Tissue Culture

Plant Tissue Culture

2010-02-09T22:51:00.000-08:00

Plant Tissue Culture

Presenter: Lydiane (Ann) Kyte
Host: Kathy Liu
Discussion

Did you ever have a plant that was so unique or so beautiful that you wished you had hundreds or thousands of them to enjoy or to sell? Plant tissue culture (micropropagation) is a technique which will do just that for us. We are going to discuss this tool which is used so extensively in the nursery business and in plant biotechnology. It is a fascinating and useful tool which allows the rapid production of many genetically identical plants using relatively small amounts of space, supplies and time.

Basically the technique consists of taking a piece of a plant (such as a stem tip, node, meristem, embryo, or even a seed) and placing it in a sterile, (usually gel-based) nutrient medium where it multiplies. The formulation of the growth medium is changed depending upon whether you are trying to get the plant to produce undifferentiated callus tissue, multiply the number of plantlets, grow roots, or multiply embryos for "artificial seed".

For many who become superficially aware of the technique it seems shrouded in mystery and is shrugged off as too technical to be of concern. Actually, it is no more of a mystery than taking a cutting of your favorite house plant and growing it to share with a friend. As for being technical, you can begin plant tissue culture with as little as a cookbook approach and a feeling for sterile technique.

Some people have visions of scientists doing plant tissue cultures in white gowns and masks in hospital-clean environments. Such conditions are excessive. While it is true that mold spores, bacteria, and other contaminants will grow and overrun a culture, air that is not moving has a minimum of contaminants. In addition, disinfection of implements, work surface and nearby areas helps eliminate contaminants.

The guidelines for preparation and the laboratory protocol provided here are given as a place to begin. Included with is a limited discussion of some of the many options you have as you explore micropropagation. We can discuss these in more depth if you have questions, concerns or related experiences to share. I would be particularly interested in success and challenges you may have had or are currently having in your classroom.

Some suggestions are given for the following
(a) Selecting plant sources. Some species, or even clones are easier to grow in culture than others. Some respond reluctantly to culture, some do not respond at all, and many plants have never been tried.

(b) Choosing a growth medium (price, convenience, type of plant and purpose of the micropropagation all enter into this decision.) How important are the kinds of hormones used? On limited scale, media ingredients are available at the grocery and health food stores.

(c) Suggestions for media preparation and sterilization. There are alternatives to sterilization in a pressure cooker or an autoclave.

(d) Methods for cleaning, storing and manipulating explants (plant pieces to be cultured).

Given certain basics there are many options for procedure, equipment and supplies for plant tissue culture. Some of your decisions will be based upon the amount of time, money and space you have. Other decisions will be based upon why you are doing plant tissue culture and what you expect as a result (more plants?) . Catalogs, such as Sigma, Carolina Biological, or Edmund Scientific are good reference and they are for purchasing needed materials..

I look forward to sharing tissue culture experiences with you.

References:
Debergh, P.C. and R.H. Zimmerman, eds. 1991. Micropropagation, Technology and Application. Kluwer Academic Publishers. $61.50. Lab design, info on labs worldwide, in depth discussions of problems. Not for the beginner.

Donnelly, D.J., and W.E.Vidaver, 1988. Glossary of Plant Tissue Culture, Portland, OR. Timber Press, $22.95. Good definitions of tissue culture terms.

Kyte, Lydiane and J. Kleyn, 1996. Plants from Test Tubes: An Introduction to Micropropagation, 3rd ed., Timber Press, 1996 $29.95. Good basics for the beginning amateur or grower.

Smith, Roberta H., 1992. Plant Tissue Culture-Techniques and Experiments. Academic Press. $35.00. Good introduction and broad base for college course.

Trigiano, Robert N, and Dennis J. Gray, eds.1996,Plant Tissue Culture Concepts and Laboratory Exercises. CRC Press. $65.00. For the advanced student.

Sources of supplies:

Carolina Biological

Edmond Scientific

PhytoTechnology Laboratories
This company specializes in plant tissue culture supplies. Downloadable documents (choose MS Word or PDF format) on Media Preparation, Setting Up a Tissue Culture Lab, Basic Laboratory Procedures, and more are available in the "Technical" section of the web site.

Sigma, 1996. "Plant Tissue Culture Catalog".
In addition to media ingredients, premixes, equipment and supplies, this catalog contains a media comparison chart, procedures for media preparations.references and other valuable data.

REPEREN :http://www.accessexcellence.org/LC/ST/st2bgplant.php

Plant Tissue Culture Media

2010-02-09T22:41:00.000-08:00

Plant Tissue Culture Media

Plant Tissue Culture refers to the technique of growing plant cells, tissues, organs, seeds or other plant parts in a sterile environment on a nutrient medium. Culture media used for in vitro cultivation of plant cells are composed of following basic components:

	Complex Mixture of Salts: Essential elements, or mineral ions
	Organic Supplement: vitamins and/or amino acids
	Carbon Source: usually sugar sucrose
	Gelling Agents
	Plant Growth Regulators
	Antibiotics

Complex Mixture of Salts

These include essential elements or mineral ions important for plant nutrition and their physiological function. The essential elements can further be divided into the following categories:

	Macroelements (or macronutrients)
	Microelements (or micronutrients)
	Iron source

Macroelements	:-	These elements are required in large amounts for plant growth and development. Nitrogen, phosphorus, potassium, magnesium, calcium and sulphur (and carbon, which is added separately) are regarded as macroelements. These elements comprise at least 0.1% of the dry weight of plants.

Microelements	:-	These elements are required in trace amounts for plant growth and development. Manganese, iodine, copper, cobalt, boron, molybdenum, iron and zinc are regarded as microelements, although other elements like aluminium and nickel are frequently found in some formulations.


Iron Source	:-	Iron is usually added in the medium as iron sulphate, although iron citrate can also be used. Ethylenediaminetetraacetic acid (EDTA) is usually used in conjunction with the iron sulphate. The EDTA complexes with the iron so as to allow the slow and continuous release of iron into the medium. Uncomplexed iron can precipitate out of the medium as ferric oxide.

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Organic Supplements

These include vitamins and amino acids. Two vitamins, i.e., thiamine (vitamin B1) and myoinositol (a vitamin B) are essential for the culture of plant cells in vitro. However, other vitamins are often added to for historical reasons. The most commonly used amino acid is glycine. However, arginine, asparagine, aspartic acid, alanine, glutamic acid, glutamine and proline are also used. Amino acids provide a source of reduced nitrogen and, like ammonium ions, uptake causes acidification of the medium. Casein hydrolysate can be used as a source of a mixture of amino acids.

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Carbon Source

The most commonly used carbon source is sucrose. It is readily assimilated and relatively stable. Other carbohydrates like glucose, maltose, galactose and sorbitol can also be used and may prove better than sucrose in specialized circumstances.

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Gelling Agents

Plant tissue culture media can be used in either liquid or ‘solid’ forms, depending on the type of culture being grown. Agar, produced from seaweed, is the most common type of gelling agent, and is ideal for routine applications. For more demanding applications, a range of purer gelling agents are available. Purified agar or agarose can be used, as can a variety of gellan gums.

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Plant Growth Regulators

Specific media manipulations can be used to direct the development of plant cells in culture due to plasticity and totipotency. Plant growth regulators are the critical media components in determining the developmental pathway of the plant cells. There are five main classes of plant growth regulator used in plant cell culture, namely:

	Auxins
	Cytokinins
	Gibberellins
	Abscisic Acid
	Ethylene


Auxins	:-	Auxins promote both cell division and cell growth. IAA (indole-3-acetic acid) is the most important naturally occurring auxin but its use in plant tissue culture media is limited because it is unstable to both heat and light. 2,4-Dichlorophenoxyacetic acid (2,4-D) is the most commonly used auxin and is extremely effective in most circumstances.

Cytokinins	:-	Cytokinins promote cell division. Of the naturally occurring cytokinins, only zeatin and 2iP (2-isopentyl adenine have some use in plant tissue culture media. The synthetic analogues, kinetin and BAP (benzylaminopurine), are used more frequently. Non-purine-based chemicals, such as substituted phenylureas, are also used as cytokinins in plant tissue culture media.

Auxins	:-	Auxins promote both cell division and cell growth. IAA (indole-3-acetic acid) is the most important naturally occurring auxin but its use in plant tissue culture media is limited because it is unstable to both heat and light. 2,4-Dichlorophenoxyacetic acid (2,4-D) is the most commonly used auxin and is extremely effective in most circumstances.


Cytokinins	:-	Cytokinins promote cell division. Of the naturally occurring cytokinins, only zeatin and 2iP (2-isopentyl adenine have some use in plant tissue culture media. The synthetic analogues, kinetin and BAP (benzylaminopurine), are used more frequently. Non-purine-based chemicals, such as substituted phenylureas, are also used as cytokinins in plant tissue culture media.

Gibberellins	:-	Gibberellins are involved in regulating cell elongation, in determining plant height and fruit-set. Only a few of the gibberellins like GA3 are used in plant tissue culture media.
Abscisic Acid	:-	It is used in plant tissue culture to promote distinct developmental pathways such as somatic embryogenesis. Abscisic acid (ABA) inhibits cell division.

Ethylene	:-	Ethylene is associated with controlling fruit ripening in climacteric fruits, and its use in plant tissue culture is not widespread. Some plant cell cultures produce ethylene, which, if it builds up sufficiently, can inhibit the growth and development of the culture.

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Antibiotics

Antibiotics are substances produced by certain microorganisms that suppress the growth of other microorganisms and eventually destroy them. Their applications include:

A.	Suppresses bacterial infections in plant cell and tissue culture.
B.	Suppresses mould and yeast infections in cell cultures.
C.	Eliminates Agrobacterium species after the transformation of plant tissue.

These antibiotics can be divided into different classes on the basis of chemical structure and their mechanism of action:

	Inhibitors of Bacterial Cell Wall Synthesis
	e.g. β-lactam antibiotics, Penicillins and Cephalosporins.
	Antibiotics that affect Cell Membrane permeability.
	• Antibacterial e.g. Colistin Sulphate, Polymixin B Sulphate, Gramicidin • Antifungal e.g. Amphotericin B, Nystatin, Pimaricin
	Bacteriostatic Inhibitors of Protein

		Antibiotics that affect the function of 30 S or 50 S ribosomal subunits to cause a reversible inhibition of protein synthesis. e.g. Chloramphenicol, Chlortetracycline HCl, Clindamycin HCl, Doxycycline HCl, Erythromycin, Lincomycin HCl, Oxytetracycline HCl, Spectinomycin sulphate, Tetracycline HCl, Tylosin tartrate, Lincomycin HCl
		Bactericide Inhibitors of Protein Synthesis
		Antibiotics that bind to the 30 S ribosomal subunit and alter protein synthesis which eventually leads to cell death. This group includes:
		Aminoglycosides: Apramycin, Butirosine, Gentamicin, Kanamycin, Neomycin, Streptomycin, Tobramycin. Inhibitors of Nucleic Acid Metabolism: e.g. Rifampicin, Mitomycin C and Nalidixic acid Antimetabolites: Antibiotics, which block specific metabolic steps that are essential to microorganisms e.g. Metronidazole, Miconazole, Nitrofurantoin, Trimethoprim and Sulphomethoxazole. Nucleic Acid Analogs, which inhibit enzymes essential for DNA synthesis. e.g. 5-Fluorouracil, Mercaptopurine.

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Preparation of Plant Tissue Culture Medium

	Measure approximately 90% of the required volume of the deionized-distilled water in a flask/container of double the size of the required volume.

	Add the dehydrated medium into the water and stir to dissolve the medium completely. Gentle heating of the solution may be required to bring powder into solution.

	Add desired heat stable supplements to the medium solution.

	Add additional deionized-distilled water to the medium solution to obtain the final required volume.

	Set the desired pH with NaOH or HCl.

	Dispense the medium into culture vessels.

	Sterilize the medium by autoclaving at 15 psi (121^οC) for appropriate time period. Higher temperature
	may result in poor cell growth.

	Add heat labile supplements after autoclaving.

referens by: http://www.planttissueculturemedia.com/

Cell culture

2010-02-09T22:36:00.000-08:00

Cell culture

Cell culture is the process by which 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 common laboratory technique in the mid-1900s,^[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.

[edit] Concepts in mammalian cell culture

[edit] 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 tumors, 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.

[edit] 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, particularly in biotechnology medical applications. Current practice is to minimize or eliminate the use of these ingredients wherever possible, but this cannot always be accomplished. Alternative strategies involve sourcing the animal blood from countries with minimum BSE/TSE risk such as Australia and New Zealand, and using purified nutrient concentrates derived from serum in place of whole animal serum for cell culture.^[5]
Cells can be grown in suspension or adherent cultures. Some cells naturally live in suspension, without being attached 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 or microcarrier, 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 (e.g. diffusion).

[edit] Cell line cross-contamination

Cell line cross-contamination can be a problem for scientists working with cultured cells. Studies suggest that anywhere from 15–20% of the time, cells used in experiments have been misidentified or contaminated with another cell line.^[6]^[7]^[8] Problems with cell line cross contamination have even been detected in lines from the NCI-60 panel, which are used routinely for drug-screening studies.^[9]^[10] Major cell line repositories including the American Type Culture Collection (ATCC) and the German Collection of Microorganisms and Cell Cultures (DSMZ) have received cell line submissions from researchers that were misidentified by the researcher.^[9]^[11] Such contamination poses a problem for the quality of research produced using cell culture lines, and the major repositories are now authenticating all cell line submissions.^[12] ATCC uses short tandem repeat (STR) DNA fingerprinting to authenticate its cell lines.^[13]
To address this problem of cell line cross-contamination, researchers are encouraged to authenticate their cell lines at an early passage to establish the identity of the cell line. Authentication should be repeated before freezing cell line stocks, every two months during active culturing and before any publication of research data generated using the cell lines. There are many methods for identifying cell lines including isoenzyme analysis, human lymphocyte antigen (HLA) typing and STR analysis.^[13]
One significant cell-line cross contaminant is the immortal HeLa cell line.

[edit] 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 (e.g. penicillin and streptomycin) and antifungals (e.g. Amphotericin B) can also be added to the growth media.
As cells undergo metabolic processes, acid is produced and the pH decreases. Often, a pH indicator is added to the medium in order to measure nutrient depletion.

[edit] Media changes

In the case of adherent cultures, the media can be removed directly by aspiration and replaced.

[edit] Passaging cells

Main article: Passaging

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.

[edit] Transfection and transduction

Main article: transfection

Main article: transformation (genetics)

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.

[edit] 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.

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.^[14]

[edit] 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 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 lymphoctye 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.

[edit] 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, 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 practicle 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.

[edit] 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. The major application of human cell culture is in stem cell industry where mesenchymal stem cells can be cultured and cryopreserved for future use.

[edit] 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,^[15]^[16] or the use of adjuvants.^[17]

[edit] Culture of non-mammalian cells

[edit] Plant cell culture methods

[edit] Bacterial/Yeast culture methods

Main article: microbiological culture

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.

[edit] Viral culture methods

Main article: Viral culture

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.

[edit] Common cell lines

Human cell lines

National Cancer Institute's 60 cancer cell lines
ESTDAB database http://www.ebi.ac.uk/ipd/estdab/directory.html

DU145 (Prostate cancer)
Lncap (Prostate cancer)
MCF-7 (breast cancer)
MDA-MB-438 (breast cancer)
PC3 (Prostate cancer)
T47D (breast cancer)
THP-1 (acute myeloid leukemia)
U87 (glioblastoma)
SHSY5Y Human neuroblastoma cells, cloned from a myeloma
Saos-2 cells (bone cancer)

Primate cell lines

Vero (African green monkey Chlorocebus kidney epithelial cell line initiated 1962)

Rat tumor cell lines

GH3 (pituitary tumor)
PC12 (pheochromocytoma)

Mouse cell lines

MC3T3 (embryonic calvarial)

Plant cell lines

Tobacco BY-2 cells (kept as cell suspension culture, they are model system of plant cell)

Other species cell lines

zebrafish ZF4 and AB9 cells.
Madin-Darby Canine Kidney (MDCK) epithelial cell line
Xenopus A6 kidney epithelial cells.

[edit] List of cell lines

Cell line	Meaning	Organism	Origin tissue	Morphology	Link
293-T		Human	kidney (embryonic)		Derivative of HEK 293 ECACC
3T3 cells	"3-day transfer, inoculum 3 x 105 cells"	Mouse	embryonic fibroblast		Also known as NIH 3T3 ECACC
721		Human	melanoma
9L		Rat	glioblastoma
A2780		Human	Ovary	Ovarian Cancer	ECACC
A2780ADR		Human	Ovary	Adriamycin-resistant derivative	ECACC
A2780cis		Human	Ovary	Cisplatin-resistant derivative	ECACC
A172		human	glioblastoma	malignant glioma	ECACC
A20		murine	B lymphoma	B lymphocyte
A253		human	head and neck carcinoma	submandibular duct
A431		human	skin epithelium	squamous carcinoma	ECACC Cell Line Data Base
A-549		human	lungcarcinoma	epithelium	DSMZ ECACC
ALC		murine	bone marrow	stroma	PubMed
B16		murine	Melanoma		ECCAC
B35		rat	Neuroblastoma		ATCC
BCP-1 cells		Human	PBMC	HIV+ lymphoma	ATCC
BEAS-2B	bronchial epithelium + adenovirus 12-SV40 virus hybrid (Ad12SV40)	Human	lung	epithelial	ATCC
bEnd.3	brain endothelial	mouse	brain / cerebral cortex	endothelium	ATCC
BHK-21	"Baby Hamster Kidney Fibroblast cells"	Hamster	kidney	fibroblast	ECACC Olympus
BR 293		human	breast	breast cancer
BxPC3	Biopsy xenograph of pancreatic carcinoma line 3	human	pancreatic adenocarcinoma	epithelial	ATCC
C3H-10T1/2		Mouse	Embryonic mesenchymal cell line		ECACC
C6/36		Asian tiger mosquito	larval tissue		ECACC
Cal-27		human	tongue	squamous cell carcinoma
CHO	Chinese hamster ovary	hamster	Ovary	epithelium	ECACC ICLC
COR-L23		Human	Lung		ECACC
COR-L23/CPR		Human	Lung		ECACC
COR-L23/5010		Human	Lung		ECACC
COR-L23/R23		Human	Lung	Epithelial	ECACC
COS-7	Cercopithecus aethiops, origin-defective SV-40	ape - Cercopithecus aethiops (Chlorocebus)	kidney	fibroblast	ECACC ATCC
CML T1	Chronic Myelod Leukaemia T-lymphocyte 1	human	CML acute phase	T cell leukaemia	Blood
CMT	canine mammary tumor	dog	mammary gland	epithelium
CT26		murine	Colorectal Carcinoma	Colon
D17		canine	osteosarcoma		ECACC
DH82		canine	histiocytosis	monocyte/macrophage	ECACC J Vir Meth
DU145		human	Androgen insensitive carcinoma	Prostate
DuCaP	Dura mater Cancer of the Prostate	human	Metastatic Prostate Cancer	epithelial	PubMed
EL4		mouse		T cell leukaemia	ECACC
EM2		human	CML blast crisis	Ph+ CML line	Cell Line Data Base
EM3		human	CML blast crisis	Ph+ CML line	Cell Line Data Base
EMT6/AR1		mouse	Breast	Epithelial-like	ECACC
EMT6/AR10.0		Mouse	Breast	Epithelial-like	ECACC
FM3		human	Metastatic lymph node	melanoma
H1299		human	lung	lung cancer
H69		Human	Lung		ECACC
HB54		hybridoma	hybridoma	secretes L243 mAb (against HLA-DR)	Human Immunology
HB55		hybridoma	hybridoma	secretes MA2.1 mAb (against HLA-A2 and HLA-B17)	Journal of Immunology
HCA2		human	fibroblast		Journal of General Virology
HEK-293	human embryonic kidney	human	kidney (embryonic)	epithelium	ATCC
HeLa	Henrietta Lacks	human	Cervical cancer	epithelium	DSMZ ECACC
Hepa1c1c7	clone 7 of clone 1 hepatoma line 1	mouse	Hepatoma	epithelial	ECACC ATCC
HL-60	human leukemia	human	Myeloblast	bloodcells	ECACC DSMZ
HMEC	human mammary epithelial cell	human		epithelium	ECACC
HT-29		human	colon epithelium	adenocarcinoma	ECACC Cell Line Data Base
Jurkat		human	T-Cell-Leukemia	white blood cells	ECACC DSMZ
JY cells		human	lymphoblastoid	EBV immortalised B cell
K562 cells		human	lymphoblastoid	CML blast crisis	ECACC
Ku812		human	lymphoblastoid	erythroleukemia	ECACC LGCstandards
KCL22		human	lymphoblastoid	CML
KG1		human	lymphoblastoid	AML
KYO1	Kyoto 1	human	lymphoblastoid	CML	DSMZ
LNCap	Lymph node Cancer of the Prostate	human	prostatic adenocarcinoma	epithelial	ECACC ATCC
Ma-Mel 1, 2, 3....48		human		a range of melanoma cell lines
MC-38		mouse		adenocarcinoma
MCF-7	Michigan Cancer Foundation-7	human	mammary gland	invasive breast ductal carcinoma	ER+, PR+
MCF-10A	Michigan Cancer Foundation	human	mammary gland	epithelium	ATCC
MDA-MB-231	M.D. Anderson - Metastatic Breast	human	breast	cancer	ECACC
MDA-MB-468	M.D. Anderson - Metastatic Breast	human	breast	cancer	ECACC
MDA-MB-435	M.D. Anderson - Metastatic Breast	human	breast	melanoma or carcinoma (disputed)	Cambridge Pathology ECACC
MDCK II	Madin Darby canine kidney	dog	kidney	epithelium	ECACC ATCC
MDCK II	Madin Darby canine kidney	dog	kidney	epithelium	[2] ATCC
MOR/0.2R		Human	Lung		ECACC
MONO-MAC 6		human	WBC	myeloid metaplasic AML	Cell Line Data Base
MTD-1A		mouse		epithelium
MyEnd	myocardial endothelial	mouse		endothelium
NCI-H69/CPR		Human	Lung		ECACC
NCI-H69/LX10		Human	Lung		ECACC
NCI-H69/LX20		Human	Lung		ECACC
NCI-H69/LX4		Human	Lung		ECACC
NIH-3T3	NIH, 3-day transfer, inoculum 3 x 10⁵ cells	mouse	embryo	fibroblast	ECACC ATCC
NALM-1			peripheral blood	blast-crisis CML	Cancer Genetics and Cytogenetics
NW-145				melanoma	ESTDAB
OPCN / OPCT cell lines	Onyvax[3] Prostate Cancer....			Range of prostate tumour lines	Asterand
Peer		human	T cell leukemia		DSMZ
PNT-1A / PNT 2				Prostate tumour lines	ECACC
RenCa	Renal Carcinoma	mouse		renal carcinoma
RIN-5F		mouse	pancreas
RMA/RMAS		mouse		T cell tumour
Saos-2 cells		human		Osteosarcoma	ECACC
Sf-9	Spodoptera frugiperda	insect - Spodoptera frugiperda (moth)	Ovary		DSMZ ECACC
SkBr3		human		breast carcinoma
T2		human		T cell leukemia/B cell line hybridoma	DSMZ
T-47D		human	mammary gland	ductal carcinoma
T84		human	colorectal Carcinoma / lungmetastasis	epithelium	ECACC ATCC
THP1 cell line		human	monocyte	AML	ECACC
U373		human	glioblastoma-astrocytoma	epithelium
U87		human	glioblastoma-astrocytoma	epithelial-like	Abcam
U937		human	leukaemic monocytic lymphoma		ECACC
VCaP	Vertebra Prostate Cancer	human	metastatic prostate cancer	epithelial	ECACC ATCC
Vero cells	'Vera Reno' ('green kidney') / 'Vero' ('truth')	African Green Monkey	kidney epithelium		ECACC
WM39		human	skin	primary melanoma
WT-49		human	lymphoblastoid
X63		mouse	melanoma
YAC-1		mouse	lymphoma		Cell Line Data Base ECACC
YAR		human	B-cell	EBV transofrmed	[4] Human Immunology

Note: this list is a sample of available cell lines, and is not comprehensive

[edit] See also

[edit] References and notes

^ ""Cell Culture"". http://www.bioteach.ubc.ca/Bioengineering/CellCulture/index.htm. Retrieved 2006-04-19.
^ ""Some landmarks in the development of tissue and cell culture."". http://www.ncbi.nlm.nih.gov/books/bv.fcgi?db=Books&rid=mboc4.table.1516. Retrieved 2006-04-19.
^ ""Animals and alternatives in testing."". http://caat.jhsph.edu/pubs/animal_alts/appendix_c.htm. Retrieved 2006-04-19.
^ Schiff, Judith Ann. ""An unsung hero of medical research."". http://www.yalealumnimagazine.com/issues/02_02/old_yale.html. Retrieved 2006-04-19. Yale Alumni Magazine, February 2002.
^ "LipiMAX purified lipoprotein solution from bovine serum". Selborne Biological Services. 2006. http://www.selbornebiological.com/products/lipimax.htm. Retrieved 2010-02-02.
^ Drexler, HG; Dirks; Macleod (Oct 1999). "False human hematopoietic cell lines: cross-contaminations and misinterpretations". Leukemia 13 (10): 1601–7. doi:10.1038/sj/leu/2401510. ISSN 0887-6924. PMID 10516762.
^ Drexler, HG; Macleod; Dirks (Dec 2001). "Cross-contamination: HS-Sultan is not a myeloma but a Burkitt lymphoma cell line" (Free full text). Blood 98 (12): 3495–6. doi:10.1182/blood.V98.12.3495. ISSN 0006-4971. PMID 11732505. http://www.bloodjournal.org/cgi/pmidlookup?view=long&pmid=11732505.
^ Cabrera, CM; Cobo, F; Nieto, A; Cortés, JL; Montes, RM; Catalina, P; Concha, A (Jun 2006). "Identity tests: determination of cell line cross-contamination". Cytotechnology 51 (2): 45–50. doi:10.1007/s10616-006-9013-8. ISSN 0920-9069. PMID 19002894.
^ ^a ^b Chatterjee, R (Feb 2007). "Cell biology. Cases of mistaken identity.". Science (New York, N.Y.) 315 (5814): 928–31. doi:10.1126/science.315.5814.928. ISSN 0036-8075. PMID 17303729.
^ Liscovitch, M; Ravid (Jan 2007). "A case study in misidentification of cancer cell lines: MCF-7/AdrR cells (re-designated NCI/ADR-RES) are derived from OVCAR-8 human ovarian carcinoma cells.". Cancer letters 245 (1-2): 350–2. doi:10.1016/j.canlet.2006.01.013. ISSN 0304-3835. PMID 16504380.
^ Macleod, RA; Dirks; Matsuo; Kaufmann; Milch; Drexler (Nov 1999). "Widespread intraspecies cross-contamination of human tumor cell lines arising at source.". International journal of cancer. Journal international du cancer 83 (4): 555–63. ISSN 0020-7136. PMID 10508494.
^ Masters, JR (Apr 2002). "HeLa cells 50 years on: the good, the bad and the ugly.". Nature reviews. Cancer 2 (4): 315–9. doi:10.1038/nrc775. ISSN 1474-175X. PMID 12001993.
^ ^a ^b Dunham, J.H. and Guthmiller, P. (2008) Doing good science: Authenticating cell line identity. Cell Notes 22, 15–17.
^ Ceb.com
^ Reuters (2006-01-26). Wired.com "Quickie Bird Flu Vaccine Created". Wired. http://wired.com/news/wireservice/0,70102-0.html?tw=wn_index_7 Wired.com. Retrieved 2010-01-31.
^ Gao W, Soloff AC, Lu X, Montecalvo A, Nguyen DC, Matsuoka Y, Robbins PD, Swayne DE, Donis RO, Katz JM, Barratt-Boyes SM, Gambotto A. (February 2006). "Protection of mice and poultry from lethal H5N1 avian influenza virus through adenovirus-based immunization". Journal of Virology (United States: American Society for Microbiology) 80 (4): 1959–1964. doi:10.1128/JVI.80.4.1959-1964.2006. ISSN 0022-538X. http://jvi.asm.org/cgi/content/abstract/80/4/1959. Retrieved 2010-01-31.
^ "NIAID Taps Chiron to Develop Vaccine Against H9N2 Avian Influenza". National Institute of Allergy and Infectious Diseases (NIAID). 2004-08-17. http://www3.niaid.nih.gov/news/newsreleases/2004/h9n2.htm. Retrieved 2010-01-31.

Hpacultures.org.uk, Health Protection Agency Culture Collections (ECACC)
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.

[edit] External links

Health Protection Agency Culture Collections - including ECACC (European Collection of Cell Cultures)
Witkowski JA. Experimental pathology and the origins of tissue culture: Leo Loeb's contribution. Med Hist. 1983 July; 27(3): 269–288.
atcc
Coriell Cell Repositories
The National Centre for Cell Science (NCCS), Pune, India; national repository for cell lines/hybridomas etc.
Neural Stem Cell Culture: Neurosphere generation, microscopical analysis and cryopreservation (a protocol)
Rat Chromaffin cells primary cultures: Standardization and quality assessment for single-cell assays (a protocol)
Table of common cell lines from Alberts 4th ed.
Cancer Cells in Culture
Hypertext version of the Cell Line Data Base

Tissue Culture Methods

2010-02-09T22:21:00.000-08:00

I. TYPES OF CELLS GROWN IN CULTURE
Tissue culture is often a generic term that refers to both organ culture and cell culture and the terms are often used interchangeably. Cell cultures are derived from either primary tissue explants or cell suspensions. Primary cell cultures typically will have a finite life span in culture whereas continuous cell lines are, by definition, abnormal and are often transformed cell lines.
II. WORK AREA AND EQUIPMENT
A. Laminar flow hoods. There are two types of laminar flow hoods, vertical and horizontal. The vertical hood, also known as a biology safety cabinet, is best for working with hazardous organisms since the aerosols that are generated in the hood are filtered out before they are released into the surrounding environment. Horizontal hoods are designed such that the air flows directly at the operator hence they are not useful for working with hazardous organisms but are the best protection for your cultures. Both types of hoods have continuous displacement of air that passes through a HEPA (high efficiency particle) filter that removes particulates from the air. In a vertical hood, the filtered air blows down from the top of the cabinet; in a horizontal hood, the filtered air blows out at the operator in a horizontal fashion. NOTE: these are not fume hoods and should not be used for volatile or explosive chemicals. They should also never be used for bacterial or fungal work. The hoods are equipped with a short-wave UV light that can be turned on for a few minutes to sterilize the surfaces of the hood, but be aware that only exposed surfaces will be accessible to the UV light. Do not put your hands or face near the hood when the UV light is on as the short wave light can cause skin and eye damage. The hoods should be turned on about 10-20 minutes before being used. Wipe down all surfaces with ethanol before and after each use. Keep the hood as free of clutter as possible because this will interfere with the laminar flow air pattern.
B. CO₂Incubators. The cells are grown in an atmosphere of 5-10% CO₂ because the medium used is buffered with sodium bicarbonate/carbonic acid and the pH must be strictly maintained. Culture flasks should have loosened caps to allow for sufficient gas exchange. Cells should be left out of the incubator for as little time as possible and the incubator doors should not be opened for very long. The humidity must also be maintained for those cells growing in tissue culture dishes so a pan of water is kept filled at all times.
C. Microscopes. Inverted phase contrast microscopes are used for visualizing the cells. Microscopes should be kept covered and the lights turned down when not in use. Before using the microscope or whenever an objective is changed, check that the phase rings are aligned.
D. Preservation. Cells are stored in liquid nitrogen (see Section III- Preservation and storage).
E. Vessels. Anchorage dependent cells require a nontoxic, biologically inert, and optically transparent surface that will allow cells to attach and allow movement for growth. The most convenient vessels are specially-treated polystyrene plastic that are supplied sterile and are disposable. These include petri dishes, multi-well plates, microtiter plates, roller bottles, and screwcap flasks - T-25, T-75, T-150 (cm² of surface area). Suspension cells are either shaken, stirred, or grown in vessels identical to those used for anchorage-dependent cells.
III. PRESERVATION AND STORAGE. Liquid N₂ is used to preserve tissue culture cells, either in the liquid phase (-196°C) or in the vapor phase (-156°C). Freezing can be lethal to cells due to the effects of damage by ice crystals, alterations in the concentration of electrolytes, dehydration, and changes in pH. To minimize the effects of freezing, several precautions are taken. First, a cryoprotective agent which lowers the freezing point, such as glycerol or DMSO, is added. A typical freezing medium is 90% serum, 10% DMSO. In addition, it is best to use healthy cells that are growing in log phase and to replace the medium 24 hours before freezing. Also, the cells are slowly cooled from room temperature to -80°C to allow the water to move out of the cells before it freezes. The optimal rate of cooling is 1°-3°C per minute. Some labs have fancy freezing chambers to regulate the freezing at the optimal rate by periodically pulsing in liquid nitrogen. We use a low tech device called a Mr. Frosty (C#1562 -Nalgene, available from Sigma). The Mr. Frosty is filled with 200 ml of isopropanol at room temperature and the freezing vials containing the cells are placed in the container and the container is placed in the -80°C freezer. The effect of the isopropanol is to allow the tubes to come to the temperature of the freezer slowly, at about 1°C per minute. Once the container has reached -80°C (about 4 hours or, more conveniently, overnight) the vials are removed from the Mr. Frosty and immediately placed in the liquid nitrogen storage tank. Cells are stored at liquid nitrogen temperatures because the growth of ice crystals is retarded below -130°C. To maximize recovery of the cells when thawing, the cells are warmed very quickly by placing the tube directly from the liquid nitrogen container into a 37°C water bath with moderate shaking. As soon as the last ice crystal is melted, the cells are immediately diluted into prewarmed medium.
IV. MAINTENANCE
Cultures should be examined daily, observing the morphology, the color of the medium and the density of the cells. A tissue culture log should be maintained that is separate from your regular laboratory notebook. The log should contain: the name of the cell line, the medium components and any alterations to the standard medium, the dates on which the cells were split and/or fed, a calculation of the doubling time of the culture (this should be done at least once during the semester), and any observations relative to the morphology, etc.
A. Growth pattern. Cells will initially go through a quiescent or lag phase that depends on the cell type, the seeding density, the media components, and previous handling. The cells will then go into exponential growth where they have the highest metabolic activity. The cells will then enter into stationary phase where the number of cells is constant, this is characteristic of a confluent population (where all growth surfaces are covered).
B. Harvesting. Cells are harvested when the cells have reached a population density which suppresses growth. Ideally, cells are harvested when they are in a semi-confluent state and are still in log phase. Cells that are not passaged and are allowed to grow to a confluent state can sometime lag for a long period of time and some may never recover. It is also essential to keep your cells as happy as possible to maximize the efficiency of transformation. Most cells are passaged (or at least fed) three times a week.
1. Suspension culture. Suspension cultures are fed by dilution into fresh medium.
2. Adherent cultures. Adherent cultures that do not need to be divided can simply be fed by removing the old medium and replacing it with fresh medium.
When the cells become semi-confluent, several methods are used to remove the cells from the growing surface so that they can be diluted:

Mechanical - A rubber spatula can be used to physically remove the cells from the growth surface. This method is quick and easy but is also disruptive to the cells and may result in significant cell death. This method is best when harvesting many different samples of cells for preparing extracts, i.e., when viability is not important.
Proteolytic enzymes - Trypsin, collagenase, or pronase, usually in combination with EDTA, causes cells to detach from the growth surface. This method is fast and reliable but can damage the cell surface by digesting exposed cell surface proteins. The proteolysis reaction can be quickly terminated by the addition of complete medium containing serum
EDTA - EDTA alone can also be used to detach cells and seems to be gentler on the cells than trypsin. The standard procedure for detaching adherent cells is as follows:

1. Visually inspect daily

2. Release cells from monolayer surface

a. wash once with a buffer solution
b. treat with dissociating agent
c. observe cells under the microscope. Incubate until cells become rounded and loosen when flask is gently tapped with the side of the hand.
d. Transfer cells to a culture tube and dilute with medium containing serum.
e. Spin down cells, remove supernatant and replace with fresh medium.
f. Count the cells in a hemacytometer, and dilute as appropriate into fresh medium.

C. Media and growth requirements
1. Physiological parameters

A. temperature - 37C for cells from homeother

B. pH - 7.2-7.5 and osmolality of medium must be maintained

C. humidity is required

D. gas phase - bicarbonate conc. and CO₂ tension in equilibrium

E. visible light - can have an adverse effect on cells; light induced production of toxic compounds can occur in some media; cells should be cultured in the dark and exposed to room light as little as possible;

2. Medium requirements: (often empirical)

A. Bulk ions - Na, K, Ca, Mg, Cl, P, Bicarb or CO₂
B. Trace elements - iron, zinc, selenium
C. sugars - glucose is the most common
D. amino acids - 13 essential
E. vitamins - B, etc.
F. choline, inositol
G. serum - contains a large number of growth promoting activities such as buffering toxic nutrients by binding them, neutralizes trypsin and other proteases, has undefined effects on the interaction between cells and substrate, and contains peptide hormones or hormone-like growth factors that promote healthy growth.
H. antibiotics - although not required for cell growth, antibiotics are often used to control the growth of bacterial and fungal contaminants.

3. Feeding - 2-3 times/week.
4. Measurement of growth and viability. The viability of cells can be observed visually using an inverted phase contrast microscope. Live cells are phase bright; suspension cells are typically rounded and somewhat symmetrical; adherent cells will form projections when they attach to the growth surface. Viability can also be assessed using the vital dye, trypan blue, which is excluded by live cells but accumulates in dead cells. Cell numbers are determined using a hemocytometer.
V. SAFETY CONSIDERATIONS

	Assume all cultures are hazardous since they may harbor latent viruses or other organisms that are uncharacterized. The following safety precautions should also be observed:
	pipetting: use pipette aids to prevent ingestion and keep aerosols down to a minimum
	no eating, drinking, or smoking
	wash hands after handling cultures and before leaving the lab
	decontaminate work surfaces with disinfectant (before and after)
	autoclave all waste
	use biological safety cabinet (laminar flow hood) when working with hazardous organisms. The cabinet protects worker by preventing airborne cells and viruses released during experimental activity from escaping the cabinet; there is an air barrier at the front opening and exhaust air is filtered with a HEPA filter make sure cabinet is not overloaded and leave exhaust grills in the front and the back clear (helps to maintain a uniform airflow)
	use aseptic technique
	dispose of all liquid waste after each experiment and treat with bleach

REFERENCES:
R. Ian Freshney, Culture of Animal cells: A manual of basic techniques, Wiley-Liss, 1987.

VI. TISSUE CULTURE PROCEDURES
Each student should maintain his own cells throughout the course of the experiment. These cells should be monitored daily for morphology and growth characteristics, fed every 2 to 3 days, and subcultured when necessary. A minimum of two 25 cm² flasks should be carried for each cell line; these cells should be expanded as necessary for the transfection experiments. Each time the cells are subcultured, a viable cell count should be done, the subculture dilutions should be noted, and, after several passages, a doubling time determined. As soon as you have enough cells, several vials should be frozen away and stored in liquid N₂. One vial from each freeze down should be thawed 1-2 weeks after freezing to check for viability. These frozen stocks will prove to be vital if any of your cultures become contaminated.
Procedures:1. Media preparation. Each student will be responsible for maintaining his own stock of cell culture media; the particular type of media, the sera type and concentration, and other supplements will depend on the cell line. Do not share media with you partner (or anyone else) because if a culture or a bottle of media gets contaminated, you have no back-up. Most of the media components will be purchased prepared and sterile. In general, all you need to do is sterily combine several sterile solutions. To test for sterility after adding all components, pipet several mls from each media bottle into a small sterile petri dish or culture tube and incubate at 37EC for several days. Use only media that has been sterility tested. For this reason, you must anticipate your culture needs in advance so you can prepare the reagents necessary. But, please try not to waste media. Anticipate your needs but don't make more than you need. Tissue culture reagents are very expensive; for example, bovine fetal calf serum cost ~ $200/500 ml. Some cell culture additives will be provided in a powdered form. These should be reconstituted to the appropriate concentration with double-distilled water (or medium, as appropriate) and filtered (in a sterile hood) through a 0-22 μm filter.
All media preparation and other cell culture work must be performed in a laminar flow hood. Before beginning your work, turn on blower for several minutes, wipe down all surfaces with 70% ethanol, and ethanol wash your clean hands. Use only sterile pipets, disposable test tubes and autoclaved pipet tips for cell culture. All culture vessels, test tubes, pipet tip boxes, stocks of sterile eppendorfs, etc. should be opened only in the laminar flow hood. If something is opened elsewhere in the lab by accident, you can probably assume its contaminated. If something does become contaminated, immediately discard the contaminated materials into the biohazard container and notify the instructor.
2. Growth and morphology. Visually inspect cells frequently. Cell culture is sometimes more an art than a science. Get to know what makes your cells happy. Frequent feeding is important for maintaining the pH balance of the medium and for eliminating waste products. Cells do not typically like to be too confluent so they should be subcultured when they are in a semi-confluent state. In general, mammalian cells should be handled gently. They should not be vortexed, vigorously pipetted or centrifuged at greater than 1500 g.
3. Cell feeding. Use prewarmed media and have cells out of the incubator for as little time as possible. Use 10-15 ml for T-25's, 25-35 ml for T-75's and 50-60 ml for T-150's. a. Suspension cultures. Feeding and subculturing suspension cultures are done simultaneously. About every 2-3 days, dilute the cells into fresh media. The dilution you use will depend on the density of the cells and how quickly they divide, which only you can determine. Typically 1:4 to 1:20 dilutions are appropriate for most cell lines. b. Adherent cells. About every 2-3 days, pour off old media from culture flasks and replace with fresh media. Subculture cells as described below before confluency is reached.
4. Subculturing adherent cells. When adherent cells become semi-confluent, subculture using 2 mM EDTA or trypsin/EDTA.
Trypsin-EDTA :

	a. Remove medium from culture dish and wash cells in a balanced salt solution without Ca++ or Mg++. Remove the wash solution.
	b. Add enough trypsin-EDTA solution to cover the bottom of the culture vessel and then pour off the excess.
	c. Place culture in the 37°C incubator for 2 minutes.
	d. Monitor cells under microscope. Cells are beginning to detach when they appear rounded.
	e. As soon as cells are in suspension, immediately add culture medium containing serum. Wash cells once with serum containing medium and dilute as appropriate (generally 4-20 fold).

EDTA alone:

	a. Prepare a 2 mM EDTA solution in a balanced salt solution (i.e., PBS without Ca⁺⁺ or Mg⁺⁺).
	b. Remove medium from culture vessel by aspiration and wash the monolayer to remove all traces of serum. Remove salt solution by aspiration.
	c. Dispense enough EDTA solution into culture vessels to completely cover the monolayer of cells.
	d. The coated cells are allowed to incubate until cells detach from the surface. Progress can be checked by examination with an inverted microscope. Cells can be gently nudged by banging the side of the flask against the palm of the hand.
	e. Dilute cells with fresh medium and transfer to a sterile centrifuge tube.
	f. Spin cells down, remove supernatant, and resuspend in culture medium (or freezing medium if cells are to be frozen). Dilute as appropriate into culture flasks.

5. Thawing frozen cells.

	a. Remove cells from frozen storage and quickly thaw in a 37°C waterbath by gently agitating vial.
	b. As soon as the ice crystals melt, pipet gently into a culture flask containing prewarmed growth medium.
	c. Log out cells in the "Liquid Nitrogen Freezer Log" Book.

6. Freezing cells.

	a. Harvest cells as usual and wash once with complete medium.
	b. Resuspend cells in complete medium and determine cell count/viability.
	c. Centrifuge and resuspend in ice-cold freezing medium: 90% calf serum/10% DMSO, at 10⁶ - 10⁷ cells/ml. Keep cells on ice.
	d. Transfer 1 ml aliquots to freezer vials on ice.
	e. Place in a Mr. Frosty container that is at room temperature and that has sufficient isopropanol.
	f. Place the Mr. Frosty in the -70°C freezer overnight. Note: Cells should be exposed to freezing medium for as little time as possible prior to freezing
	g Next day, transfer to liquid nitrogen (DON'T FORGET) and log in the "Liquid Nitrogen Freezer Log" Book.

7. Viable cell counts. USING A HEMOCYTOMETER TO DETERMINE TOTAL CELL COUNTS AND VIABLE CELL NUMBERS (Reference: Sigma catalogue)Trypan blue is one of several stains recommended for use in dye exclusion procedures for viable cell counting. This method is based on the principle that live cells do not take up certain dyes, whereas dead cells do.
1. Prepare a cell suspension, either directly from a cell culture or from a concentrated or diluted suspension (depending on the cell density) and combine 20 μl of cells with 20 μl of trypan blue suspension (0.4%). Mix thoroughly and allow to stand for 5-15 minutes.
2. With the cover slip in place, transfer a small amount of trypan blue-cell suspension to both chambers of the hemocytometer by carefully touching the edge of the cover slip with the pipette tip and allowing each chamber to fill by capillary action. Do not overfill or underfill the chambers.3. Starting with 1 chamber of the hemocytometer, count all the cells in the 1 mm center square and four 1 mm corner square. Keep a separate count of viable and non-viable cells.4. If there are too many or too few cells to count, repeat the procedure either concentrating or diluting the original suspension as appropriate.5. The circle indicates the approximate area covered at 100X microscope magnification (10X ocular and 10X objective). Include cells on top and left touching middle line. Do not count cells touching middle line at bottom and right. Count 4 corner squares and middle square in both chambers and calculate the average.6. Each large square of the hemocytometer, with cover-slip in place, represents a total volume of 0.1 mm³ or 10^-4 cm³. Since 1 cm³ is equivalent to approximately 1 ml, the total number of cells per ml will be determined using the following calculations:Cells/ml = average cell count per square x dilution factor x 10⁴;
Total cells = cells/ml x the original volume of fluid from which the cell sample was removed; % Cell viability = total viable cells (unstained)/total cells x 100.

This Web page is maintained by Julie B. Wolf, UMBC;
Last updated on 10/02/210

2010-02-08T08:55:00.000-08:00

KULTUR JARINGAN

Kultur Jaringan adalah teknik memperbanyak tanaman dengan memperbanyak jaringan mikro tanaman yang ditumbuhkan secara invitro menjadi tanaman yang sempurna dalam jumlah yang tidak terbatas. Yang menjadi dasar kultur jaringan ini adalah teori totipotensi sel yang berbunyi “setiap sel organ tanaman akan mampu tumbuh menjadi tanaman yang sempurna jika ditempatkan di lingkungan yang sesuai. Tujuan dari teknik ini adalah untuk memperbanyak tanaman dengan waktu yang lebih singkat.

Begitu banyak tanaman yang dapat dibudidayakan dengan kultur jaringan ini seperti Acasia sp, Eucalyptus sp, jati, jelutung, gaharu, sengon, sonokeling, berbagai jenis pisang, berbagai jenis anggrek, dsb.

Penanaman Jati dengan Metode Kultur Jaringan

Jati (Tectona grandis) merupakan famili dari Verbenacea. Merupakan penghasil kayu yang berkualitas, terkenal dengan keawetan dan kekuatannya, dan keindahan teksturnya membuatnya menjadi bahan furniture. Peluang pasar jati amat tinggi, akibatnya permintaan akan bahan kayu jati pun amat tinggi. Akan tetapi sayangnya permintaan tersebut belum dapat diimbangi dengan permintaan bahan kayu jati. Penghasilan baru bahan jati Indonesia adalah 2,5 juta m3/tahun. Harga jati sendiri cukup tinggi. Harganya di dalam negeri sekitar 8-9 juta /m3 sedangkan di luar negeri sekitar 15 juta/m3. akan tetapi walaupun tanaman jati merupakan tanaman yang potensial masih tetap ada kendala dalam hal produksi jati, diantaranya adalah:

™ Jati memerlukan investasi jangka panjang.

™ Masyarakat dan perusahaan swasta kurang meminati bidang produksi jati.

™ Sulit didapatnya bibit yang berkualitas dalam skala banyak dan seragam.

Seperti yang kita singgung sedikit tentang teori totipotensi yang menyebutkan bahwa secara teoritis tiap sel organ tanaman akan bisa tumbuh menjadi tanaman yang sempurna jika ditempatkan dalam lingkungan yang sesuai. Maka digunakanlah metode kultur jaringan ini untuk membudidayakan pohon jati.

Media untuk kultur jaringan ini mengandung:

™ Unsur hara makro dan mikro

™ Vitamin

™ Gula

™ Agar (untuk memadatkan larutan)

™ Zat pengatur tumbuh:

o Auksin (pertumbuhan tinggi dan akar)

o Sitokinin (penggandaan tunas)

Proses pembuatan media kultur itu sendiri adalah sebagai berikut:

Bahan kimia ditimbang, dilarutkan dalam air destilasi (air bebas mineral), lalu PH larutan diukur, campurkan agar kemudian dimasaka hingga mendididh, lalu tuangkan media kedalam botol ukur, setelah itu berikan label media dan disterilkan dengan autoclare.

Proses selanjutnya adalah sterilisasi eksplan jati, yang caranya adalah sebagai berikut:

™ Siapkan pucuk tunas muda jati.

™ Lalu rendam didalam larutan fungisida dan bakterisida.

™ Lalu rendam dalam larutan disinfektan (Clorox/baydin)

™ Dicuci dengan air steril hingga bersih dari desifektan.

™ Lalu tanam didalam media inisiasi tunas invitro.

Tunas-tunas yang ditanam dalam media invitro, disimpan di ruang steril. Botol steril disimpan pada rak kultur yang diberi cahaya lampu TL dengan intensitas cahaya 1000-4000 lux. Lampu TL diatur 16 jam menyala dan 8 jam padam agar sesuai seperti keadaan siang dan malam di bumi. Ruangan tempat penyimpanan dijaga suhunya di temperatur 250-280 C dengan menggunakan AC. Dan secara berkala ruang kultur disteril dengan menggunakan formalin. Inisiasi In vitro pertama adalah saat tunas berusia 3 minggu dan pemanjangan tunas 3-4 minggu.

Setelah itu akan ada proses aklimatisasi yaitu pembiasaan tanaman eksplan dari media botol ke media tanah. Proses aklimatisasi dilanjutkan dengan pembesaran bibit di polybag.

Kelebihan bibit hasil kutur jaringan antara lain :

™ Kontinuitas ketersediaan bibit dalam jumlah besar akan terjaga sepanjang waktu.

™ Bibit yang sama memiliki sifat yang sama dengan induknya.

™ Bibit yang dihasilkan bebas dari penyakit dan virus.

™ Lebih cepat tumbuh.

Cara Melakukan Pemindahan Tanaman Eksplan, Mempersihkan Kalusnya, dan Proses Aklimitasi

1. memindahkan tanaman eksplan & membersihkan kalusnya.

™ Alat dan bahan:

o Pinset steril.

o Pisau khusus steril.

o Kapas steril.

o Alat laminar.

o Tanaman eksplan.

o Dua buah botol dengan media agar didalamnya.

o Spiritus

o Korek api.

o Wadah pinset dan pisau.

o Alkohol.

™ Cara kerja:

o Sterilkan tangan dengan menyemprotkan alkohol ke tangan.

o Keluarkan tanaman eksplan yang akan dibersihkan kalusnya dengan menggunakan pinset.

o Letakkan di sebuah wadah dengan kapas diatasnya.

o Jepit bagian batang eksplan dengan pinset kemudian potong bagian kalusnya menggunkan pinset denganhati-hati. Potong kalus dari keempat sisinya. Jangan sampai kalus tersebut terpotong semua.

o Setelah selesai proses pemotongannya, bersihkan kalus tersebut dari media dengan menggunkan kapas steril.

o Pindahkan tanaman eksplan yang telah bersih dengan menggunakan pinset ke dalam media agar pada botol yang baru.

o Tutup botol tersebut, jaga agar tetap steril.

o Setelah selesai, celupkan pisau dan pinset kedalam alcohol kemudian bakar dengan api dan lekas letakkan kembali pada wadahnya.

Proses pensterilan selalu dilakukan secara rutin tiap sebulan sekali selama 24 jam. Botol-botl berisi tanaman eksplan disimpan di rak-rak dengan suhu 240-260 C selama 24 jam (setiap botol harus diberi label). Vitamin yang diberikan untuk eksplan yaitu C, B2, & B3 kemudian diaduk dengan gula dan agar-agar. Waktu tumbuh tanaman eksplan yaitu: induksi (3 minggu), multipikasi (3 minggu), aklimitasi (3 minggu). Biasanya tanaman diberi “bapitrof” (obat yang diberikan setelah proses aklimitasi yang berfungsi untuk merangsang pertumbuhan akar).

2. Proses Aklimitasi.

Proses aklimitasi mmerlukan kadar kelembaban 80%. Di perkebunan & Greenhouse biasanya digunakan suatu alat yang disebut sonic level fungsinya antara lain:

1. mengusir serangga dengan getarannya.

2. merangsang pertumbuhan tanaman.

Untuk mengukur PH tanaman menggunakan PH meter, ukuran PH tanaman biasanya ± 5,7-5,8 PH. Apabila PH tinggi diberi KOH, NaOh, apabila PH rendah diberi HCL.

Tanaman-tanaman yang terdapat di Greeen House di antaranya:

™ pohon kelengkeng.

™ Zodia

™ Pohon meranti.

™ Pohon jelutung.

™ Pohon jati.

™ Pohon buah merah.

™ Pohon mahoni.

™ Pohon gaharu.

™ Lalu pisang ABACA (Musa textilis Nec) yang seratnya diambil untuk:

o Tissue

o Kertas uang.

o Dokumen.

o Cheque

o Plester.

o Kertas mimeograph.

o Kantung teh.

Download versi pdf (bergambar) disini

Tissue Culture

Plant Tissue Culture

Plant Tissue Culture

Presenter: Lydiane (Ann) Kyte Host: Kathy Liu Discussion

Plant Tissue Culture Media

Cell culture

Cell culture

History

[edit] Concepts in mammalian cell culture

[edit] Isolation of cells

[edit] Maintaining cells in culture

[edit] Cell line cross-contamination

[edit] Manipulation of cultured cells

[edit] Media changes

[edit] Passaging cells

[edit] Transfection and transduction

[edit] Established human cell lines

[edit] Generation of hybridomas

[edit] Applications of cell culture

[edit] Tissue culture and engineering

[edit] Vaccines

[edit] Culture of non-mammalian cells

[edit] Plant cell culture methods

[edit] Bacterial/Yeast culture methods

[edit] Viral culture methods

[edit] Common cell lines

[edit] List of cell lines

[edit] See also

[edit] References and notes

[edit] External links

Tissue Culture Methods

Presenter: Lydiane (Ann) Kyte
Host: Kathy Liu
Discussion