In Vitro and in Vivo Research

I n vitro and in vivo research

In vitro research is generally referred to as the manipulation of organs, tissues, cells, and biomolecules in a controlled, artificial environment. The characterization and analysis of biomolecules and biological systems in the context of intact organisms is known as in vivo research.

The basic unit of living organisms is the cell, which in terms of scale and dimension is at the interface between the molecular and the microscopic level. The living cell is in turn divided into functional and structural domains such as the nucleus , the cytoplasm , and the secretory pathway, which are composed of a vast array of biomolecules. These molecules of life carry out the chemical reactions that enable a cell to interact with its environment, use and store energy, reproduce, and grow. The structure of each biomolecule and its subcellular localization determines in which chemical reactions it is able to participate and hence what role it plays in the cell's life process. Any manipulation that breaks down this unit of life, that is, the cell into its non-living components is, considered an in vitro approach. Thus, in vitro, which literally means "in glass," refers to the experimental manipulation conducted using cell-free extracts and purified or partially purified biomolecules in test tubes. Most of the biochemical and molecular biological approaches and techniques are considered genetic manipulation research. Molecular cloning of a gene with the aim of expressing its protein product includes some steps that are considered in vitro experiments such as the PCR amplification of the gene and the ligation of that gene to the expression vector. The expression of that gene in a host cell is considered an in vivo procedure. What characterizes an in vitro experiment is in principle the fact the conditions are artificial and are reconstructions of what might happen in vivo. Many in vitro assays are approximate reconstitutions of biological processes by mixing the necessary components and reagents under controlled conditions. Examples of biological processes that can be reconstituted in vitro are enzymatic reactions, folding and refolding of proteins and DNA , and the replication of DNA in the PCR reaction.

The definition of in vitro and in vivo research depends on the experimental model used. Microbiologists and yeast geneticists working with single cells or cell populations are conducting in vivo research while an immunologist who works with purified lymphocytes in tissue culture usually considers his experiments as an in vitro approach. The in vivo approach involves experiments performed in the context of the large system of the body of an experimental animal. In the case of in vitro fertilization, physicians and reproductive biologists are manipulating living systems, and many of the biological processes involved take place inside the living egg and sperm. This procedure is considered an in vitro process in order to distinguish it from the natural fertilization of the egg in the intact body of the female.

In vivo experimental research became widespread with the use microorganisms and animal models in genetic manipulation experiments as well as the use of animal models to study drug toxicity in pharmacology. Geneticists have used prokaryotic, unicellular eukaryotes like yeast, and whole organisms like Drosophila, frogs, and mice to study genetics, molecular biology and toxicology. The function of genes has been studied by observing the effects of spontaneous mutations in whole organisms or by introducing targeted mutations in cultured cells. The introduction of gene cloning and in vitro mutagenesis has made it possible to produce specific mutations in whole animals thus considerably facilitating in vivo research. Mice with extra copies or altered copies of a gene in their genome can be generated by transgenesis, which is now a well-established technique. In many cases, the function of a particular gene can be fully understood only if a mutant animal that does not express the gene can be obtained. This is now achieved by gene knock-out technology, which involves first isolating a gene of interest and then replacing it in vivo with a defective copy.

Both in vitro and in vivo approaches are usually combined to obtain detailed information about structure-function relationships in genes and their protein products, either in cultured cells and test tubes or in the whole organism.

See also Immunogenetics; Immunologic therapies; Immunological analysis techniques; Laboratory techniques in immunology; Laboratory techniques in microbiology; Molecular biology and molecular genetics