Environmental Contamination

Another area of concern in regards to cell cultures is environmental contamination. This kind of contamination is impossible to avoid as the potential sources are everywhere. This can occur from the plastic dishes used during cultures, from the purity of the water used, unintentional effects from certain light sources, or even from cleaning the incubators used for cell growth:

Reproducibility: Respect your cells!

The cell’s physical environment is a profound influence. Researchers at the Wyss Institute in Boston, Massachusetts, found that mechanical peristalsis-like deformations and fluid flow changes alone could, without any alterations to the growth media, induce functional villi from cells that otherwise grow flat.

Lab dishes of different brands leach different chemicals into cell-culture media, and can confound studies of cell metabolites. Deliberate additives can change cell metabolism in unappreciated ways: antibiotics in particular frequently impair mitochondrial activity. Even a glass door on a lab refrigerator can ruin experiments, because some chemicals in growth media are sensitive to light. Just changing the laboratory plates, and thus the height of media in which cells are sitting, can alter how cells behave. What’s more, cells growing in a given culture are not identical, and the subset of cells that thrives the most can quickly dominate a population. That means cells may not revert back to former behaviour if a researcher decides to restore previous experimental conditions.”

https://www.nature.com/articles/537433a

Let’s look a little more in-depth on some of these contaminants:

PLASTIC WARE:

“Control of the cellular environment is a principal attribute of in vitro cell cultures. Unintentional exposure to environmental compounds can adversely affect cultures and, therefore, experimental results. Estrogenic compounds arising from common plastic ware have been found during cell culture.”

https://pubmed.ncbi.nlm.nih.gov/17316062/

“Plastic – who cares ?

One might think so initially, but cell culture plastic ware may make a difference in your cell culture especially in your assays. Depending on the manufacturer, the starting material does not differ (all cell culture flasks, dishes and multiwell plates to date are manufactured from polystyrene) but the surface modifications, the design, air flow, evaporation and other features may differ greatly.

The base material polystryrol without further treatment is not sufficient for cell adhesion in most cases. Therefore, all suppliers of cell culture plastic ware treat the surfaces of flasks, dishes and multiwell plates with ionized gas (corona-treatment) to produce surface modifications that make the surface more polar or charged. The mixture of plasma gases is secret and therefore, each supplier has a different surface modification.

The following surface modifications are used:

  • positive charges
  • negative charges
  • positive and negative charges mixed
  • polar groups
  • additional hydrophobic modifications (for e.g. suspension cells)

Depending on the quality and quality control of the plasma treatment, the surfaces may be activated for adhesion either homegeneously or artefacts may be introduced such as regions like edges are not treated or parts in the middle area or over or underactivated as shown in the images below.”

‘Another crucial point when choosing your cell culture consumable supplier is that the construction design of especially dishes and multiwell plates strongly influences the adhesion (general and areas), the distribution of cells and the evaporation from the plate or dish. In multiwell plates, this can prove problematic as the outer rows and columns usually give results totally different from the other wells and thereby increase the standard deviation of the results.

“Plastic may contain certain chemicals that are used in the process of production or convey certain attributes to the plastic (e.g. release aid or softeners). These may leak out or may be extracted from the plastic during the culture period. Then, cells or tissues may take up these compounds and eventually they are transplanted with the ATMP / TEP into the patient.”

https://incelligence.de/en/cell-culture/cell-culture-plastic-ware

To summarize the contamination problems with plastics:

  • Estrogenic (female hormone) compounds have been found in plastic ware used for culturing
  • Chemicals in the plastic can affect assays
  • The modifications done to each type of plastic ware can differ greatly
  • All plastic ware is treated with ionized gas
  • The mixtures used are secret and all suppliers have their own surface modifications
  • Artefacts may be introduced due to surface plasma treatment
  • Construction design of dishes and multiwell plates strongly influences the adhesion (general and areas), the distribution of cells and the evaporation from the plate or dish
  • Different results are usually obtained from the outer rows/columns of multiwell plates
  • The chemicals in plastic ware can leak out or be extracted during the cell culture and be taken up by the cells

WATER:

“Water is a ubiquitous solvent used throughout cell isolation and culture. Therefore, water contamination may affect experimental outcomes. The use of ultrapure water is advisable for culturing of cells sensitive to environmental contaminants, such as endotoxins and bacteria. Cardiomycyte isolation experiments showed that using water contaminated with bacteria and endotoxins decreases cell viability, while using freshly produced ultrapure water dramatically increases it.”

“Water is used in many steps of the tissue culture process, including buffer and media preparation as well as glassware washing. Thus, water quality may play an important role in experimental outcomes.”

“High resistivity purified water (18.2 MΩ.cm) with low organic content (total organic carbon ≤ 15 ppb) and no endotoxins should be used when performing tissue culture experiments. Storage of the water after purification should be avoided as the water quality may degrade and bacterial contamination may occur, which leads to the production of endotoxins. Endotoxins may cause obvious changes in cell growth and morphology, as was the case for cardiomyocytes. They also may have more subtle effects and only influence cell function. Unknowingly working with endotoxin-contaminated cultures may lead not only to inaccurate or erroneous results, but also to loss of time, money and effort.”

https://www.labmanager.com/white-papers-and-application-notes/endotoxin-free-water-for-tissue-culture-19637

Contaminated cultures and cell death are a major problem that can negatively impact on downstream experiments. Water is used in many steps of the cell culture process – it is the main component of buffers and media, and may be used for dissolution of additives and drugs. The use of purified water is essential for successful experimental outcomes, as cultures are adversely affected by contaminating microorganisms, biologically active cell debris and by-products, and organic and inorganic compounds.”

What Types Of Contaminants In Water Can Affect Cell Culture Results?

“The main types of impurity that affect the performance of cell culture techniques are bacteria, endotoxins, organic compounds and ionic contaminants. 

1. Bacteria 
Bacteria thrive in typical cell culturing conditions, and can quickly outgrow the cells of interest, causing nutrient levels to fall and toxic by-products to increase. Bacterial contamination can also lead to sudden changes in media pH and the contamination of previously pure cultures.

2. Endotoxins 
Endotoxins are released by most Gram-negative bacteria. These endotoxins affect various cell types, even those lacking CD14 endotoxin receptors, and stimulate macrophages and mononuclear phagocytes to release a variety of pro-inflammatory cytokines. The resulting adverse effects include changes in cell growth and function, the production of recombinant proteins and a reduction in the efficiency of cloning.

3. Organic Compounds 
Small organic compounds commonly found in water – such as humic acids, tannins, pesticides and endocrine disruptors – can affect cell development. They provide an uncontrolled source of nutrients for bacterial growth, and should be removed from water used for preparation of materials for cell culture. 

4. Ions 
Ionic contaminants, particularly multivalent ions and heavy metals, must be kept low. Heavy metals – for example, mercury and lead – are known to be cytotoxic to a range of cell types.”

https://www.elgalabwater.com/cell-tissue-cultures

To summarize the contamination issues related to water:

  • Water is the most used solvent in cell culture and is the main component of media and buffers
  • Water contamination can affect experimental outcomes and downstream applications
  • Using water contaminated with bacteria and endotoxins decreases cell viability
  • Storage of purified water should be avoided as it can degrade and become contaminated
  • Endotoxins in contaminated water may change cell growth and morphology or may influence cell functions
  • Unknowingly working with contaminated water leads to inaccurate and erroneous results
  • Bacteria, endotoxins, organic compounds, and ions are the main contaminants found in water

LIGHT:

“However, photo-sensitive molecules inside cells and in standard cell culture media generate toxic by-products that interfere with cellular functions and cell viability when exposed to light.”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5429800/

The sharp decline in growth-supporting capacity of DMEM exposed to fluorescent light has been attributed to two mechanisms: the photoactivation of riboflavin leading to tryptophan free radical production accompanied by peroxide formation (5, 6) and the formation of photoadducts of riboflavin and tryptophan (7). Our results show that cell yields in DMEM that had been exposed to light were 10% to 40% of those in non-exposed DMEM. The loss of growth capacity was dose-dependent, with greater declines observed with either higher light intensities or longer exposure periods.

Furthermore, we examined the effect of exposure to laboratory light on FBS. The data indicate that FBS as compared with DMEM is relatively stable to light exposure and partially stabilizes cell culture medium against the light-induced loss of growth capacity. Some of the protective effect of serum can be attributed to the catalase activity in serum (5), which could interrupt free radical-mediated reactions. Both light exposure and prolonged storage at room temperature resulted in declines in cell culture performance of FBS when performance was measured by cloning efficiency. In fact, the decline attributable to exposure to light for 28 days was nearly equal to the decline attributable to storage at room temperature (22°C) rather than at refrigerator temperature (4°C) for 28 days. Both conditions resulted in approximately 40% reductions in relative cloning efficiency.

As demonstrated here, the deleterious effects of laboratory lights on cell culture medium performance can be reduced by keeping medium in the dark or in protective yellow bags. Further precautions may include covering fluorescent lights in storage areas and cell culture hoods with yellow plastic films. These same procedures are recommended as ways to limit the harmful effects of light on serum where serum can be exposed for an extended time or to repeat short exposures.”

https://www.google.com/url?sa=t&source=web&rct=j&url=https://www.cytivalifesciences.co.kr/wp-content/uploads/2020/04/Effect-of-laboratory-lights-on-cell-culture-media.pdf&ved=2ahUKEwiRzZ_6h_nuAhUbXM0KHeXaAxcQFjADegQIGBAC&usg=AOvVaw1_F5uRUJD-XZUkGaArjU8J

Fluorescent Lights

“An important but often overlooked source of chemical contamination results from the exposure of media containing HEPES (N-[2-hydroxylethyl] piperazine-N’-[2-ethanesulfonic acid]) — an organic buffer commonly used to supplement bicarbonate-based buffers), riboflavin or tryptophan to normal fluorescent lighting. These media components can be photoactivated producing hydrogen peroxide and free radicals that are toxic to cells; the longer the exposure the greater the toxicity (4,5). Short term exposure of media to room or hood lighting when feeding cultures is usually not a significant problem; but leaving media on lab benches for extended periods, storing media in walk-in cold rooms with the lights on, or using refrigerators with glass doors where fluorescent light exposure is more extensive, will lead to a gradual deterioration in the quality of the media.”

https://www.google.com/url?sa=t&source=web&rct=j&url=https://safety.fsu.edu/safety_manual/supporting_docs/Understanding%2520and%2520Managing%2520Cell%2520Culture%2520Contamination.pdf&ved=2ahUKEwiVz7CHxPTuAhUKCM0KHXh0AD4QFjAAegQIARAC&usg=AOvVaw3M6StZvPbYyw30IqM9YhAY

To summarize the contamination effects of light:

  • Photo-sensitive molecules create toxic by-products interfere with cellular functions and cell viability when exposed to light
  • Media components can be photoactivated producing hydrogen peroxide and free radicals that are toxic to cells; the longer the exposure the greater the toxicity
  • DMEM has a sharp decline in growth-promoting capacity when exposed to light
  • The loss of growth capacity was dose-dependent, with greater declines observed with either higher light intensities or longer exposure periods
  • Both light exposure and prolonged storage at room temperature resulted in declines in cell culture performance of Fetal Bovine Serum

INCUBATORS:

“The cell culture incubator is designed to artificially replicate in vitro the conditions essential to in vivo physiology typical of human and animal models. Cell growth outside of a natural environment presents a myriad of challenges associated with exposure to microorganisms that are not present in the in vivo state.”

“Contamination of a cell culture in vitro is usually caused by the inadvertent introduction of one or more organisms that can damage or destroy the cell culture in progress. These organisms include:

• Bacteria (including Thermophilic Bacteria) and Mycoplasma
• Molds and Yeasts
• Viruses

Other contaminants include dust, VOCs from adjacent instrumentation or processes, cross contaminants from other cultures in a shared incubator environment and particulates found in the natural environment.”

“When the incubator door is opened, however, the conditioned bubble is lost. Accessing cell culture labware for transport to a biological safety cabinet (BSC) or other processes is a normal part of laboratory workflow. Opening the door exposes the incubator interior walls, shelves, humidity pan water and culture vessels to ambient conditions that carry the potential for contamination from molds, yeasts, fungi or other microorganisms such as mycoplasma and viruses. In a practical sense, unless the incubator is installed in a clean room, this exposure cannot be avoided.”

https://www.google.com/url?sa=t&source=web&rct=j&url=https://www.labrepco.com/wp-content/uploads/2018/11/Mitigating-Contamination-in-the-Cell-Culture-Incubator.pdf&ved=2ahUKEwjE2a_bot_yAhWzGVkFHeHiBhQ4ChAWegQIGxAB&usg=AOvVaw3NItipb7KMrEtsbWWgdcYH

Reducing contamination

“Cleanliness is critical for preventing contamination in cell cultures. Dust and dirt can be carried by air currents created by movement in the lab. Normal indoor room air contains 100–1000 microorganisms per cubic meter,1 all circulating at any given moment, and most of which come from the trillions of normal flora that live in and on the skin. This means that contaminants can enter each time the incubator door is opened. The lab should be cleaned at least once a month, including cleaning and disinfecting the biological safety cabinet, water bath, centrifuge, microscope and all corners of the lab and around equipment. Cardboard storage in or around refrigerators and freezers should be eliminated as cardboard can get wet and breed fungi. Items should not be stored on top of the incubator because dust and dirt could be swept inside the chamber via air currents created during door opening.

Incubator disinfectants

While many disinfectants are available, not all are safe for cells. Some strong disinfectants emit fumes that enter the incubator and affect cell growth. These fumes contain volatile organic chemicals (VOCs) that can induce expression of heat shock and other stress proteins. Common laboratory chemicals such as phenol, isoamyl alcohol and beta-mercaptoethanol are VOCs, but laboratory cleaning products and disinfectants, and even floor cleaners and waxes, produce harmful vapors.”

https://www.americanlaboratory.com/914-Application-Notes/189342-Best-Practices-for-CO2-Incubator-Maintenance/

The incubator, often considered a major source of biological contamination, can also be a source of chemical contamination. The gas mixtures (usually containing carbon dioxide to help regulate media pH) perfused through some incubators may contain toxic impurities, especially oils or other gases such as carbon monoxide, that may have been previously used in the same storage cylinder or tank. This problem is very rare in medical grade gases, but more common in the less expensive industrial grade gas mixtures. Care must also be taken when installing new cylinders to make sure the correct gas cylinder is used. Other potential chemical contaminants are the toxic, volatile residues left behind after cleaning and disinfecting incubators. Disinfectant odors should not be detectable in a freshly cleaned incubator when it is placed back into use.

Keep in mind that chemical contaminants tend to be additive in cell culture; small amounts contributed from several different sources that are individually nontoxic, when combined together in medium, may end up overloading the detoxification capabilities of the cell culture resulting in toxicity-induced stress effects or even culture loss.”

https://www.google.com/url?sa=t&source=web&rct=j&url=https://safety.fsu.edu/safety_manual/supporting_docs/Understanding%2520and%2520Managing%2520Cell%2520Culture%2520Contamination.pdf&ved=2ahUKEwiVz7CHxPTuAhUKCM0KHXh0AD4QFjAAegQIARAC&usg=AOvVaw3M6StZvPbYyw30IqM9YhAY

To summarize the contamination issues related to incubators:

  • The incubator is considered a major source of biological contamination but is also a source of chemical contamination
  • Potential contaminates include dust, VOCs from adjacent instrumentation or processes, cross contaminants from other cultures in a shared incubator environment and particulates found in the natural environment
  • Opening the door exposes the incubator interior walls, shelves, humidity pan water and culture vessels to ambient conditions that carry the potential for contamination from molds, yeasts, fungi or other microorganisms such as mycoplasma and “viruses”
  • Exposure to contaminants from opening the incubator door can not be avoided
  • Dust and dirt carried by air currents created by movement in the lab is a contamination concern
  • Strong incubator disinfectants emit fumes that enter the incubator and affect cell growth
  • Fumes containing volatile organic chemicals (VOCs) can induce expression of heat shock and other stress proteins
  • Common laboratory chemicals such as phenol, isoamyl alcohol and beta-mercaptoethanol are VOCs, but laboratory cleaning products and disinfectants, and even floor cleaners and waxes, produce harmful vapors
  • The gas mixtures (usually containing carbon dioxide to help regulate media pH) perfused through some incubators may contain toxic impurities, especially oils or other gases such as carbon monoxide

Plastic, water, and light are all environmental factors which can impact cell cultures in negative ways. Even something as simple as opening the incubator door or just the act of routine cleaning can be sources of contamination and have direct impacts on the results of the cell culture experiments. This is just the tip of the iceberg as we haven’t even touched on how room temperature, atmospheric conditions, and pH levels affect cells. There are so many sources of contamination with cell cultures as well as many variables which effect the cell growth, performance, genomic stability, viability, etc. that it seems one would need an impossible 100% completely sterile environment in order to be able to trust any end product derived from the culture. Contamination isn’t the exception in cell culture experiments, it is the rule.

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