Cell culture Totally Explained

Everything about Cell Culture totally explained

Cell culture is the process by which prokaryotic, eukaryotic or plant 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, but the concept of maintaining live cell lines separated from their original tissue source was discovered in the 19th century.

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.(External Link

) 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. 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.
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 Prize for 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 cells are 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 for mammalian cells) 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 nutrients. The growth factors used to supplement media are often derived from animal blood, such as calf serum. One complication of these blood-derived ingredients is the potential for contamination of the culture with viruses or prions. Current practice is to minimize or eliminate the use of these ingredients where possible.
Cells can be grown in suspension or adherent cultures. Some cells naturally live in suspension, without being attaching to a surface, such as cells that exist in the bloodstream. There are also cell lines that have been modified to be able to survive in suspension cultures so that they can be grown to a higher density than adherent conditions would allow. Adherent cells require a surface, such as tissue culture plastic, which may be coated with extracellular matrix components to increase adhesion properties and provide other signals needed for growth and differentiation. Most cells derived from solid tissues are adherent. Another type of adherent culture is organotypic culture which involves growing cells in a three-dimensional environment as opposed to two-dimensional culture dishes. This 3D culture system is biochemically and physiologically more similar to in vivo tissue, but is technically challenging to maintain because of many factors (for example diffusion).

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:

Nutrient depletion in the growth media
Accumulation of apoptotic/necrotic (dead) cells.
Cell-to-cell contact can stimulate cell cycle arrest, causing cells to stop dividing known as contact inhibition or senescence.
Cell-to-cell contact can stimulate cellular differentiation.

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 sterile technique. Sterile technique aims to avoid contamination with bacteria, yeast, or other cell lines. 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.

Media changes

Media changes 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 resuspension in fresh media. In the case of adherent cultures, the media can be removed directly by aspiration and replaced.

Passaging cells

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're 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.

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 realized 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

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. It is estimated that about 20% of human cell lines are not the kind of cells they were generally assumed to be. 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's highly relevant for example 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.

Generation of hybridomas

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 specifity of the primary lymphoctye and the immortality of the myleoma. 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.

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), 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.

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.

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 influenza 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, or the use of adjuvants.

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. Whole wild 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

National Cancer Institute's 60 cancer cell lines

A172 (glioma)

A549 (lung cancer)

BCP-1 cells (PEL)

HEK 293 cells (kidney - original HEK line is contaminated with HeLa)

HeLa (cervical cancer)

HL60 (promyelocytic leukemia)

K562 (chronic myeloid leukemia)

KG-1 (myelogenous leukaemia)

Jurkat cell line, derived from a patient with T cell leukemia

Lncap (Prostate Cancer)

MCF-7 (breast cancer)

MDA-MB-438 (breast cancer)

THP-1 (acute myeloid leukemia)

U87 (glioblastoma)

SHSY5Y Human neuroblastoma cells, cloned from a myeloma

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

GH3 (pituitary tumor)

9L (glioblastoma)

PC12 (pheochromocytoma)

Mouse cell lines

3T3 cells (embryonic fibroblast)

MC3T3 (embryonic calvarial)

C3H-10T1/2 (embryonic mesenchymal)

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're model system of plant cell)

Other species cell lines

zebrafish ZF4 and AB9 cells.

Madin-Darby Canine Kidney (MDCK) epithelial cell line

Chinese Hamster Ovary CHO cells

Xenopus A6 kidney epithelial cells.

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.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
BxPC3	Biopsy xenograph of pancreatic carcinoma line 3	human	pancreatic adenocarcinoma	epithelial	ATCC
Hepa1c1c7	clone 7 of clone 1 hepatoma line 1	mouse	Hepatoma	epithelial	ATCC

Note: this list is a sample of available cell lines, and isn't comprehensive

Further Information

Get more info on 'Cell Culture'.

External Link Exchanges

Do you know how hard it is to get a link from a large encyclopaedia? Well we're different and will prove it. To get a link from us just add the following HTML to your site on a relevant page:

<a href="http://cell_culture.totallyexplained.com">Cell culture Totally Explained</a>

Then simply click through this link from your web page. Our crawlers will verify your link, extract the title of your web page and instantly add a link back to it. If you like you can remove the words Totally Explained and embed the link in article text.
As long as your link remains in place, we'll keep our link to you right here. Please play fair - our crawlers are watching. Your site must be closely related to this one's topic. Any kind of spamming, dubious practises or removing the link will result in your link from us being dropped and, potentially, your whole site being banned.