In vitro

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Cloned plants in vitro Laboratoriia mikroklonal'nogo rozmnozhennia roslin.jpg
Cloned plants 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.

Contents

Definition

In vitro (Latin : in glass; often not italicized in English usage [1] [2] [3] ) 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.

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. [7] 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 an aerosol for an in vitro study of the effects Vitrocell mammalian exposure module-smoking robot.jpg
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 an aerosol for an in 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. [8] [9]

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. [10]

Convenience, automation

In vitro methods can be miniaturized and automated, yielding high-throughput screening methods for testing molecules in pharmacology or toxicology. [11]

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. [12]

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. [13]

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:

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.[ citation needed ]

Extrapolating in pharmacology

In pharmacology, IVIVE can be used to approximate pharmacokinetics (PK) or pharmacodynamics (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 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. [16]

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. [17]

See also

Related Research Articles

Studies that are in vivo are those in which the effects of various biological entities are tested on whole, living organisms or cells, usually animals, including humans, and plants, as opposed to a tissue extract or dead organism. This is not to be confused with experiments done in vitro, i.e., in a laboratory environment using test tubes, Petri dishes, etc. Examples of investigations in vivo include: the pathogenesis of disease by comparing the effects of bacterial infection with the effects of purified bacterial toxins; the development of non-antibiotics, antiviral drugs, and new drugs generally; and new surgical procedures. Consequently, animal testing and clinical trials are major elements 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. In drug discovery, for example, verification of efficacy in vivo is crucial, because in vitro assays can sometimes yield misleading results with drug candidate molecules that are irrelevant in vivo.

Model organism Organisms used to study biology across species

A model organism is a non-human species that is extensively studied to understand particular biological phenomena, with the expectation that discoveries made in the model organism will provide insight into the workings of other organisms. Model organisms are widely used to research human disease when human experimentation would be unfeasible or unethical. This strategy is made possible by the common descent of all living organisms, and the conservation of metabolic and developmental pathways and genetic material over the course of evolution.

Protein Biological molecule consisting of chains of amino acid residues

Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, responding to stimuli, providing structure to cells and organisms, and transporting molecules from one location to another. Proteins differ from one another primarily in their sequence of amino acids, which is dictated by the nucleotide sequence of their genes, and which usually results in protein folding into a specific 3D structure that determines its activity.

Toxicology Study of substances harmful to living organisms

Toxicology is a scientific discipline, overlapping with biology, chemistry, pharmacology, and medicine, that involves the study of the adverse effects of chemical substances on living organisms and the practice of diagnosing and treating exposures to toxins and toxicants. The relationship between dose and its effects on the exposed organism is of high significance in toxicology. Factors that influence chemical toxicity include the dosage, duration of exposure, route of exposure, species, age, sex, and environment. Toxicologists are experts on poisons and poisoning. There is a movement for evidence-based toxicology as part of the larger movement towards evidence-based practices. Toxicology is currently contributing to the field of Cancer research, since some toxins can be used as drugs for killing tumor cells. One prime example of this is Ribosome Inactivating Proteins, tested in the treatment of Leukemia.

In vitro toxicity testing is the scientific analysis of the effects of toxic chemical substances on cultured bacteria or mammalian cells. In vitro testing methods are employed primarily to identify potentially hazardous chemicals and/or to confirm the lack of certain toxic properties in the early stages of the development of potentially useful new substances such as therapeutic drugs, agricultural chemicals and food additives.

Systems biology Computational and mathematical modeling of complex biological systems

Systems biology is the computational and mathematical analysis and modeling of complex biological systems. It is a biology-based interdisciplinary field of study that focuses on complex interactions within biological systems, using a holistic approach to biological research.

Behavioral neuroscience Field of study

Behavioral neuroscience, also known as biological psychology, biopsychology, or psychobiology, is the application of the principles of biology to the study of physiological, genetic, and developmental mechanisms of behavior in humans and other animals.

Pharmacodynamics Area of Academic Study

Pharmacodynamics (PD) is the study of the biochemical and physiologic effects of drugs. The effects can include those manifested within animals, microorganisms, or combinations of organisms.

In silico Latin phrase in biology

In biology and other experimental sciences, an in silico experiment is one performed on computer or via computer simulation. The phrase is pseudo-Latin for 'in silicon', referring to silicon in computer chips. It was coined in 1987 as an allusion to the Latin phrases in vivo, in vitro, and in situ, which are commonly used in biology. The latter phrases refer, respectively, to experiments done in living organisms, outside living organisms, and where they are found in nature.

Bioinorganic chemistry is a field that examines the role of metals in biology. Bioinorganic chemistry includes the study of both natural phenomena such as the behavior of metalloproteins as well as artificially introduced metals, including those that are non-essential, in medicine and toxicology. Many biological processes such as respiration depend upon molecules that fall within the realm of inorganic chemistry. The discipline also includes the study of inorganic models or mimics that imitate the behaviour of metalloproteins.

<i>Ex vivo</i> Process of testing biological interventions on extracted fragments of organisms

Ex vivo literally means that which takes place outside an organism. In science, ex vivo refers to experimentation or measurements done in or on tissue from an organism in an external environment with minimal alteration of natural conditions. Ex vivo conditions allow experimentation on an organism's cells or tissues under more controlled conditions than is possible in in vivo experiments, at the expense of altering the "natural" environment.

Matrigel is the trade name for the solubilized basement membrane matrix secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells produced by Corning Life Sciences. Matrigel resembles the laminin/collagen IV-rich basement membrane extracellular environment found in many tissues and is used by cell biologists as a substrate for culturing cells.

High-content screening (HCS), also known as high-content analysis (HCA) or cellomics, is a method that is used in biological research and drug discovery to identify substances such as small molecules, peptides, or RNAi that alter the phenotype of a cell in a desired manner. Hence high content screening is a type of phenotypic screen conducted in cells involving the analysis of whole cells or components of cells with simultaneous readout of several parameters. HCS is related to high-throughput screening (HTS), in which thousands of compounds are tested in parallel for their activity in one or more biological assays, but involves assays of more complex cellular phenotypes as outputs. Phenotypic changes may include increases or decreases in the production of cellular products such as proteins and/or changes in the morphology of the cell. Hence HCA typically involves automated microscopy and image analysis. Unlike high-content analysis, high-content screening implies a level of throughput which is why the term "screening" differentiates HCS from HCA, which may be high in content but low in throughput.

Intravital microscopy Form of microscopy that allows observing biological processes in live animals (in vivo) at a high resolution that makes distinguishing between individual cells of a tissue possible

Intravital microscopy is a form of microscopy that allows observing biological processes in live animals at a high resolution that makes distinguishing between individual cells of a tissue possible. Before an animal can be used for intravital microscopy imaging it has to undergo a surgery involving implantation of an imaging window. For example, if researchers want to visualize liver cells of a live mouse they will implant an imaging window into mouse’s abdomen. Mice are the most common choice of animals for intravital microscopy but in special cases other rodents such as rats might be more suitable. Animals are always anesthetized throughout surgeries and imaging sessions. Intravital microscopy is used in several areas of research including neurology, immunology, stem cell studies and others. This technique is particularly useful to assess a progression of a disease or an effect of a drug.

An organ-on-a-chip (OOC) is a multi-channel 3-D microfluidic cell culture, integrated circuit (chip) that simulates the activities, mechanics and physiological response of an entire organ or an organ system, a type of artificial organ. It constitutes the subject matter of significant biomedical engineering research, more precisely in bio-MEMS. The convergence of labs-on-chips (LOCs) and cell biology has permitted the study of human physiology in an organ-specific context, introducing a novel model of in vitro multicellular human organisms. One day, they will perhaps abolish the need for animals in drug development and toxin testing.

Phenotypic screening is a type of screening used in biological research and drug discovery to identify substances such as small molecules, peptides, or RNAi that alter the phenotype of a cell or an organism in a desired manner. Phenotypic screening must be followed up with target identification and validation campaigns, often through the use of chemoproteomics, to identify the mechanisms through which a phenotypic hit works.

A 3D cell culture is an artificially created environment in which biological cells are permitted to grow or interact with their surroundings in all three dimensions. Unlike 2D environments, a 3D cell culture allows cells in vitro to grow in all directions, similar to how they would in vivo. These three-dimensional cultures are usually grown in bioreactors, small capsules in which the cells can grow into spheroids, or 3D cell colonies. Approximately 300 spheroids are usually cultured per bioreactor.

In vitro to in vivo extrapolation (IVIVE) refers to the qualitative or quantitative transposition of experimental results or observations made in vitro to predict phenomena in vivo, biological organisms.

Bioassay

A bioassay is an analytical method to determine the concentration or potency of a substance by its effect on living animals or plants, or on living cells or tissues(in vitro). A bioassay can be either quantal or quantitative, direct or indirect. If the measured response is binary, the assay is quantal, if not, it is quantitative.

References

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