Cell culture

Jump to: navigation, search
Epithelial cells in culture, stained for keratin (red) and DNA (green)

WikiDoc Resources for Cell culture

Articles

Most recent articles on Cell culture

Most cited articles on Cell culture

Review articles on Cell culture

Articles on Cell culture in N Eng J Med, Lancet, BMJ

Media

Powerpoint slides on Cell culture

Images of Cell culture

Photos of Cell culture

Podcasts & MP3s on Cell culture

Videos on Cell culture

Evidence Based Medicine

Cochrane Collaboration on Cell culture

Bandolier on Cell culture

TRIP on Cell culture

Clinical Trials

Ongoing Trials on Cell culture at Clinical Trials.gov

Trial results on Cell culture

Clinical Trials on Cell culture at Google

Guidelines / Policies / Govt

US National Guidelines Clearinghouse on Cell culture

NICE Guidance on Cell culture

NHS PRODIGY Guidance

FDA on Cell culture

CDC on Cell culture

Books

Books on Cell culture

News

Cell culture in the news

Be alerted to news on Cell culture

News trends on Cell culture

Commentary

Blogs on Cell culture

Definitions

Definitions of Cell culture

Patient Resources / Community

Patient resources on Cell culture

Discussion groups on Cell culture

Patient Handouts on Cell culture

Directions to Hospitals Treating Cell culture

Risk calculators and risk factors for Cell culture

Healthcare Provider Resources

Symptoms of Cell culture

Causes & Risk Factors for Cell culture

Diagnostic studies for Cell culture

Treatment of Cell culture

Continuing Medical Education (CME)

CME Programs on Cell culture

International

Cell culture en Espanol

Cell culture en Francais

Business

Cell culture in the Marketplace

Patents on Cell culture

Experimental / Informatics

List of terms related to Cell culture


Overview

Cell culture is the process by which either prokaryotic or eukaryotic cells are grown under controlled conditions. In practice the term "cell culture" has come to refer to the culturing of cells derived from multicellular eukaryotes, especially animal cells. The historical development and methods of cell culture are closely interrelated to those of tissue culture and organ culture.

Animal cell culture became a routine laboratory technique in the 1950s,[1] but the concept of maintaining live cell lines separated from their original tissue source was discovered in the 19th century.[2]

History

The 19th-century English physiologist Sydney Ringer developed salt solutions containing the chlorides of sodium, potassium, calcium and magnesium suitable for maintaining the beating of an isolated animal heart outside of the body.[1] In 1885 Wilhelm Roux removed a portion of the medullary plate of an embryonic chicken and maintained it in a warm saline solution for several days, establishing the principle of tissue culture.[3] Ross Granville Harrison, working at Johns Hopkins Medical School and then at Yale University, published results of his experiments from 1907-1910, establishing the methodology of tissue culture.[4]

Cell culture techniques were advanced significantly in the 1940s and 1950s to support research in virology. Growing viruses in cell cultures allowed preparation of purified viruses for the manufacture of vaccines. The Salk polio vaccine was one of the first products mass-produced using cell culture techniques. This vaccine was made possible by the cell culture research of John Franklin Enders, Thomas Huckle Weller, and Frederick Chapman Robbins, who were awarded a Nobel Prizefor their discovery of a method of growing the virus in monkey kidney cell cultures.

Concepts in mammalian cell culture

Isolation of cells

Cells can be isolated from tissues for ex vivo culture in several ways. Cells can be easily purified from blood, however only the white cellsare capable of growth in culture. Mononuclear cells can be released from soft tissues by enzymatic digestion with enzymes such as collagenase, trypsin, or pronase, which break down the extracellular matrix. Alternatively, pieces of tissue can be placed in growth media, and the cells that grow out are available for culture. This method is known as explant culture.

Cells that are cultured directly from a subject are known as primary cells. With the exception of some derived from tumours, most primary cell cultures have limited lifespan. After a certain number of population doublings cells undergo the process of senescence and stop dividing, while generally retaining viability.

An established or immortalised cell line has acquired the ability to proliferate indefinitely either through random mutation or deliberate modification, such as artificial expression of the telomerase gene. There are numerous well established cell lines representative of particular cell types.

Maintaining cells in culture

Cells are grown and maintained at an appropriate temperature and gas mixture (typically, 37°C, 5% CO2) in a cell incubator. Culture conditions vary widely for each cell type, and variation of conditions for a particular cell type can result in different phenotypes being expressed.

Aside from temperature and gas mixture, the most commonly varied factor in culture systems is the growth medium. Recipes for growth media can vary in pH, glucose concentration, growth factors, and the presence of other nutrient components. The growth factors used to supplement media are often derived from animal blood, such as calf serum. These blood-derived ingredients pose the potential for contamination of derived pharmaceutical products with viruses or prions. Current practice is to minimize or eliminate the use of these ingredients where possible.

Some cells naturally live without attaching to a surface, such as cells that exist in the bloodstream. Others require a surface, such as most cells derived from solid tissues. Cells grown unattached to a surface are referred to as suspension cultures. Other adherent cultures cells can be grown on tissue culture plastic, which may be coated with extracellular matrix components (e.g. collagen or fibronectin) to increase its adhesion properties and provide other signals needed for growth.

Manipulation of cultured cells

As cells generally continue to divide in culture, they generally grow to fill the available area or volume. This can generate several issues:

These issues can be dealt with using tissue culture methods that rely on sterile technique. These methods aim to avoid contamination with bacteria or yeast that will compete with mammalian cells for nutrients and/or cause cell infection and cell death. Manipulations are typically carried out in a biosafety hood or laminar flow cabinet to exclude contaminating micro-organisms. Antibiotics can also be added to the growth media.

Amongst the common manipulations carried out on culture cells are media changes, passaging cells, and transfecting cells.

Media changes

The purpose of media changes is to replenish nutrients and avoid the build up of potentially harmful metabolic byproducts and dead cells. In the case of suspension cultures, cells can be separated from the media by centrifugation and resuspended in fresh media. In the case of adherent cultures, the media can be removed directly by aspiration and replaced.

Passaging cells

Passaging 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 was historically 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.

Transfection and transduction

Another common method for manipulating cells involves the introduction of foreign DNA by transfection. This is often performed to cause cells to express a protein of interest. More recently, the transfection of RNAi constructs have been realised as a convenient mechanism for suppressing the expression of a particular gene/protein.

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.

Established human cell lines

One of the earliest human cell lines, the cultured HeLa cells shown here have been stained with Hoechst turning their nuclei blue.

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 1990 that human patients have no property rights in cell lines derived from organs removed with their consent. [5]

It is estimated that about 20% of human cell lines are not the kind of cells they were generally assumed to be.[6] The reason for this is that some cell lines exhibit vigorous growth and thus can cross-contaminate cultures of other cell lines, in time overgrowing and displacing the original cells. The most common contaminant is the HeLa cell line. While this may not be of significance when general properties such as cell metabolism are researched, it is highly relevant e.g. in medical research focusing on a specific type of cell. Results of such research will be at least flawed, if not outright wrong in their conclusion, with possible consequences if therapeutic approaches are developed based on it. [7]

Generation of hybridomas

For more details on this topic, see Hybridoma.

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 blood of an immunised animal are combined with hybridoma cell lines in a selective growth medium: only the fused cells survive.

Applications of cell culture

Mass culture of animal cell lines is fundamental to the manufacture of viral vaccines and many products of biotechnology. Biological products produced by recombinant DNA (rDNA) technology in animal cell cultures include enzymes, hormones, immunobiologicals (monoclonal antibodies, interleukins, lymphokines), andanticancer 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.

Tissue culture and engineering

Cell culture is a fundamental component of tissue culture and tissue engineering, as it establishes the basics of growing and maintaining cells ex vivo. I

Vaccines

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 flu vaccines is being funded by the United States government. Novel ideas in the field include recombinant DNA-based vaccines, such as one made using human adenovirus (a common cold virus) as a vector,[8][9] or the use of adjuvants.[10]

Culture of non-mammalian cells

Plant cell culture methods

Plant cell cultures are typically grown as cell suspension cultures in liquid medium or as callus cultures on solid medium. The culturing of undifferentiated plant cells and calli requires the proper balance of the plant growth hormones auxin and cytokinin.

Bacterial/Yeast culture methods

For bacteria and yeast, small quantities of cells are usually grown on a solid support that contains nutrients embedded in it, usually a gel such as agar, while large-scale cultures are grown with the cells suspended in a nutrient broth.

Viral culture methods

The culture of viruses requires the culture of cells of mammalian, plant, fungal or bacterial origin as hosts for the growth and replication of the virus. Wholewild type viruses, recombinant viruses or viral products may be generated in cell types other than their natural hosts under the right conditions. Depending on the species of the virus, infection and viral replication may result in host cell lysis and formation of a viral plaque.

Common cell lines

Human cell lines
Primate cell lines
  • Vero (African green monkey Chlorocebus kidney epithelial cell line initiated 1962)
  • COS-7 (African Green Monkey Kidney Cells)
Rat tumor cell lines
Mouse cell lines
Invertebrate cell lines
  • C6/36 Aedes albopictus (Asian tiger mosquito) larva
  • Insect cell line Sf21
Plant cell lines
  • Tobacco BY-2 cells (kept as cell suspension culture, they are model system of plant cell)
Other species cell lines

List of cell lines

cellline meaning Organism origin tissue morphology link
HEK-293 human embryonic kidney human kidney (embryonic) epithelium ATCC
HeLa Henrietta Lacks human Cervical cancer epithelium DSMZ
CHO Chinese hamster ovary hamster Ovary epithelium ICLC
Sf-9 Spodoptera frugiperda insect - Spodoptera frugiperda (moth) Ovary DSMZ
NIH-3T3 NIH, 3-day transfer, inoculum 3 x 105 cells mouse embryo fibroblast ATCC
MTD-1A mouse epithelium
bEnd.5 brain endothelial mouse brain endothelium
bEnd.3 brain endothelial mouse brain / cerebral Cortex endothelium ATCC
MCF-10A Michigan Cancer Foundation human mammary gland epithelium ATCC
T84 human colorectal Carcinoma / lungmetastasis epithelium ATCC
HUVEC human umbilical vein endothelial cells human Umbilical cord vein endothelium ICLC
HMEC human mammary epithelial cell human epithelium
Peer human T cell leukemia DSMZ
MDCK II Madin Darby canine kidney dog kidney epithelium ATCC
CMT canine mammary tumor dog mammary gland epithelium
MyEnd myocardial endothelial mouse endothelium
COS-7 Cercopithecus aethiops, origin-defective SV-40 ape - Cercopithecus aethiops (Chlorocebus) kidney fibroblast ATCC
HL-60 human leukemia human Myeloblast bloodcells DSMZ
A-549 human lungcarcinoma epithelium DSMZ
Jurkat human T-Cell-Leukemia bloodcells DSMZ
LNCap human prostatic adenocarcinoma epithelium ATCC

See also

References and notes

  1. ""Cell Culture"". Retrieved 2006-04-19. 
  2. ""Some landmarks in the development of tissue and cell culture."". Retrieved 2006-04-19. 
  3. ""Animals and alternatives in testing."". Retrieved 2006-04-19. 
  4. Schiff, Judith Ann. ""An unsung hero of medical research."". Retrieved 2006-04-19.  Yale Alumni Magazine, February 2002.
  5. ceb
  6. (MacLeoud et al. 1999)
  7. (Masters 2002). See List of contaminated cell lines.
  8. cold as vector news
  9. cold as vector research abstract
  10. adjuvant
  • MacLeod, R. A. F. et al. (1999): Widespread intraspecies cross-contamination of human tumour cell lines. International Journal of Cancer 83:555–563.
  • Masters, John R. (2002): HeLa cells 50 years on: the good, the bad and the ugly. Nature Reviews Cancer 2:315-319.

External links


Linked-in.jpg