Please give us your feedback so we can improve the information on the page. Thank you in advance for your help. Please add your email address if you would like a reply. This third edition of Human Cell Culture Protocols expands upon the previous editions with current, detailed protocols for the isolation and culture of a range of primary cells from human tissues. With new chapters on pancreatic cells needed for basic studies on the pathogenesis of diabetes and for their application for islet transplantation, the book also delves into protocols for hepatocytes, skin cells, lung cells, parathyroid cells, gastric cells, renal cells, adipocytes, ovarian cells, bone cells, vascular smooth muscle cells, vascular endothelial cells, regulatory T cells, blood mononuclear cells, as well as new techniques being applied to human cell culture, particularly the use of biocompatible scaffolds to grow cells, the in vitro use of laser microdissection to isolate cells from culture, and automated cell culture.
Yes, I found it! 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. 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.
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.
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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  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.
Aside from Petri dishes, scientists have long been growing cells within biologically derived matrices such as collagen or fibrin, and more recently, on synthetic hydrogels such as polyacrylamide or PEG.
They do this in order to elicit phenotypes that are not expressed on conventionally rigid substrates. There is growing interest in controlling matrix stiffness ,  a concept that has led to discoveries in fields such as:. Cell culture in three dimensions has been touted as "Biology's New Dimension". Scaffold based cultures utilize an acellular 3D matrix or a liquid matrix. Scaffold-free methods are normally generated in suspensions. Eric Simon, in a NIH SBIR grant report, showed that electrospinning could be used to produced nano- and submicron-scale polystyrene and polycarbonate fibrous scaffolds specifically intended for use as in vitro cell substrates.
It was noted that, as opposed to the flattened morphology typically seen in 2D culture, cells grown on the electrospun fibers exhibited a more histotypic rounded 3-dimensional morphology generally observed in vivo.
As the natural extracellular matrix ECM is important in the survival, proliferation, differentiation and migration of cells, different hydrogel culture matrices mimicking natural ECM structure are seen as potential approaches to in vivo —like cell culturing. Several different types of hydrogels from natural and synthetic materials are available for 3D cell culture, including animal ECM extract hydrogels, protein hydrogels, peptide hydrogels, polymer hydrogels, and wood-based nanocellulose hydrogel.
The magnetic nanoparticle assemblies consist of magnetic iron oxide nanoparticles, gold nanoparticles, and the polymer polylysine. Cell culture is a fundamental component of tissue culture and tissue engineering , as it establishes the basics of growing and maintaining cells in vitro. The major application of human cell culture is in stem cell industry, where mesenchymal stem cells can be cultured and cryopreserved for future use.
Tissue engineering potentially offers dramatic improvements in low cost medical care for hundreds of thousands of patients annually. Vaccines for polio , measles , mumps , rubella , and chickenpox are currently made in cell cultures. Due to the H5N1 pandemic threat, research into using cell culture for influenza vaccines is being funded by the United States government.
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Novel ideas in the field include recombinant DNA -based vaccines, such as one made using human adenovirus a common cold virus as a vector,   and novel adjuvants. Besides the culture of well-established immortalised cell lines, cells from primary explants of a plethora of organisms can be cultured for a limited period of time before sensecence occurs see Hayflick's limit. Cultured primary cells have been extensively used in research, as is the case of fish keratocytes in cell migration studies.
Plant cell cultures are typically grown as cell suspension cultures in a liquid medium or as callus cultures on a solid medium. The culturing of undifferentiated plant cells and calli requires the proper balance of the plant growth hormones auxin and cytokinin. Cells derived from Drosophila melanogaster most prominently, Schneider 2 cells can be used for experiments which may be hard to do on live flies or larvae, such as biochemical studies or studies using siRNA.
A convenient protocol for establishing a human cell culture model of the outer retina.
FAQ Policy. About this book Human cell culture is not a new topic, but the development of new molecular techniques and reagents which can be used to investigate cell function and the responsible intracellular mechanisms make it a continuing requirement. Show all. Show next xx. Read this book on SpringerLink. Recommended for you. Mitry Robin D.