In Vitro

''In vitro'' (meaning in glass, or ''in the glass'') studies are performed with microorganisms, cells, or biological molecules outside their normal biological context. Colloquially called " test-tube experiments", these studies in biology and its subdisciplines are traditionally done in labware such as test tubes, flasks, Petri dishes, and microtiter plates. Studies conducted using components of an organism that have been isolated from their usual biological surroundings permit a more detailed or more convenient analysis than can be done with whole organisms; however, results obtained from ''in vitro'' experiments may not fully or accurately predict the effects on a whole organism. In contrast to ''in vitro'' experiments, '' in vivo'' studies are those conducted in living organisms, including humans, and whole plants.

Definition

''In vitro'' ( la, in glass; often not italicized in English usage) studies are conducted using components of an organism that have been isolated from their usual biological surroundings, such as microorganisms, cells, or biological molecules. For example, microorganisms or cells can be studied in artificial culture media, and proteins can be examined in solutions. Colloquially called "test-tube experiments", these studies in biology, medicine, and their subdisciplines are traditionally done in test tubes, flasks, Petri dishes, etc. They now involve the full range of techniques used in molecular biology, such as the omics.

In contrast, studies conducted in living beings (microorganisms, animals, humans, or whole plants) are called ''in vivo''.

Examples

Examples of ''in vitro'' studies include: the isolation, growth and identification of cells derived from multicellular organisms (in cell or tissue culture); subcellular components (e.g. mitochondria or ribosomes); cellular or subcellular extracts (e.g. wheat germ or reticulocyte extracts); purified molecules (such as proteins, DNA, or RNA); and the commercial production of antibiotics and other pharmaceutical products. Viruses, which only replicate in living cells, are studied in the laboratory in cell or tissue culture, and many animal virologists refer to such work as being ''in vitro'' to distinguish it from ''in vivo'' work in whole animals.

* Polymerase chain reaction is a method for selective replication of specific DNA and RNA sequences in the test tube.
* Protein purification involves the isolation of a specific protein of interest from a complex mixture of proteins, often obtained from homogenized cells or tissues.
* ''In vitro'' fertilization is used to allow spermatozoa to fertilize eggs in a culture dish before implanting the resulting embryo or embryos into the uterus of the prospective mother.
* ''In vitro'' diagnostics refers to a wide range of medical and veterinary laboratory tests that are used to diagnose diseases and monitor the clinical status of patients using samples of blood, cells, or other tissues obtained from a patient.
* ''In vitro'' testing has been used to characterize specific adsorption, distribution, metabolism, and excretion processes of drugs or general chemicals inside a living organism; for example, Caco-2 cell experiments can be performed to estimate the absorption of compounds through the lining of the gastrointestinal tract; The partitioning of the compounds between organs can be determined to study distribution mechanisms; Suspension or plated cultures of primary hepatocytes or hepatocyte-like cell lines (HepG2, HepaRG) can be used to study and quantify metabolism of chemicals. These ADME process parameters can then be integrated into so called "physiologically based pharmacokinetic models" or PBPK.

Advantages

''In vitro'' studies permit a species-specific, simpler, more convenient, and more detailed analysis than can be done with the whole organism. Just as studies in whole animals more and more replace human trials, so are ''in vitro'' studies replacing studies in whole animals.

Simplicity

Living organisms are extremely complex functional systems that are made up of, at a minimum, many tens of thousands of genes, protein molecules, RNA molecules, small organic compounds, inorganic ions, and complexes in an environment that is spatially organized by membranes, and in the case of multicellular organisms, organ systems. These myriad components interact with each other and with their environment in a way that processes food, removes waste, moves components to the correct location, and is responsive to signalling molecules, other organisms, light, sound, heat, taste, touch, and balance.

This complexity makes it difficult to identify the interactions between individual components and to explore their basic biological functions. ''In vitro'' work simplifies the system under study, so the investigator can focus on a small number of components.

For example, the identity of proteins of the immune system (e.g. antibodies), and the mechanism by which they recognize and bind to foreign antigens would remain very obscure if not for the extensive use of ''in vitro'' work to isolate the proteins, identify the cells and genes that produce them, study the physical properties of their interaction with antigens, and identify how those interactions lead to cellular signals that activate other components of the immune system.

Species specificity

Another advantage of ''in vitro'' methods is that human cells can be studied without "extrapolation" from an experimental animal's cellular response.

Convenience, automation

''In vitro'' methods can be miniaturized and automated, yielding high-throughput screening methods for testing molecules in pharmacology or toxicology.

Disadvantages

The primary disadvantage of ''in vitro'' experimental studies is that it may be challenging to extrapolate from the results of ''in vitro'' work back to the biology of the intact organism. Investigators doing ''in vitro'' work must be careful to avoid over-interpretation of their results, which can lead to erroneous conclusions about organismal and systems biology.

For example, scientists developing a new viral drug to treat an infection with a pathogenic virus (e.g., HIV-1) may find that a candidate drug functions to prevent viral replication in an ''in vitro'' setting (typically cell culture). However, before this drug is used in the clinic, it must progress through a series of ''in vivo'' trials to determine if it is safe and effective in intact organisms (typically small animals, primates, and humans in succession). Typically, most candidate drugs that are effective ''in vitro'' prove to be ineffective ''in vivo'' because of issues associated with delivery of the drug to the affected tissues, toxicity towards essential parts of the organism that were not represented in the initial ''in vitro'' studies, or other issues.

''In vitro'' to ''in vivo'' extrapolation

Results obtained from ''in vitro'' experiments cannot usually be transposed, as is, to predict the reaction of an entire organism ''in vivo''. Building a consistent and reliable extrapolation procedure from ''in vitro'' results to ''in vivo'' is therefore extremely important. Solutions include:

*Increasing the complexity of ''in vitro'' systems to reproduce tissues and interactions between them (as in "human on chip" systems)
*Using mathematical modeling to numerically simulate the behavior of the complex system, where the ''in vitro'' data provide model parameter values

These two approaches are not incompatible; better ''in vitro'' systems provide better data to mathematical models. However, increasingly sophisticated ''in vitro'' experiments collect increasingly numerous, complex, and challenging data to integrate. Mathematical models, such as systems biology models, are much needed here.

Extrapolating in pharmacology

In pharmacology, IVIVE can be used to approximate pharmacokinetics (PK) or pharmacodynamics (PD).
Since the timing and intensity of effects on a given target depend on the concentration time course of candidate drug (parent molecule or metabolites) at that target site, ''in vivo'' tissue and organ sensitivities can be completely different or even inverse of those observed on cells cultured and exposed ''in vitro''. That indicates that extrapolating effects observed ''in vitro'' needs a quantitative model of ''in vivo'' PK. Physiologically based PK ( PBPK) models are generally accepted to be central to the extrapolations.

In the case of early effects or those without intercellular communications, the same cellular exposure concentration is assumed to cause the same effects, both qualitatively and quantitatively, ''in vitro'' and '' in vivo''. In these conditions, developing a simple PD model of the dose–response relationship observed ''in vitro'', and transposing it without changes to predict '' in vivo'' effects is not enough.

See also

* Animal testing
*''Ex vivo''
*''In situ''
*''In utero''
*'' In vivo''
*'' In silico''
*''In papyro''
*'' In natura''
* Animal ''in vitro'' cellular and developmental biology
* Plant ''in vitro'' cellular and developmental biology
* ''In vitro'' toxicology
*In vitro to in vivo extrapolation
*Slice preparation

References

External links

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{{DEFAULTSORT:In Vitro
Latin biological phrases
Alternatives to animal testing
Animal test conditions
Laboratory techniques