2D or not, 3D? That is the Question…

There is an underlying issue for cell cultures that goes beyond the lack of purification and proper isolation of particles assumed to be “virus” as well as the toxic nature of the chemicals used in “growing” them. This is in regards to the unnatural environments used to culture the cells in. What is most often used to culture “viruses” is known as the 2D culture system.

The problem? 2D Cell Cultures can not mimic or accurately represent the IN VIVO (associated with various biological processes that are made to occur WITHIN THE LIVING ORGANISM) physiology and complexity of a living organism.

In other words, what is taken from within a living organism and is then studied outside of it in controlled laboratory conditions can not accurately reflect what actually occurs when the studied material is within a living organism.

The fact that 2D Cell Culture can not accurately reflect these conditions should give pause to anyone reading any virology paper. When one then considers the numerous alterations to the starting material through the use of animal cells/chemicals/antibiotics/fetal bovine serum/DMEM etc., it is clear to see the results have absolutely no relation to reality whatsoever.

The inability of the 2D culture system to mimic the natural microenvironment to culture the cell in has given rise to the use of 3D Cell Cultures which aims to more accurately reflect what occurs within the living organism. These cultures, while claimed to be a better reflection, have their own problems but that is a whole different post entirely. It is clear, when comparing and contrasting 2D/3D Cell Cultures, that the current crop of “evidence” for “SARS-COV-2” and any other “virus” should be immediately thrown out.

Highlights from two recent articles help to explain the various problems with the 2D culture system:

3D cell culture stimulates the secretion of in vivo like extracellular vesicles

“Although the 2D culture system is simple and easily accessible, THE CULTURE ENVIRONMENT IS UNABLE TO REPRESENT IN VIVO EXTRACELLULAR MATRIX (ECM) MICROENVIRONMENT. Our study observed that 2D- culture derived EVs SHOWED SIGNIFICANTLY DIFFERENT PROFILES IN TERMS OF SECRETION DYNAMICS AND ESSENTIAL SIGNALING MOLECULAR CONTENTS (RNAs and DNAs), when compared to the three-dimensional (3D) culture derived EVs.”

“For investigating cellular communications and behaviors EX VIVO, presently, the two-dimensional (2D) CELL CULTURE MODEL IS WIDELY USED AS THE “GOLD STANDARD”29. This 2D culture system serves as an essential model for investigating tissue physiology and complex biological activity, from cell differentiation to tissue morphogenesis30. Many 2D cell culture systems have also been widely employed for studying EV RNA expression profiles from tumor cells31, roles in promoting tumor growth17 and tumor biomarker discovery32,33,34. Although the 2D culture system provides simple cell attachment and nutrients supply, THE FLAT AND HARD SURFACE FROM PLASTIC OR GLASS SUBSTRATES ARE UNABLE TO REPRESENT THE IN VIVO EXTRACELLULAR MATRIX (ECM) MICROENVIRONMENT IN TISSUE OR ORGANS 35. The monolayer cells UNDER 2D CULTURE CONDITION COMPLETELY DIFFER FROM IN VIVO STATUS where cells grow in three dimensions (3D), IN TERMS OF CELL MORPHOLOGY, CELL-TO-CELL INTERACTIONS, GROWTH BEHAVIOR, AND INTERACTIONS WITH EXTRACELLULAR MATRIX 30. It has been well demonstrated that 2D cell monolayer is UNABLE TO REPRESENT THE PHYSIOLOGY OF IN VIVO 3D TISSUES OR ORGANS, due to the substantially different microenvironment (e.g., mechanical and biochemical properties) in tissue architecture36,37,38,39,40,41”

“Unlike the 2D culture, 3D CELL CULTURE IS MORE RECOGNIZED FOR MIMICKING IN VIVO CELLULAR BEHAVIOR 41.”

“More importantly, we observed that 3D cell-derived EV sample was clustered together with two in vivo cervical cancer patient plasma sample derived EV miRNAs. It supports that 3D cell culture is necessary for reproducing the EV miRNA content sorted by in vivo cells and establishing an accurate disease model57,58. On the other hand, the 2D-DERIVED SAMPLES WERE CLUSTERED AWAY FROM THE IN VIVO SAMPLES, INDICATING THAT 2D CELLS CULTURE WAS UNABLE TO REPRESENT IN VIVO BIOLOGICAL STATUS. This observation supports the statement from other studies56,58 that 3D CULTURE SYSTEM WOULD BE MORE USEFUL AND ACCURATE FOR MIMICKING IN VIVO PHYSIOLOGICAL ENVIRONMENT in studying EV functions.”

https://www.nature.com/articles/s41598-019-49671-3#ref-CR9

3D culture models to study SARS-CoV-2 infectivity and antiviral candidates: From spheroids to bioprinting

“Despite its importance, 2D CELL CULTURE MODELS FAIL TO RECAPITULATE THE COMPLEXITY OF LIVING ORGANISMS AND OFTEN ACQUIRE PHENOTYPES THAT DIFFER SIGNIFICANTLY FROM NATIVE TISSUES, WHICH LEADS TO POOR PREDICTIONS OF RESULTS [7]. Therefore, the use of platforms that provide INCREASED SIMILARITY TO THE IN VIVO PHYSIOLOGY AND PATHOLOGY can contribute to advances in the treatments of COVID-19.”

“Due to the seriousness of the pandemic situation caused by COVID-19, the rapid development of in vitro models to study SARS-CoV-2 infection was necessary in order to assist clinical approaches and treatments through the knowledge acquired in basic research, almost in real time. For these purposes, 2D MONOLAYERS HAVE BEEN EXTENSIVELY USED TO STUDY SARS-CoV-2 LIFE CYCLE AND PATHOGENESIS ANALYSIS, drug screening and preclinical evaluation of antiviral potential, and CYTOPATHIC EFFECT OF CANDIDATE MOLECULES [35].

Vero cells E6 cells, ISOLATED FROM AFRICAN GREEN MONKEYS KIDNEYS, are susceptible to many types of viruses, including the SARS-CoV [36] and SARS-CoV-2 [37]. They produce high viral titers, probably due to the expressive presence of ACE-2 in their apical region, and because these cells do not produce type I interferons (IFN) when infected by several viruses. This phenomenon is due to a deletion of ∼9 Mbp deletion on chromosome 12, which when in homozygosis, results in a more permissive phenotype for viruses. Thus, the IFN deficiency allows SARS-CoV-2 to replicate sustainably in Vero cells [38]. THIS CELL LINE WAS USED IN SOME IMPORTANT STUDIES INVOLVING SARS-Cov-2, such as for identifying the ACE2 as the functional receptor of SARS-CoV, for demonstrating that anti-ACE2 acted as an inhibitor of viral replication in these cells [5], for identifying other potential routes of infection [31], and for testing the inhibition potential of antiviral candidates [6]. HOWEVER, THE HIGHLY PERMISSIVE PHENOTYPE VERO CELLS HAVE SOME LIMITATIONS, AS IT DOES NOT ACCURATELY REPRESENT THE PATHOGENESIS OF COVID-19, as its initial target organs are the air and pulmonary epithelia and the venous endothelium. Therefore, other cell types seem to serve as in vitro models THAT MAY BETTER RECAPITULATE THE REAL PHYSIOLOGY OF THE DISEASE.”

“Conventional 2D cell cultures have greatly contributed to the understanding of host cell–virus interactions, mechanisms of virus transmission, replication, and adaptation, as well as screening of antiviral drugs [6,37]. However, THIS MODEL HAVE SOME LIMITATIONS THAT RELY ON THE DIFFICULTY OF RECONSTITUTING THE ACCURATE AND COMPLEXITY MICROENVIRONMENT FOUND IN LIVING ORGANISMS. Cell–cell junctions, apical-basal polarity, and cell communication through gradients of endogenous growth factors, chemokines, and nutrients MAY BE INADEQUATE TO GUARANTEE THE SIMILARITY WITH AN IN VIVO SYSTEM [7]. THESE LIMITATIONS EXEMPLIFY THE NECESSITY TO DEVELOP NEW PLATFORMS FOR IN VITRO MODELING [7,49].”

“Although 2D conventional cell culture has shown to be an important tool in virology studies, THIS MODEL FAILS IN REPLICATING THE CELLULAR MICROENVIRONMENT IN TERMS OF ARCHITECTURE, COMPOSITION, PHYSIOLOGICAL FUNCTIONS, AND MECHANICAL STIMULUS, WHICH MAY LEAD TO LOW PREDICTION OF RESULTS [7]. The establishment of 3D cell culture and the biofabrication of tissue-like structures can mimic the complex microenvironment found in the many organs affected by SARS-CoV-2 with higher accuracy, providing robust data to elucidate cellular and molecular mechanisms of virus infection, replication kinetics, and host–virus interaction.”

https://www.sciencedirect.com/science/article/pii/S2319417020302079

IN SUMMARY, 2D CELL CULTURES:

-are unable to respresent the in vivo extracellular matrix microenvironment

-differ completely in terms of cell morphology, cell-to-cell interactions, growth behavior, and interactions with the extracellular matrix

-are unable to represent in vivo physiology

-are unable to represent in vivo biological status

-often acquire phenotypes that differ significantly from native tissues

-lead to poor prediction of results

-can not replicate the cellular microenvironment in terms of architecture, composition, physiological functions, and mechanical stimulus

It’s time to realize that the “evidence” we are presented regarding “viruses” is highly flawed, based on guesswork and assumptions, and does not reflect reality. It is time to throw Virology out into the trash with the rest of the pseudosciences.

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