In vitro (meaningin glass, orin the glass)studies are performed withmicroorganisms,cells, orbiological molecules outside their normal biological context. Colloquially called "test-tube experiments", these studies inbiology and its subdisciplines are traditionally done in labware such as test tubes, flasks,Petri dishes, andmicrotiter plates. Studies conducted using components of anorganism 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 fromin vitro experiments may not fully or accurately predict the effects on a whole organism. In contrast toin vitro experiments,in vivo studies are those conducted in living organisms, including humans, known asclinical trials, and whole plants.[1][2]
In vitro (Latin for "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 ofin vitro studies include: the isolation, growth and identification of cells derived frommulticellular organisms (incell ortissue culture); subcellular components (e.g.mitochondria orribosomes); cellular or subcellular extracts (e.g.wheat germ orreticulocyte extracts); 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 vitro to distinguish it fromin vivo work in whole animals.[14][15]
Polymerase chain reaction is a method for selective replication of specific DNA and RNA sequences in the test tube.[16]
Protein purification involves the isolation of a specific protein of interest from a complex mixture of proteins, often obtained from homogenized cells or tissues.[17]
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.[18]
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.[19]
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;[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 (Hep G2,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.
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 arein vitro studies replacing studies in whole animals.
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 anaerosol for anin vitro study of the effects
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.[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 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.
Another advantage ofin vitro methods is that human cells can be studied without "extrapolation" from an experimental animal's cellular response.[27][28][29]
In vitro methods can be miniaturized and automated, yielding high-throughput screening methods for testing molecules in pharmacology or toxicology.[30]
The primary disadvantage ofin vitro experimental studies is that it may be challenging to extrapolate from the results ofin vitro work back to the biology of the intact organism. Investigators doingin vitro work 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 vitro setting (typically cell culture). However, before this drug is used in the clinic, it must progress through a series ofin 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 effectivein vitro prove to be ineffectivein 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 initialin vitro studies, or other issues.[33]
A method which could help decrease animal testing is the use ofin vitro batteries, where severalin vitro assays are compiled to cover multiple endpoints. Within developmentalneurotoxicity and 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 vitro test batteries are already in use for regulatory purpose and for toxicological evaluation of chemicals.[38]In vitro tests can also be combined within vivo testing to make ain vitro in vivo test battery, for example for pharmaceutical testing.[39]
Results obtained fromin vitro experiments cannot usually be transposed, as is, to predict the reaction of an entire organismin vivo. Building a consistent and reliable extrapolation procedure fromin vitro results toin vivo is therefore extremely important. Solutions include:
Increasing the complexity ofin vitro systems 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 vitro data provide model parameter values[41]
These two approaches are not incompatible; betterin vitro systems provide better data to mathematical models. However, increasingly sophisticatedin vitro experiments collect increasingly numerous, complex, and challenging data to integrate. Mathematical models, such assystems biology models, are much needed here.[42]
In pharmacology, IVIVE can be used to approximatepharmacokinetics (PK) orpharmacodynamics (PD).[43][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 vivo tissue 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 vitro needs a quantitative model ofin vivo PK. Physiologically based PK (PBPK) models are generally accepted to be central to the extrapolations.[44]
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 andin vivo. In these conditions, developing a simple PD model of thedose–response relationship observedin vitro, and transposing it without changes to predictin vivo effects is not enough.[45]
^Artursson P.; Palm K.; Luthman K. (2001). "Caco-2 monolayers in experimental and theoretical predictions of drug transport".Advanced Drug Delivery Reviews.46 (1–3):27–43.doi:10.1016/s0169-409x(00)00128-9.PMID11259831.
^Pelkonen O.; Turpeinen M. (2007). "In vitro-in vivo extrapolation of hepatic clearance: biological tools, scaling factors, model assumptions and correct concentrations".Xenobiotica.37 (10–11):1066–1089.doi:10.1080/00498250701620726.PMID17968737.S2CID3043750.
^Alberts, Bruce (2008).Molecular biology of the cell. New York: Garland Science.ISBN978-0-8153-4105-5.
^Vignais, Paulette M.; Pierre Vignais (2010).Discovering Life, Manufacturing Life: How the experimental method shaped life sciences. Berlin: Springer.ISBN978-90-481-3766-4.
^Jacqueline Nairn; Price, Nicholas C. (2009).Exploring proteins: a student's guide to experimental skills and methods. Oxford [Oxfordshire]: Oxford University Press.ISBN978-0-19-920570-7.
^Quignot N.; Hamon J.; Bois F. (2014).Extrapolating in vitro results to predict human toxicity, in In Vitro Toxicology Systems, Bal-Price A., Jennings P., Eds, Methods in Pharmacology and Toxicology series. New York, US: Springer Science. pp. 531–550.
^Sung, JH; Esch, MB; Shuler, ML (2010). "Integration of in silico and in vitro platforms for pharmacokinetic-pharmacodynamic modeling".Expert Opinion on Drug Metabolism & Toxicology.6 (9):1063–1081.doi:10.1517/17425255.2010.496251.PMID20540627.S2CID30583735.
^Yoon M, Campbell JL, Andersen ME, Clewell HJ (2012). "Quantitative in vitro to in vivo extrapolation of cell-based toxicity assay results".Critical Reviews in Toxicology.42 (8):633–652.doi:10.3109/10408444.2012.692115.PMID22667820.S2CID3083574.
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