While I have already done a post breaking down some of the sources of contamination in cell cultures, I wanted to present even more information on this subject as it is crucial to hammering home how invalid this method truly is. The process itself is the very opposite of purification (free of contaminants) and isolation (separated from everything else), two essential components needed to be satisfied in order to show that the particles assumed to be “viruses” actually exist and come from sick humans. The problem for cell culture, however, is that contamination is a serious and unavoidable problem that can only be mitigated rather than eliminated. This is due to the numerous sources of contamination such as from the equipment, water, and reagents used for the culture as well as any bacteria, fungi, “viruses,” amoebas, parasites, etc. which could be present in the sample itself or even the media used. As the problem of contamination is pervasive in cell cultures, this is reason enough to question the evidence for “SARS-COV-2” and any other “virus” said to be “isolated” from this toxic cell culture process.
Highlights from a guide on Cell Culture Contamination provides an excellent breakdown of the seriousness of the problem. I have pointed out the major sources of contamination from the article but I did edit out some environmental sources which I will go into more in-depth in a separate post. This one source is such a plethora of information and for editing purposes (it’s 24 pages in length), I had to leave out quite a bit, but hopefully this condensed version will provide a good overview of the seriousness and inevitable problem of contamination in cell cultures:
Understanding and Managing
Cell Culture Contamination
“No cell culture problem is as universal as that of culture loss due to contamination. All cell culture laboratories and cell culture workers have experienced it. Culture contaminants may be biological or chemical, seen or unseen, destructive or seemingly benign, But in all cases they adversely affect both the use of your cell cultures and the quality of your research. Contamination problems can be divided into three classes:
- MINOR ANNOYANCES – when up to several plates or flasks are occasionally lost to contamination;
- SERIOUS PROBLEMS – when contamination frequency increases or entire experiments or cell cultures are lost;
- MAJOR CATASTROPHES – contaminants (usually other cell lines or mycoplasma) are discovered that call into doubt the validity of your past or current work.
The most obvious consequence of cell culture contamination is the loss of your time, money (for cells, culture vessels, media, and sera), and effort spent developing cultures and setting up experiments. However, the less obvious consequences are often more serious (Table 1). First there are the adverse effects on cultures suffering from undetected chemical or biological contaminants. These hidden
(cryptic) contaminants can achieve high densities altering the growth and characteristics of the cultures. Worse yet are the potentially inaccurate or erroneous results obtained by unknowingly working with these cryptically contaminated cultures. Products, such as vaccines, drugs or monoclonal antibodies, manufactured by these cultures will probably be useless. For some researchers the most serious consequence of contamination is suffering the embarrassment and damage to their reputation that results when they notify collaborators or journals that their experimental results are faulty and must be retracted due to contaminants in their cultures.
Preventing all cell culture contamination has long been the dream of many researchers, but it is an impractical, if not impossible, dream. Contamination cannot be totally eliminated, but it can be managed to reduce both its frequency of occurrence and the seriousness of its consequences.”
“A cell culture contaminant can be defined as some element in the culture system that is undesirable because of its possible adverse effects on either the system or its use. These elements can be divided into two main categories: chemical contaminants and biological contaminants.
Chemical contamination is best described as the presence of any nonliving substance that results in undesirable effects on the culture system. To define further is difficult; even essential nutrients become toxic at high enough concentrations. Nor is toxicity the only concern since hormones and other growth factors found in serum can cause changes that, while not necessarily harmful to cultures, may be unwanted by researchers using the system. (Reviewed in references 1-3.)
The majority of chemical contaminants are found in cell culture media and come either from the reagents and water used to make them, or the additives, such as sera, used to supplement them.”
“Sera used in media have long been a
source of both biological and chemical contaminants. Due to cell culture-based screening programs currently used by good sera manufacturers, it is unusual to find a lot of fetal bovine sera that is toxic to a majority of cell cultures. However, it is common to find substantial variations in the growth promoting abilities of different lots of sera for particular cell culture systems, especially for cultures that have specialized or differentiated characteristics. Uncontrollable lot-to-lot variation in hormone and growth factor concentrations makes this problem inevitable; careful testing of sera before purchase, or switching to serum-free media can avoid these problems.”
“The water used for making media and
washing glassware is a frequent source of chemical contamination and requires special care to ensure its quality.”
“Because of its aggressive solvent characteristics, highly purified water can leach potentially toxic metal ions from glassware or metal pipes, and plasticizers from plastic storage vessels or tubing. These contaminants can then end up in media or deposited on storage vessels and pipettes during washing and rinsing.”
“Endotoxins, the lipopolysaccaride-containing by-products of gram negative bacteria, are another source of chemical contaminants in cell culture systems. Endotoxins are commonly found in water, sera and some culture additives (especially those manufactured using microbial fermentation)“
“These highly biologically reactive molecules have major influences in vivo on humoral and cellular systems. Studies of endotoxins using in vitro systems have shown that they may affect the growth or performance of cultures and are a significant source of experimental variability.”
“Biological contaminants can be subdivided into two groups based on the difficulty of detecting them in cultures:
◗ those that are usually easy to detect —
bacteria, molds and yeast;
◗ those that are more difficult to detect,
and as a result potentially more serious
culture problems, — viruses, protozoa,
insects, mycoplasmas and other cell lines.”
Bacteria, Molds, and Yeasts
“Bacteria, molds and yeasts are found virtually everywhere and are able to quickly colonize and flourish in the rich and relatively undefended environment provided by cell cultures. Because of their size and fast growth rates, these microbes are the most commonly encountered cell culture contaminants.”
“However, when antibiotics are routinely used in culture, resistant organisms may develop into slow growing, low level infections that are very difficult to detect by direct visual observation. Similar detection problems can occur with naturally slow growing organisms or very small or intracellular bacteria that are difficult to see during routine microscopic culture observation. These cryptic contaminants may persist indefinitely in cultures causing subtle but significant alterations in their behavior. By the time these cryptic contaminants are discovered, many experiments and cultures may have been compromised.”
“Due to their extremely small size, viruses are the most difficult cell culture contaminants to detect in culture, requiring methods that are impractical for most research laboratories. Their small size also makes them very difficult to remove from media, sera, and other solutions of biological origin. However, most viruses have stringent requirements for their original host species’ cellular machinery (may also be tissue specific) which greatly limits their ability to infect cell cultures from other species. Thus, although viruses may be more common in cell cultures than many researchers realize, they are usually not a serious problem unless they have cytopathic or other adverse effects on the cultures. (Reviewed in Ref. 7, 40.) Since cytopathic viruses usually destroy the cultures they infect, they tend to be self-limiting. Thus, when cultures self-destruct for no apparent reason and no evidence of common biological contaminants can be found, cryptic viruses are often blamed. (See Figures 3a and 3b.) They are perfect culprits, unseen and undetectable; guilty without direct evidence. This is unfortunate, since the real cause of this culture destruction may be something else, possibly mycoplasma or a chemical contaminant, and as a result will go undetected to become a more serious problem.”
“Both parasitic and free-living, single-celled protozoa, such as amoebas, have occasionally been identified as cell culture contaminants. Usually of soil origin, amoebas can form spores and are readily isolated from the air, occasionally from tissues, as well as throat and nose swabs of laboratory personnel. They can cause cytopathic effects resembling viral damage and completely destroy a culture within ten days. Because of their slow growth and morphological similarities to cultured cells, amoebas are somewhat difficult to detect in culture, unless already suspected as contaminants (7).”
“Insects and arachnids commonly found in laboratory areas, especially flies, ants, cockroaches and mites, can both be culture contaminants as well as important sources of microbial contamination. Warm rooms are common sites of infestation.”
“Unfortunately, mycoplasmas are not relatively benign culture contaminants but have the ability to alter their host culture’s cell function, growth, metabolism, morphology, attachment, membranes, virus propagation and yield, interferon induction and yield, cause chromosomal aberrations and damage, and cytopathic effects including plaque formation (12). Thus, the validity of any research done using these unknowingly infected cultures is questionable at best.”
“Their small size and lack of a cell wall allow mycoplasmas to grow to very high densities in cell culture (107 to 109 colony forming units/mL are common) often without any visible signs of contamination — no turbidity, pH changes or even cytopathic effects. (See Figures 4a and 4b.) Even careful microscopic observation of live cell cultures cannot detect their presence. These same two characteristics also make mycoplasmas, like viruses, very difficult to completely remove from sera by membrane filtration. In addition, their fastidious growth requirements (unfortunately, easily provided for by cell cultures) make them very difficult to grow and detect using standard microbiological cultivation methods.”
“Unfortunately, even with the advances in detection methods (discussed in detail later) mycoplasma infection rates (Table 3) have not changed noticeably since they were first detected in cell cultures.”
Cross-Contamination by Other Cell Cultures
“The seriousness of cross-contamination, while not as common as microbial contamination, cannot be overstated. The validity of experimental results from cultures having inter- or intraspecies contamination is, at the very least, questionable. Furthermore, their use can lead to the embarrassment of having to retract published results. Whenever the invading cell is better adapted to the culture conditions and thus faster growing than the original cells, it will almost always completely replace them. Because of the outward physical similarities of different cell lines and the wide morphological variations that can be caused by the culture environment, it is impossible to rely only on microscopic observation to screen for cross-contamination of cultures. Simple accidents are one of the most common means by which other cell lines gain entry into cultures and will be discussed separately in the next section.
Remember, the seriousness of any culture contaminant is usually directly proportional to the difficulty of detecting it; those that go undetected the longest have the most serious consequences. Cultures containing nonlethal (but not harmless), cryptic chemical or biological contaminants are sometimes used in research for months or even years before being uncovered; during this time the quality and validity of all research done with those cultures is compromised, as is the reputation of the researchers using them.”
Strategic Use of Antibiotics
“When used intelligently, antibiotics are a useful tool in cell culture, but they can be very dangerous when overused or used incorrectly. Experienced cell culture users have recommended for many years that antibiotics never be used routinely in culture media (3,7,12,17,18,26,27). In a major study, Barile found that 72% of cultures grown continuously in antibiotics were contaminated by mycoplasma, but only 7% grown without antibiotics were contaminated, a ten-fold difference (37). Similar results are common: workers who routinely and continuously use antibiotics in their media tend to have higher contamination problems, including mycoplasma, than workers who don’t.”
A Final Warning
“In the United States alone, losses due to
cell culture contamination, especially by mycoplasma, cost cell culture users millions of dollars annually; this is money that could otherwise be used for additional research. Unfortunately this serious problem does not appear to be getting any better. As shown by the survey results in Table 7 (page 19), contamination is a problem for most cell culture workers. At least 23% of respondents have experienced mycoplasma contamination of their cultures, but an additional 44% suspected mycoplasma contamination but were not sure. The reason for their uncertainty is clarified by the response to the next question: 50% of all respondents do not test for mycoplasma, as a result they are unaware of the status of their cultures. The answer to the last question points out one important reason for widespread contamination problems – the over use of antibiotics. With 65% of respondents using antibiotics on a regular basis, the continued frequent occurrence of cryptic contaminants, especially mycoplasmas, is likely.“
“Cell culture contamination will never be totally eliminated, but through a better understanding of the nature of contamination and the implementation of some basic concepts it can be better controlled and its damage greatly reduced.”
In Summary, contamination comes in the form of:
- Chemical contaminants
- Biological contaminants
- Cross-contamination from other cells
- Environmental contaminants
As previously stated, contamination in cell culture is not an “if” but a “how much” problem. It is unavoidable and the effects on the cell culture leads to erroneous and irreproducible results. There are far too many sources and far too many variables to be contained. At best, Virologists attempt to mitigate the issue. However, the cell culture itself is one gigantic pool of contamination so the issue will remain as the foundation of the process itself is built upon it.
I highly recommend taking the time to read this manual as there is far too much information to copy/paste here. It is a great resource if you want to understand the many faults associated with cell cultures and why any “virus” said to be “isolated” from one should raise an immediate red flag.