Animal Cell Biotechnology: Methods and Protocols (Methods in Biotechnology)

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Cell culture

Seller Inventory LIE Book Description Humana. Seller Inventory ING Book Description Humana Pr Inc, Condition: Brand New. Ed , Ringeling, F. Publishing With Us. Book Authors Journal Authors. Methods in Molecular Biology. Each protocol is presented in readily-reproducible step-by-step fashion, beginning with an introductory overview of the method, a comprehensive list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section that provides expert tips and tricks of the trade along with relevant troubleshooting advice.

Animal Cell Biotechnology

These hallmark features, introduced by series editor Dr. Tested and trusted, comprehensive and reliable, Methods in Molecular Biology provides researchers with the tools they need for successful experiment implementation. All protocols from the series are indexed in PubMed.

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Animal Cell Biotechnology Methods and Protocols Methods in Biotechnology

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Animal Cell Biotechnology - Methods and Protocols | Ralf Pörtner | Springer

Many methods are used to identify cell lines, including isoenzyme analysis, human lymphocyte antigen HLA typing, chromosomal analysis, karyotyping, morphology and STR analysis. One significant cell-line cross contaminant is the immortal HeLa cell line. As cells generally continue to divide in culture, they generally grow to fill the available area or volume. This can generate several issues:. Among the common manipulations carried out on culture cells are media changes, passaging cells, and transfecting cells.

These are generally performed using tissue culture methods that rely on aseptic technique. Aseptic technique aims to avoid contamination with bacteria, yeast, or other cell lines. Manipulations are typically carried out in a biosafety cabinet or laminar flow cabinet to exclude contaminating micro-organisms. Antibiotics e. As cells undergo metabolic processes, acid is produced and the pH decreases. Often, a pH indicator is added to the medium to measure nutrient depletion. In the case of adherent cultures, the media can be removed directly by aspiration, and then is replaced.

Media changes in non-adherent cultures involve centrifuging the culture and resuspending the cells in fresh media. Passaging also known as subculture or splitting cells involves transferring a small number of cells into a new vessel. Cells can be cultured for a longer time if they are split regularly, as it avoids the senescence associated with prolonged high cell density. Suspension cultures are easily passaged with a small amount of culture containing a few cells diluted in a larger volume of fresh media. For adherent cultures, cells first need to be detached; this is commonly done with a mixture of trypsin - EDTA ; however, other enzyme mixes are now available for this purpose.

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A small number of detached cells can then be used to seed a new culture. Some cell cultures, such as RAW cells are mechanically scraped from the surface of their vessel with rubber scrapers. Another common method for manipulating cells involves the introduction of foreign DNA by transfection.

This is often performed to cause cells to express a gene of interest. DNA can also be inserted into cells using viruses, in methods referred to as transduction , infection or transformation. Viruses, as parasitic agents, are well suited to introducing DNA into cells, as this is a part of their normal course of reproduction. Cell lines that originate with humans have been somewhat controversial in bioethics , as they may outlive their parent organism and later be used in the discovery of lucrative medical treatments.

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In the pioneering decision in this area, the Supreme Court of California held in Moore v. Regents of the University of California that human patients have no property rights in cell lines derived from organs removed with their consent. It is possible to fuse normal cells with an immortalised cell line. This method is used to produce monoclonal antibodies.

In brief, lymphocytes isolated from the spleen or possibly blood of an immunised animal are combined with an immortal myeloma cell line B cell lineage to produce a hybridoma which has the antibody specificity of the primary lymphocyte and the immortality of the myeloma. Selective growth medium HA or HAT is used to select against unfused myeloma cells; primary lymphoctyes die quickly in culture and only the fused cells survive. These are screened for production of the required antibody, generally in pools to start with and then after single cloning.

A cell strain is derived either from a primary culture or a cell line by the selection or cloning of cells having specific properties or characteristics which must be defined. Cell strains are cells that have been adapted to culture but, unlike cell lines, have a finite division potential. Non-immortalized cells stop dividing after 40 to 60 population doublings [24] and, after this, they lose their ability to proliferate a genetically determined event known as senescence. Mass culture of animal cell lines is fundamental to the manufacture of viral vaccines and other products of biotechnology.

Culture of human stem cells is used to expand the number of cells and differentiate the cells into various somatic cell types for transplantation. Biological products produced by recombinant DNA rDNA technology in animal cell cultures include enzymes , synthetic hormones , immunobiologicals monoclonal antibodies , interleukins , lymphokines , and anticancer agents. Although many simpler proteins can be produced using rDNA in bacterial cultures, more complex proteins that are glycosylated carbohydrate-modified currently must be made in animal cells.

An important example of such a complex protein is the hormone erythropoietin. The cost of growing mammalian cell cultures is high, so research is underway to produce such complex proteins in insect cells or in higher plants, use of single embryonic cell and somatic embryos as a source for direct gene transfer via particle bombardment, transit gene expression and confocal microscopy observation is one of its applications. It also offers to confirm single cell origin of somatic embryos and the asymmetry of the first cell division, which starts the process.

Cell culture is also a key technique for cellular agriculture , which aims to provide both new products and new ways of producing existing agricultural products like milk, cultured meat , fragrances, and rhino horn from cells and microorganisms. It is therefore considered one means of achieving animal-free agriculture. It is also a central tool for teaching cell biology. Research in tissue engineering , stem cells and molecular biology primarily involves cultures of cells on flat plastic dishes.

This technique is known as two-dimensional 2D cell culture, and was first developed by Wilhelm Roux who, in , removed a portion of the medullary plate of an embryonic chicken and maintained it in warm saline for several days on a flat glass plate. From the advance of polymer technology arose today's standard plastic dish for 2D cell culture, commonly known as the Petri dish.

Julius Richard Petri , a German bacteriologist , is generally credited with this invention while working as an assistant to Robert Koch. Various researchers today also utilize culturing laboratory flasks , conicals, and even disposable bags like those used in single-use bioreactors.