Differences between in vitro, in vivo, and in silico studies

There are three broad categories of experiments: in vitro studies, in vivo studies, and in silico studies. Each study type has conveniences and liabilities. Understanding the liabilities of study types offers insight into the validity of researchers' conclusions.

In vitro studies

In vitro (Latin for within the glass) refers to the technique of performing a given procedure in a controlled environment outside of a living organism. Many experiments in cellular biology are conducted outside of organisms or cells. One of the abiding weaknesses of in vitro experiments is that they fail to replicate the precise cellular conditions of an organism, particularly a microbe. To cite one example among many, the lysates or extracts from culture-grown spirochetes do not reflect antigens expressed in the mammalian Borrelia:

Because of this, in vitro studies may lead to results that do not correspond to the circumstances occuring around a living organism.

Until the last several years, efforts to detect and identify microorganisms in the human body have depended almost exclusively on in vitro studies. As a result, many researchers began to assume that chronic diseases were not caused by microbes. The net effect of all this was that the understanding of pathogens in disease was driven by the study of well-known, easy-to-culture microbes–which, as it turns out, represent the vast minority of bacteria in the human body. By one estimate, 99.6% of the species in the human microbiota have not or cannot be characterized through in vitro techniques.2)

Another example of a shortcoming of in vitro studies relates to concentrations of molecules, especially as they compete for nuclear receptors. For example, the vitamin D metabolite, 1,25-D, exerts its effects at 30 picograms per milliliter, or 0.000000000003 grams per milliliter.

In vivo studies

In vivo (Latin for “within the living”) refers to experimentation using a whole, living organism as opposed to a partial or dead organism. Animal studies and clinical trials are two forms of in vivo research. In vivo testing is often employed over in vitro because it is better suited for observing the overall effects of an experiment on a living subject.

While there are many reasons to believe in vivo studies have the potential to offer conclusive insights about the nature of medicine and disease, there is a number of ways that these conclusions can be misleading. For example, a therapy can offer a short-term benefit, but a long-term harm.

In silico studies

In silico is an expression used to mean “performed on computer or via computer simulation.” The expression in silico was first used in public in 1989 in the workshop “Cellular Automata: Theory and Applications” in Los Alamos, New Mexico. Pedro Miramontes, a mathematician from National Autonomous University of Mexico (UNAM), presented the report “DNA and RNA Physicochemical Constraints, Cellular Automata and Molecular Evolution.” In his talk, Miramontes used the term “in silico” to characterize biological experiments carried out entirely in a computer.

Although in silico studies represent a relatively new avenue of inquiry, it has begun to be used widely in studies which predict how drugs interact with the body and with pathogens. For example, a 2009 study used software emulations to predict how certain drugs already on the market could treat multiple-drug-resistant and extensively drug-resistant strains of tuberculosis.3)

There is a variety of in silico techniques, but the two that are discussed the most in connection with the Marshall Protocol are:

Bacterial sequencing techniques – As an alternative to in vitro methods for identifying bacteria, various in silico methods which sequence bacterial DNA and RNA have been developed. The most commonly used use is polymerase chain reaction (PCR). PCR takes a single or few copies of a piece of DNA and increases it across several orders of magnitude, generating millions or more copies of a particular DNA sequence. PCR has allowed researchers to detect bacteria associated with a variety of conditions with increasingly high sensitivity.

Molecular modeling – Part of the Marshall Pathogenesis is based on in silico work, demonstrating how drugs and other substances interact with the nuclear receptors of cells. In particular, Trevor Marshall, PhD, has used computer-based emulations to show that 25-D, one of the vitamin D metabolites, and Capnine, a substance produced by bacteria, turn off the Vitamin D Receptor. These conclusions have since been validated by clinical observations.

Whole cell simulations – As described here, researchers have built a computer model of the crowded interior of a bacterial cell that—in a test of its response to sugar in its environment—accurately simulates the behavior of living cells