, unwelcome cells and chemical impurities, which can create costly issues in both bench research and manufacturing. Cell culture contamination is a problem on many levels, creating immediate implications for experiments and wider issues for the scientific community.Consequences of cell culture contaminationContaminants can affect all cell characteristics (e.g. growth, metabolism, and morphology) and contribute to unreliable or erroneous experimental results. Cell culture contamination will likely create a need for experiments to be repeated, resulting in frustrating time delays and costly reagent wastage. Data derived from undetected contaminated cultures can end up published in scientific journals, allowing others to build hypotheses from dubious results. The pervasiveness of cross-contaminated and misidentified cell lines is a decades-long issue; in 1967, cell lines thought to be derived from various tissues were shown to be HeLa cells, a human cervical adenocarcinoma cell line.1 However, studies involving these misidentified cell lines continued to feature in hundreds of citations during the early 2000s.2This pattern is a well-acknowledged problem and threatens to undermine scientific integrity. The first published retraction in Nature Methods was due to cell line contamination3, and one conservative estimate of contaminated literature in 2017 found 32,755 articles reporting on research with misidentified cells.4 While many scientists may have been blissfully ignorant in the past, awareness of misidentified cell lines is growing.Deciding how best to deal with this knowledge is not straightforward and has been discussed extensively.4 In the interest of preventing further data contamination, a certificate of authentication of the origin and identity of human cells is now required by the International Journal of Cancer, and encouraged by funding agencies. Others have questioned whether mandatory testing really is the best way forward.3But what should be done about existing contaminated literature? Mass retraction of affected articles may disproportionately punish the careers of a few scientists, and could be a waste of resources containing potentially valuable data. One recently proposed system of self-retraction recommends replacing blame with praise in order to encourage self-correction.5 Post hoc labeling of published articles in the form of an expression of concern allows existing findings to remain accessible, while giving readers a chance to form their own judgement.

Lastly, pathogens carried by cells (either intentionally or accidentally) or in components of the culture medium are potential health hazards, and laboratory-acquired viral infections have been reported.6-8 Indeed, the stakes are higher when cells are to be introduced into patients, highlighting the critical importance of quality control in cell therapies.

While pipetting is a key part of everyday laboratory work, it is also one of the stages most prone to contamination. As sample contamination can affect the reliability of results, it is important to know how it can be avoided, saving both time and money. Download this poster for ten tips to avoiding contamination in pipetting.

Avoid leaving your cultures out of the incubator for extended periods

Label all cultures clearly and unambiguously

Disinfect work surfaces before and after use

Check disinfectants are effective and appropriate choices for the job

Work with only one cell culture at a time

Use separate media and reagents for each individual cell line

Quarantine new cell lines until tested negative for mycoplasma

Avoid overusing and relying on antibiotics

Record how long a cell line has been kept in cultureThe design of the laboratory can also play a role; cabinets should be placed away from through-traffic, doors and air-conditioning inlets.6 Restricting area access to allow only essential laboratory personnel to enter reduces disturbances of airflow around the microbiological safety cabinet.

Water baths, CO2 incubators, shelves and water pans are common culprits and should be cleaned or autoclaved regularly, using a chemical disinfectant where appropriate. Other routes of infection include accidental spillages, contact with non-sterile surfaces, splash-back from pipetting or pouring, microscopic aerosol, and infestation by vertebrates, dust and mites.Research groups isolating stem cells use unique cell properties to filter out undesired cells, explains Dr Mei-Ju Hsu, postdoctoral researcher in stem cell therapy at Leipzig University. Dr Hsu notes that: one of the most important features of mesenchymal stem cells is the attachment and growth on the plastic surfaces without prior coating. This step serves as a good way to eliminate the non-adherent cells (e.g. blood cells) by the removal of supernatants.

Organoid researcher Hans Clevers, from the Hubrecht Institute for Developmental Biology and Stem Cell Research at Utrecht University, assesses genetic diversity in cells through the use of single nucleotide polymorphism (SNP) genotyping. The Clevers laboratory recently branched out from their work with mammalian cells to produce snake venom gland organoids. Dr Clevers notes that: We have come to realize that contamination of organoid cultures is a serious problem. We have observed that organoid cultures that are commonly used and are fast growers contaminate slower growing organoid cultures. Typical fast growers are the original mouse mini-guts that have popped up in various human organoid cultures in the lab. We SNP-type all human samples when they come in, which allows us to follow purity of human organoid cultures over time. Cheap, fast and crucial to avoid big mistakes.

Mycoplasma is one of the most common cell culture contaminants, with six species of mycoplasma accounting for 95% of all contamination. Therefore, it is important to improve our understanding of where mycoplasma contamination can stem from and how best to prevent it. Download this infographic to discover more about mycoplasma contamination in cell culture labs.

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Meet the Culprits of Cell Culture Contamination - Technology Networks