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This article is about the type of scientific experiment. For other uses, seeIn vitro (disambiguation).
Clonedplantsin vitro

In vitro(meaningin glass,orin the glass)studiesare performed withmicroorganisms,cells,orbiological moleculesoutside their normal biological context. Colloquially called "test-tubeexperiments ", these studies inbiologyand its subdisciplines are traditionally done in labware such as test tubes, flasks,Petri dishes,andmicrotiter plates.Studies conducted using components of anorganismthat 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 fromin vitroexperiments may not fully or accurately predict the effects on a whole organism. In contrast toin vitroexperiments,in vivostudies are those conducted in living organisms, including humans, known as clinical trials, and whole plants.[1][2]

Definition

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In vitro(Latinfor "in glass"; often not italicized in English usage[3][4][5]) 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 artificialculture media,and proteins can be examined insolutions.Colloquially called "test-tube experiments", these studies in biology, medicine, and their subdisciplines are traditionally done in test tubes, flasks, Petri dishes, etc.[6][7]They now involve the full range of techniques used in molecular biology, such as theomics.[8]

In contrast, studies conducted in living beings (microorganisms, animals, humans, or whole plants) are calledin vivo.[9]

Examples

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Examples ofin vitrostudies include: the isolation, growth and identification of cells derived frommulticellular organisms(incellortissue culture); subcellular components (e.g.mitochondriaorribosomes); cellular or subcellular extracts (e.g.wheat germorreticulocyteextracts); purified molecules (such asproteins,DNA,orRNA); and the commercial production of antibiotics and other pharmaceutical products.[10][11][12][13]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 beingin vitroto distinguish it fromin vivowork in whole animals.[14][15]

  • Polymerase chain reactionis a method for selective replication of specific DNA and RNA sequences in the test tube.[16]
  • Protein purificationinvolves the isolation of a specific protein of interest from a complex mixture of proteins, often obtained from homogenized cells or tissues.[17]
  • In vitrofertilizationis 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.[18]
  • In vitrodiagnosticsrefers 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.[19]
  • In vitrotesting 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;[20]The partitioning of the compounds between organs can be determined to study distribution mechanisms;[21]Suspension or plated cultures of primary hepatocytes or hepatocyte-like cell lines (HepG2,HepaRG) can be used to study and quantify metabolism of chemicals.[22]These ADME process parameters can then be integrated into so called "physiologically based pharmacokinetic models" orPBPK.

Advantages

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In vitrostudies 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 arein vitrostudies replacing studies in whole animals.

Simplicity

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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.[23][24]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.

Top view of a Vitrocell mammalian exposure module "smoking robot", (lid removed) view of four separated wells for cell culture inserts to be exposed to tobacco smoke or anaerosolfor anin vitrostudy of the effects

This complexity makes it difficult to identify the interactions between individual components and to explore their basic biological functions.In vitrowork simplifies the system under study, so the investigator can focus on a small number of components.[25][26]

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 ofin vitrowork 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

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Another advantage ofin vitromethods is that human cells can be studied without "extrapolation" from an experimental animal's cellular response.[27][28][29]

Convenience, automation

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In vitromethods can be miniaturized and automated, yielding high-throughput screening methods for testing molecules in pharmacology or toxicology.[30]

Disadvantages

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The primary disadvantage ofin vitroexperimental studies is that it may be challenging to extrapolate from the results ofin vitrowork back to the biology of the intact organism. Investigators doingin vitrowork must be careful to avoid over-interpretation of their results, which can lead to erroneous conclusions about organismal and systems biology.[31][32]

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 anin vitrosetting (typically cell culture). However, before this drug is used in the clinic, it must progress through a series ofin vivotrials 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 effectivein vitroprove to be ineffectivein vivobecause 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 initialin vitrostudies, or other issues.[33]

In vitrotest batteries

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A method which could help decrease animal testing is the use ofin vitrobatteries, where severalin vitroassays are compiled to cover multiple endpoints. Within developmentalneurotoxicityand reproductive toxicity there are hopes for test batteries to become easy screening methods for prioritization for which chemicals to be risk assessed and in which order.[34][35][36][37]Within ecotoxicologyin vitrotest batteries are already in use for regulatory purpose and for toxicological evaluation of chemicals.[38]In vitrotests can also be combined within vivotesting to make ain vitro in vivotest battery, for example for pharmaceutical testing.[39]

In vitrotoin vivoextrapolation

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Main article:In vitro to in vivo extrapolation

Results obtained fromin vitroexperiments cannot usually be transposed, as is, to predict the reaction of an entire organismin vivo.Building a consistent and reliable extrapolation procedure fromin vitroresults toin vivois therefore extremely important. Solutions include:

  • Increasing the complexity ofin vitrosystems to reproduce tissues and interactions between them (as in "human on chip" systems)[40]
  • Using mathematical modeling to numerically simulate the behavior of the complex system, where thein vitrodata provide model parameter values[41]

These two approaches are not incompatible; betterin vitrosystems provide better data to mathematical models. However, increasingly sophisticatedin vitroexperiments collect increasingly numerous, complex, and challenging data to integrate. Mathematical models, such assystems biologymodels, are much needed here.[42]

Extrapolating in pharmacology

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In pharmacology, IVIVE can be used to approximatepharmacokinetics(PK) orpharmacodynamics(PD).[citation needed] 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 vivotissue and organ sensitivities can be completely different or even inverse of those observed on cells cultured and exposedin vitro.That indicates that extrapolating effects observedin vitroneeds a quantitative model ofin vivoPK. Physiologically based PK (PBPK) models are generally accepted to be central to the extrapolations.[43]

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 vitroandin vivo.In these conditions, developing a simple PD model of thedose–response relationshipobservedin vitro,and transposing it without changes to predictin vivoeffects is not enough.[44]

See also

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References

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  2. ^Toxicity, National Research Council (US) Subcommittee on Reproductive and Developmental (2001).Experimental Animal and In Vitro Study Designs.National Academies Press (US).
  3. ^Merriam-Webster,Merriam-Webster's Collegiate Dictionary,Merriam-Webster, archived fromthe originalon 2020-10-10,retrieved2014-04-20.
  4. ^Iverson, Cheryl; et al., eds. (2007). "12.1.1 Use of Italics".AMA Manual of Style(10th ed.). Oxford, Oxfordshire:Oxford University Press.ISBN978-0-19-517633-9.
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