“Barbara McClintock might be surprised to learn how well recent discoveries support her hypotheses. Her experiments of 60 years ago led her to propose that cells under environmental stress activate transposable elements in order to restructure the cell genome (McClintock, 1984).“
It is well known that cells under stress change and adapt due to the environmental and physical stresses placed upon them. This was discovered by Barbara McClintock in the 1960’s who won a Nobel Prize for her work. The changes that occur from stressors alter the genetic expression of the cells as they adapt to survive. The toxic antibiotics, the foreign human and animal DNA, the chemicals and “nutrients” added, etc. can all stress the cell yet there is another more physical stressor that can cause these changes on top of all of that. This is a process called Sub-Culturing, also known as Passaging:
“Subculturing, also referred to as passaging, is the removal of the medium and transfer of cells from a previous culture into fresh growth medium, a procedure that enables the further propagation of the cell line or cell strain.
The growth of cells in culture proceeds from the lag phase following seeding to the log phase, where the cells proliferate exponentially. When the cells in adherent cultures occupy all the available substrate and have no room left for expansion, or when the cells in suspension cultures exceed the capacity of the medium to support further growth, cell proliferation is greatly reduced or ceases entirely (see Figure 4.1 below). To keep them at an optimal density for continued growth and to stimulate further proliferation, the culture has to be divided and fresh medium supplied.“
Cell cultures are removed and checked on at regular intervals when looking for cytopathic effects that are supposed to indicate a “virus” is present in the sample. Once the cells grow and expand too much and/or the media needs replacing, the culture is divided and new cell-altering media/chemicals are added. This continues until they get the desired CPE that they want.
Take, for example, this paper from Korea on the “isolation” of “SARS-COV-2” which is considered one of the original papers used as evidence for its existence:
Virus Isolation from the First Patient with SARS-CoV-2 in Korea
“The patient’s oropharyngeal samples were obtained by using UTM™ kit containing 1 mL of viral transport media (Copan Diagnostics Inc., Murrieta, CA, USA) on day 7 of her illness. We inoculated monolayers of Vero cells (ATCC ® CCL-81™) with the samples and cultured the cells at 37°C in a 5% carbon dioxide atmosphere. Until 5 days after inoculation, cytopathic effects were not distinct, which is compatible with the previous findings that no specific cytopathic effects were observed in the Vero E6 cells until 6 days after inoculation in the report about first isolation of SARS-CoV-2.3 Five days after inoculation, we did blind passage of culture supernatant into T-25 culture flask (ThermoFisher Scientific Inc., Waltham, MA, USA) with monolayers of Vero cells, and cytopathic effects consisting of rounding and detachment of cells were observed in the whole area of the T-25 flask 3 days after the first blind passage (Fig. 1A and B).”
“Next-generation sequencing of BetaCoV/Korea/SNU01/2020 (GenBank accession no. MT039890) revealed 9 mutations compared to the NC_045512 reference genome isolated from Wuhan (Table 1). Most of the mutations in our isolate consisted of 70% alternative genes and 30% reference genes (NC_045512). Five variants were found in ORF1ab, one variant in S gene, two variants in ORF3a, and one variant in E gene. Of the nine mutations, six also showed changes in amino acids. When comparing our isolate with the one isolated from Korea Centers for Disease Control and Prevention (BetaCoV/Korea/KCDC03/2020), 12 variants including the above 9 mutations were found. These mutations may occur by cell culture-adaptation in that our culture isolates was obtained after first blind passage, or by micro-evolution of SARS-CoV-2 before acquisition in Wuhan. Because those genome sequences are quite homologous each other, it is difficult to validate these two hypothesis.”
From this study, they cultured the cells for 5 days and did not notice the CPE they were looking for. They then did a blind passage of the cell culture and 3 days later, finally noticed the CPE that they wanted to see. Upon sequencing their “isolate,” they noticed several mutations that differed from the original genome from China as well as variations from another genome sequenced in Korea. They could not say if these mutations/variations were due to a “micro-evolution” of the “virus” or if the cell culture went through adaptations during the blind passage. This information would seemingly be pretty important to know.
The alterations to cell lines from sub-culturing is not an unknown issue. In fact, they are supposed to keep track of the amount of times they passage the cells due to the expected changes that will occur. The problem is that, as with much regarding cell cultures, there is NO STANDARD for determining how many times a culture should be passaged or how many passages is too many:
Passage Number Effects in Cell Lines
“The ability of continuous cell lines to exist almost indefinitely in vitro has opened the possibility of questionable subculturing practices and hence, questionable scientific data. The degree of subculturing a cell line has undergone is often expressed as “passage number,” which can generally be thought of as the number of times cells have been transferred from vessel-to-vessel. A growing body of literature demonstrates passage number affects a cell line’s characteristics over time.1-6 Cell lines at high passage numbers experience alterations in morphology, response to stimuli, growth rates, protein expression, and transfection efficiency, compared to lower passage cells.
The scientific community is taking notice that cell line quality is crucial to successful experimentation and that avoiding the use of cell lines that have been in culture too long is an important step to ensure reliable and reproducible results. But while the evidence for passage number-related effects on cell lines is compelling, much less is understood about the mechanisms underlying passage dependent changes and about actions researchers can take to avoid passage number effects in their experiments.
By beginning with the concept that cells in culture are under environmental and manipulative stress, both the “why” and “how” genotypic and phenotypic changes that occur become more apparent, including the mechanisms that underlie passage effects. Cells in culture are continually subjected to the evolutionary processes of competition and natural selection.
Most cell cultures represent heterogeneous populations that compete for resources such as growth factors, salts, and nucleic acids. When given an advantage, such as a faster growth rate, one cell type may overgrow another within a single population. Such competition gives rise to extant populations that no longer correctly represent the original starting material. Events such as dedifferentiation and loss of tissue-specific function should be considered the norm as passage numbers increase.
Transformed and diseased cell lines are of special concern, since they represent abnormal starting populations in which evolutionary changes occur rapidly at both the genotypic and phenotypic levels over time. In these cell types one or all of the typical cellular checkpoint genes, such as p16/INK4a, pRB and p53, have been altered whereby the cells have become “immortal.” These alterations are often in parallel with other cellular mutations, and the continual subculture of these cell lines exacerbates genomic instability.
How many passages are too many?
A straightforward method for determining the passage number of a cell line does not exist. A review of the literature on passage-related effects in cell lines demonstrates that the effects are complex and heavily dependent on a host of factors such as the type of cell line, the tissue and species of origin, the culture conditions and the application for which the cells are used.”
“Morphology can vary between lines depending on the health of the cells and, in some cases, the differentiation state. Morphology can change with plating density as well as with different media and sera combinations.”
This is from a study that looked at the effects of passaging on gene expression. After 7-8 passages, more than 10% of the genes were differentially expressed:
“From passages 2-4, mRNA expression did not change significantly. Gene expression in RASF started to change in passages 5-6 with 7-10% differentially expressed genes. After passages 7-8, more than 10% of the genes were differentially expressed. The doubling rate was constant for up to 5 passages and decreased after passages 6-8.”
As can be seen, passaging the cells is a problem that has profound effects on the culture. To hammer this point home, here is some further information on the problems associated with passaging cells:
What’s in a Number: Getting the Right Passage in Cell Culture
“This subculture is also known as a “passage.” A passage number is the number of times a cell culture has been subcultured, and knowing the passage number can make or break an experiment.
All cell cultures start somewhere; this “somewhere” is the reference strain, or reference culture. These are fresh cells that come from a reliable source, like the ATCC. While many labs may passage cells dozens, even hundreds of times, this many passages probably results in cells that have little in common with the original reference strain. These “working cultures,” if passaged enough times, can show evidence of genetic drift—changes in genotype from the original reference strain which may or may not result in observable changes in phenotype. Other genotype changes may not show any phenotypic variation immediately, but could result in changes after further subculturing. In addition, genetic changes caused by subculturing could create epigenetic changes that could affect how genes are regulated. More passages also increase the risk of contamination. Not good.
A passage too far?
One study showed that high- and low-passage adenocarcinoma cells had different responses to androgens and retinoids, indicating alterations in gene expression. Researchers in Belgium compared how two strains of LNCaP prostate cancer cells responded to androgens and retinoids, depending on passage numbers. The cells with high passage numbers showed higher-amplitude response curves to 3H-thymidine (measuring cell proliferation), while cells with low passage numbers showed greater growth inhibition by the synthetic androgen R1881, greater PSA mRNA expression and PAP expression (prostatic acid phosphatase). For responses to retinoic acid (atRA), lower-passage cells showed a marked stimulation of 3H-thymidine incorporation in the cells, while lower passage cells only showed growth inhibition. Clearly, passage number affected cellular physiology, which in this case caused the researchers to caution about the use of prostate drugs containing these molecules!
What’s your passage number?
Good cell practice calls for starting any experiment with low-passage cell culture, and limit the number of passages you’ll accept in your experiment. But what is a good passage number (besides “zero,” that is)? The numbers have differed over the years. Some standards recommend three stock subcultures and three “working culture” subcultures—those add up to seven passages, including the original passage from the reference. Meanwhile, some cell culture producers charge more for cultures of two passages or less. However, the ATCC warns researchers to assume that a cell culture from a commercial source may be already one or two passages away from the reference strain. Generally, the ATCC recommends that cell culture should be limited to five passages, at least for use in medical and biopharmaceutical applications.”
What’s in a Number: Getting the Right Passage in Cell Culture
- In the 1960’s, Barbara McClintock discovered that cells under environmental stress activate transposable elements to restructure the cell genome
- One of the original “SARS-COV-2” papers from Korea admitted that the mutations/variations in their genome may have been due to cell culture-adaptation from the first blind passage
- Passage numbers affect a cell lines characteristics over time
- Cell lines at high passage numbers experience alterations in morphology, response to stimuli, growth rates, protein expression, and transfection efficiency, compared to lower passage cells
- Avoiding cell lines that have been cultured/passaged too long is crucial to obtaining reliable and reproducible results
- Not much is understood about the mechanisms underlying passage dependent changes and about actions researchers can take to avoid passage number effects in their experiments
- Cells in culture are under environmental and manipulative stress which leads to genotypic/phenotypic changes
- Cells compete for resources such as growth factors, salts, and nucleic acids
- Competition gives rise to extant populations that no longer correctly represent the original starting material.
- Dedifferentiation and loss of tissue-specific function should be considered the norm as passage numbers increase
- Continual subculture of transformed or diseased cell lines exacerbates genomic instability
- A straightforward method for determining the passage number of a cell line does not exist
- Passage-related effects are dependent on many factors such as the type of cell line, the tissue and species of origin, the culture conditions and the application for which the cells are used
- Cell morphology can change with plating density as well as with different media and sera combinations
- Passaged cells have little in common with the original reference strain
- Passaged cells show evidence of genetic drift
- Genetic changes caused by subculturing could create epigenetic changes that could affect how genes are regulated
- More passages also increase the risk of contamination
- The passage number clearly affects cellular physiology
- What is considered a “good” passage number has changed throughout the years
- The ATCC warns researchers to assume that a cell culture from a commercial source may be already one or two passages away from the reference strain
It’s clear that sub-culturing cells before and during cell culture experiments can alter the cell. The stress from the change in environment and the added media can and will change gene expression, alter morphology, effect growth rate, hinder stimulus response, change protein expression, increase contamination, etc. These cell culture adaptations are attributed to “natural” mutations and variations and are accepted as new variants of the same “virus” even though there are numerous other explanations for why these changes occur and why they can never get the same exact sequence twice.
omg, I am only at the beginning of this post and “Once the cells grow and expand too much and/or the media needs replacing, the culture is divided and new cell-altering media/chemicals are added. This continues until they get the desired CPE that they want.” !!! wow, amazing give away, but they think it’s fine.
It’s just so weird. I was thinking yesterday, why is it that they call the CPE “virus particles”. I mean, why would the product of death (cell debris or fragments) be suspected as a virus, EVEN IF you theorized that viruses were killing the cells??? Does my question make sense? They think they are taking virus isolate (how they think this is beyond me….) and put it onto tissue. Okay. So they think the virus is going to kill the tissue. Yes? But why would the result be a virus? Then I remembered or thought about it: Oh, they are looking for GENES not a THING. So they find genetic fragments and through the magical powers of intense denial they assume that those fragments of genetics are NOT from the thing that died (the tissue, the cells…). Okay, denial gets 1000 points. But why would the virologist even expect the viruses to “come out of the killing fields” … oh, wait, they are IN the cells at all times, right? I mean, the virus can’t exist without a cell? Argh…. but then this is cellular debris we are talking about. I am sorry for rambling, I will post this anyway, just to show my confusion.
They only engage in circular reasoning and assumptions. That’s why it hurts your head. They claim a “virus” needs a host cell to survive. If that were true, then what does a “virus” look like before it invades a body to find the host cell it needs in order to replicate? It must exist in some form. They can not show this ever. Yet I have seen they claim that they are basically just A,C,T,G’s waiting to take a form. It’s ridiculous.
wow…. That is what I mean…. what does this thing look like before and after invading or leaving a host cell? Interesting that they have said “they” (these imaginary viruses) are just “A,C,T,Gs” waiting to take a form. So this imaginary “thing” isn’t a thing, but genetic components without form yet? And yet, when it invades a cell it becomes a thing? And replicates? But it is just genes, not living…. it’s a mindless program of genes reproducing? And then when it leaves a cell it is parts of a “thing”? It breaks up? Into genetic fragments? And this is why they think they are doing real science because they are (in their minds) re-assembling a “thing”. But it is only a thing while IN the cell? See, the goal seems to be to avoid admitting that everything is intelligent. Everything means ALL OF IT. The components, the harmony of the whole… the parts and the whole are all intelligent and NOT just mechanical parts. I think what this all is is a distortion of truth. Meaning there IS a coming in and going out of form (that is what pleomorphism is, right?) but the way Scientism interprets this action or process is through the lens of materialism, and the war metaphor, and all the conceptual baggage associated with that way of interpreting reality. It’s all just mindless matter (MATTER having NOT come from MIND, this is the key…they say that MIND came from MATTER, ie big bang) and it’s all a struggle for survival (Darwin), and it’s all competitive and never (essentially anyway) cooperative in nature, and it’s all random and meaningless (Big Bang). Mutation just happens. Instead of something like, “all matter is actually action, always changing, and never “solid” or “static” like we may think… everything is conscious and alive and interconnected and communicating with all other aspects of the whole, each cell and even atom and molecule is aware of its own value, it’s own nature and has innate intelligence of the whole” — none of that kind of woo woo language allowed!!!! But that is exactly the language that is more accurate. They are denying reality, not us. Divorcing yourself from the idea or concept or belief that all of nature and reality is innately intelligent is NOT being objective and smart and facing reality. It is a denial of what is apparent, experienced, and logical. Our egoic reasoning mind has been hijacked and told to only be “rational” in a certain narrow way. So, when you go outside of those bounds, you are labeled irrational, unscientific etc. But they are so “rational” that they are exclude other ways of knowing reality, it’s a very narrow view. It’s also a depressing view. They want us thinking nature/reality and thus OUR LIVES is NOT good, benevolent, giving, and connected with a non-physical source of intelligence IN ANY WAY. It’s all about suppressing our true selves, our true nature and we live within concepts like germ theory that will try to explain reality in an artificial, surface, essentially retarded fashion. It’s just stupid. That’s why their models end up being appallingly stupid when examined. They aren’t internally consistent and since it is a cult that doesn’t matter. They are conceptually all backasswards and seeing reality through a false and limited way. Well then, we end up with weird ideas that make no sense. They are like drug addicts who will keep making shit up, covering their asses, lying, cheating, doing what they need to do to uphold the concepts that they feed upon. Something like that. Sorry this got so long…
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Wonderfully said! You are spot on that they have been pigeonholed into a narrow view/belief system. They have become trapped and encaged in an illogical illusion. They try to cram experimental results into their preconceived notions and assumptions of the fictional narratives they have created and they also rewrite/rework their fiction to fit these results. Just like “Giruses.” 😉
They never realize that what they have created in vitro has no relation whatsoever to what’s going on in vivo.
It’s no more circular than calling the effects of toxins “cytotoxic effects”…or illness of the mind “mental illness” or defects present at birth “birth defects”….that’s literally how language works.
There are tons of pictures of viruses outside cells. They’re called virions. Extracellular virions are most definitely not ATCG’s waiting to take a form (that would be a nucleic acid fragment.)
Completely separated from cells prior to microscopic analysis:https://www.researchgate.net/figure/Negative-stain-electron-micrograph-of-purified-virions-The-70-nm-particles-exhibit_fig1_318879662https://www.researchgate.net/figure/Electron-microscopy-of-purified-Sindbis-virus-Micrograph-of-nega_fig1_235415227
Next to cell (some inside some outside)https://www.researchgate.net/figure/Electron-microscopy-of-ebolavirus-infected-cells-on-day-4-post-infection-A-Large-viral_fig1_43159494
Sadly the links do not seem to work but it doesn’t really matter. Pictures of particles claimed to be “viruses,” especially from cell culture supernatant, is not proof of “viruses.” All these images show are various particles in a fixed state. Whether or not these particles are “viruses” has never been proven as they are never purified/isolated directly from a human nor have they ever been proven pathogenic in a natural way. The cell culture process is the exact opposite of purification/isolation.
YUP I have always questioned, then how did the virus come about? How did it originate? The idea is from some other host interacting with your host — but what happens in the middle?
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The more we question the dogma, the more illogical it becomes and the easier it is to see the lie. Keep questioning. 👍
CPE are not called virus particles. CPE stands for cytopathic effect. Cyto means cell and pathic is like abnormality, disease. This is an umbrella term to refer to specific observations of the cells (such as cell rounding and syncytium formation) when in a diseased state and is distinguished from cytotoxic effects, senescence, and other observations unrelated to a disease state. You can have (for instance) syncytium formation as a normal finding in certain cells, but it is pathologic in other types of cells.
Not all viruses cause cytopathic effects, and attempting to culture a virus (even a cytopathic one) in non-permissive cells will not result in a cytopathic effect. For instance Respiratory Syncytial Virus causes syncytium formation in human respiratory cells (not just cell culture but also biopsies from actual patients, hence the name) but does absolutely nothing to like, kangaroo liver cells.
I never claimed CPE was “virus” particles. CPE is what they look for to claim a “virus” is within the cell culture. There are numerous factors which cause this effect that have nothing to do with “viruses.” And as you have stated, they claim there are times where “viruses” do not cause CPE. If CPE is the effect they look for to claim a cell culture was successfu (i.e. replication of “virus”)l, they can not claim that there are “viruses” which do not cause this effect. They can’t have it both ways.
My cytopathic effect comment was directed at user forcgd, they asked “why is it that they call the CPE “virus particles””
Yeah, I saw that after I commented. Unfortunately, the notification only showed up as a comment on my post but not who it was directed to so I thought it was directed to me. Thanks for clarifying!
Sam, from what I’ve read, the monkey kidney cells are the most susceptible to producing the CPE when exposed to the SARS-COV-2 virus. Shouldn’t this mean that monkeys, which don’t wear masks and haven’t been vaccinated, should be dying from kidney failure?
So this article is incredibly important in regards to the 2015 Shi Baric study claiming gain of function. They used passaging to create a “mouse model” of SARS2. I encourage all to check it out. That study should be exposed as fraudulent.
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Do you by any chance have a link to the study? I’m slowly working on a GOF post.