Monday, January 21, 2008

cell culture


























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,[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 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) 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 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

Main article: Passaging

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 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

Main article: transfection

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. see concentration

Established human cell lines

One of the earliest human cell lines, descended from Henrietta Lacks, who died of the cancer that those cells originated from, the cultured HeLa cells shown here have been stained with Hoechst turning their nuclei blue.
One of the earliest human cell lines, descended from Henrietta Lacks, who died of the cancer that those cells originated from, 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), 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,[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

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.

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.

Cell Culture Assays


In Biomaterials Testing, a cell culture assay is any method which is used to assess the cytotoxicity of a material. This refers to the in vitro assessment of material to determine whether or not it releases toxic chemicals in sufficient quantities to kill cells either directly or indirectly through the inhibition of cell metabolic pathways. Cell culture evaluations are the precursor to whole animal studies and are a way to determine if significant cytotoxicity exists for the given material. Cell culture assays are standardized by ASTM, ISO, and BSI (British Standards Institution.)


Direct Contact Method
1. A near confluent layer of fibroblasts are prepared in a culture plate
2. Old cell culture media (agar generally) is removed
3. Fresh media is added
4. Material being tested is placed onto the cultures, which are incubated for 24 hours at 37 degrees celsius
5. The material is removed
6. The culture media is removed
7. The remaining cells are fixed and stained, dead cells are lost during fixation and only the live cells are stained
8. The toxicity of the material is indicated by the absence of stained cells around the material


Agar Diffusion Method
1. A near confluent layer of fibroblasts are prepared in a culture plate
2. Old cell culture media is removed
3. The cells are covered with a solution of 2% agar, which often contains red vital stain
4. When the agar solidifies the cells will have dispersed throughout its volume
5. The material is then placed on the surface of the agar and incubated for 24 hours at 37 degrees celsius
6. Live cells take up the vital stain and retain it, dead cells do not
7. The toxicity of the material is evaluated by the loss of vital stain under and around the material
8. Surface microscopy is also needed to evaluate the material-cell interfacae


Elution Method
1. A near confluent layer of fibroblasts are prepared in a culture plate
2. An extract of the material which is being tested is prepared using physiological saline or serum free media (the latter is generally preferred)
3. Extraction conditions are used which are appropriate for the type of exposure which the cells would receive in the in vivo environment if the material were to be implanted
4. The extract is placed on the cells and incubated for 48 hours at 37 degrees celsius
5. After 48 hours the toxicity is evaluated using either a histochemical or vital stain


Each method has its own advantages and disadvantages, and some are more suitable for certain applications than others. For example the direct contact method offers conditions which are most similar to the physiological environment but the cells are suceptible to trauma if the material moves. The agar diffusion method is good for materials with high densities and offers an even concentration gradient for potential toxicants, but there is a serious risk of the cells going into thermal shock when they are overlayed with agar. The elution method is best for applications which might require extra incubation time, but additional time and steps are required for preparing such a test.

In vitro biomaterials testing yields fundamental information about the behavior of materials in contact with living cells, but cannot qualify or even accurately predict the performance of a material in vivo.



cell,the functional unit





















Animal Cell Structure

Animal cells are typical of the eukaryotic cell, enclosed by a plasma membrane and containing a membrane-bound nucleus and organelles. Unlike the eukaryotic cells of plants and fungi, animal cells do not have a cell wall. This feature was lost in the distant past by the single-celled organisms that gave rise to the kingdom Animalia. Most cells, both animal and plant, range in size between 1 and 100 micrometers and are thus visible only with the aid of a microscope.

The lack of a rigid cell wall allowed animals to develop a greater diversity of cell types, tissues, and organs. Specialized cells that formed nerves and muscles—tissues impossible for plants to evolve—gave these organisms mobility. The ability to move about by the use of specialized muscle tissues is a hallmark of the animal world, though a few animals, primarily sponges, do not possess differentiated tissues. Notably, protozoans locomote, but it is only via nonmuscular means, in effect, using cilia, flagella, and pseudopodia.

The animal kingdom is unique among eukaryotic organisms because most animal tissues are bound together in an extracellular matrix by a triple helix of protein known as collagen. Plant and fungal cells are bound together in tissues or aggregations by other molecules, such as pectin. The fact that no other organisms utilize collagen in this manner is one of the indications that all animals arose from a common unicellular ancestor. Bones, shells, spicules, and other hardened structures are formed when the collagen-containing extracellular matrix between animal cells becomes calcified.

Animals are a large and incredibly diverse group of organisms. Making up about three-quarters of the species on Earth, they run the gamut from corals and jellyfish to ants, whales, elephants, and, of course, humans. Being mobile has given animals, which are capable of sensing and responding to their environment, the flexibility to adopt many different modes of feeding, defense, and reproduction. Unlike plants, however, animals are unable to manufacture their own food, and therefore, are always directly or indirectly dependent on plant life.

Most animal cells are diploid, meaning that their chromosomes exist in homologous pairs. Different chromosomal ploidies are also, however, known to occasionally occur. The proliferation of animal cells occurs in a variety of ways. In instances of sexual reproduction, the cellular process of meiosis is first necessary so that haploid daughter cells, or gametes, can be produced. Two haploid cells then fuse to form a diploid zygote, which develops into a new organism as its cells divide and multiply.

The earliest fossil evidence of animals dates from the Vendian Period (650 to 544 million years ago), with coelenterate-type creatures that left traces of their soft bodies in shallow-water sediments. The first mass extinction ended that period, but during the Cambrian Period which followed, an explosion of new forms began the evolutionary radiation that produced most of the major groups, or phyla, known today. Vertebrates (animals with backbones) are not known to have occurred until the early Ordovician Period (505 to 438 million years ago).

Cells were discovered in 1665 by British scientist Robert Hooke who first observed them in his crude (by today's standards) seventeenth century optical microscope. In fact, Hooke coined the term "cell", in a biological context, when he described the microscopic structure of cork like a tiny, bare room or monk's cell. Illustrated in Figure 2 are a pair of fibroblast deer skin cells that have been labeled with fluorescent probes and photographed in the microscope to reveal their internal structure. The nuclei are stained with a red probe, while the Golgi apparatus and microfilament actin network are stained green and blue, respectively. The microscope has been a fundamental tool in the field of cell biology and is often used to observe living cells in culture. Use the links below to obtain more detailed information about the various components that are found in animal cells.

  • Centrioles - Centrioles are self-replicating organelles made up of nine bundles of microtubules and are found only in animal cells. They appear to help in organizing cell division, but aren't essential to the process.

  • Cilia and Flagella - For single-celled eukaryotes, cilia and flagella are essential for the locomotion of individual organisms. In multicellular organisms, cilia function to move fluid or materials past an immobile cell as well as moving a cell or group of cells.

  • Endoplasmic Reticulum - The endoplasmic reticulum is a network of sacs that manufactures, processes, and transports chemical compounds for use inside and outside of the cell. It is connected to the double-layered nuclear envelope, providing a pipeline between the nucleus and the cytoplasm.

  • Endosomes and Endocytosis - Endosomes are membrane-bound vesicles, formed via a complex family of processes collectively known as endocytosis, and found in the cytoplasm of virtually every animal cell. The basic mechanism of endocytosis is the reverse of what occurs during exocytosis or cellular secretion. It involves the invagination (folding inward) of a cell's plasma membrane to surround macromolecules or other matter diffusing through the extracellular fluid.

  • Golgi Apparatus - The Golgi apparatus is the distribution and shipping department for the cell's chemical products. It modifies proteins and fats built in the endoplasmic reticulum and prepares them for export to the outside of the cell.

  • Intermediate Filaments - Intermediate filaments are a very broad class of fibrous proteins that play an important role as both structural and functional elements of the cytoskeleton. Ranging in size from 8 to 12 nanometers, intermediate filaments function as tension-bearing elements to help maintain cell shape and rigidity.

  • Lysosomes - The main function of these microbodies is digestion. Lysosomes break down cellular waste products and debris from outside the cell into simple compounds, which are transferred to the cytoplasm as new cell-building materials.

  • Microfilaments - Microfilaments are solid rods made of globular proteins called actin. These filaments are primarily structural in function and are an important component of the cytoskeleton.

  • Microtubules - These straight, hollow cylinders are found throughout the cytoplasm of all eukaryotic cells (prokaryotes don't have them) and carry out a variety of functions, ranging from transport to structural support.

  • Mitochondria - Mitochondria are oblong shaped organelles that are found in the cytoplasm of every eukaryotic cell. In the animal cell, they are the main power generators, converting oxygen and nutrients into energy.

  • Nucleus - The nucleus is a highly specialized organelle that serves as the information processing and administrative center of the cell. This organelle has two major functions: it stores the cell's hereditary material, or DNA, and it coordinates the cell's activities, which include growth, intermediary metabolism, protein synthesis, and reproduction (cell division).

  • Peroxisomes - Microbodies are a diverse group of organelles that are found in the cytoplasm, roughly spherical and bound by a single membrane. There are several types of microbodies but peroxisomes are the most common.

  • Plasma Membrane - All living cells have a plasma membrane that encloses their contents. In prokaryotes, the membrane is the inner layer of protection surrounded by a rigid cell wall. Eukaryotic animal cells have only the membrane to contain and protect their contents. These membranes also regulate the passage of molecules in and out of the cells.

  • Ribosomes - All living cells contain ribosomes, tiny organelles composed of approximately 60 percent RNA and 40 percent protein. In eukaryotes, ribosomes are made of four strands of RNA. In prokaryotes, they consist of three strands of RNA.

In addition the optical and electron microscope, scientists are able to use a number of other techniques to probe the mysteries of the animal cell. Cells can be disassembled by chemical methods and their individual organelles and macromolecules isolated for study. The process of cell fractionation enables the scientist to prepare specific components, the mitochondria for example, in large quantities for investigations of their composition and functions. Using this approach, cell biologists have been able to assign various functions to specific locations within the cell. However, the era of fluorescent proteins has brought microscopy to the forefront of biology by enabling scientists to target living cells with highly localized probes for studies that don't interfere with the delicate balance of life processes.