Cell culture is the process by which either prokaryotic or eukaryotic cells are grown under controlled conditions. In practice the term "cell culture" has come to refer to the culturing of cells derived from multicellular eukaryotes, especially animal cells. The historical development and methods of cell culture are closely interrelated to those of tissue culture and organ culture. The growth of microorganisms such as bacteria and yeast, or human, plant, or animal cells in the laboratory. Cell cultures may be used to diagnose infections, to test new drugs, and in research. The technique or process of keeping tissue alive and growing in a culture medium.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.

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Type of cell cultures:Type of cell cultures:1. 1. Primary cellsPrimary cells are cells taken directly from the are cells taken directly from the

live animal tissue (they typically are tough to live animal tissue (they typically are tough to keep alive in the Petri dish, and do not live keep alive in the Petri dish, and do not live very long; typical cell divides once and dies). very long; typical cell divides once and dies). They only live if they can attach to bottom of They only live if they can attach to bottom of Petri Dish (anchorage-dependent). The Petri Dish (anchorage-dependent). The bottom of the Petri Dish has a special layer bottom of the Petri Dish has a special layer that cells can bind. that cells can bind.

2. 2. Fetal cellFetal cell lines can live longer; cells dividing lines can live longer; cells dividing 20-80 times before they die. 20-80 times before they die.

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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

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Concepts in mammalian cell cultureIsolation of cellsCells 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. A 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.

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Extracellular matrixIn biology, extracellular matrix (ECM) is any material part of a tissue that is not part of any cell. Extracellular matrix is the defining feature of connective tissue.1. ComponentsThe ECM's main components are various glycoproteins, proteoglycans and hyaluronic acid. In most animals, the most abundant glycoproteins in the ECM are collagens. In fact, collagen is the most abundant protein in the human body.

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ECM also contains many other components: proteins such as fibrin, elastin, fibronectins, laminins, and nidogens, and minerals such as hydroxylapatite, or fluids such as blood plasma or serum with secreted free flowing antigens.

In addition it sequesters a wide range of cellular growth factors, and acts as a local depot for them. Changes in physiological conditions can trigger protease activities that cause local release of such depots. This allows the rapid and local activation of cellular functions, without de novo synthesis

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FunctionsGiven this diversity, ECM can serve many functions, such as providing support and anchorage for cells, providing a way of separating the tissues, and regulating intercellular communication. The ECM regulates a cell's dynamic behavior.3.Adhesion to matrixMany cells bind to components of the extracellular matrix. This cell-to-ECM adhesion is regulated by specific cell surface cellular adhesion molecules (CAM) known as integrins. The integrins transmit mechanical stimuli from the ECM to the cytoskeleton

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Senecence:The word senescence is derived from the Latin word senex, meaning "old man" or "old age."

(upper) Primary mouse embryonic fibroblast cells (MEFs) before senescence. Spindle-shaped. (lower) MEFs became senescent after passages. Cells grow larger, flatten shape and expressed senescence-associated β-galactosidase (SABG, blue areas), a marker of cellular senescence.

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Cellular senescence is the phenomenon where normal diploid differentiated cells lose the ability to divide. This phenomenon is also known as "replicative senescence", the "Hayflick phenomenon", or the Hayflick limit in honour of Dr. Leonard Hayflick who was the first to publish it in 1965. In response to DNA damage (including shortened telomeres) cells either senesce or self-destruct (apoptosis, programmed cell death) if the damage cannot be repaired. In this 'cellular suicide', the death of one, or more, cells may benefit the organism as a whole

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Maintaining cells in cultureCells 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.

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

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Growth factorThe term growth factor refers to a naturally occurring protein capable of stimulating cellular proliferation and cellular differentiation. Growth factors are important for regulating a variety of cellular processes.Growth factors typically act as signaling molecules between cells. Examples are cytokines and hormones that bind to specific receptors on the surface of their target cells.They often promote cell differentiation and maturation, which varies between growth factors. For example, bone morphogenic proteins stimulate bone cell differentiation, while vascular endothelial growth factors stimulate blood vessel differentiation.

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Example of growth factorsIndividual growth factor proteins tend to occur as members of larger

families of structurally and evolutionarily related proteins. There are dozens and dozens of growth factor families such as TGF-beta (transforming growth factor-beta), BMP (bone morphogenic protein), neurotrophins (NGF, BDNF, and NT3), fibroblast growth factor (FGF), and so on.

Several well known growth factors are:Transforming growth factor beta (TGF-β) Granulocyte-colony stimulating factor (G-CSF) Granulocyte-macrophage colony stimulating factor (GM-CSF) Nerve growth factor (NGF) Neurotrophins Platelet-derived growth factor (PDGF)

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Erythropoietin (EPO) Thrombopoietin (TPO) Myostatin (GDF-8) Growth differentiation factor-9 (GDF9) Basic fibroblast growth factor (bFGF or FGF2) Epidermal growth factor (EGF) Hepatocyte growth factor (HGF]

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Cytokines are a group of proteins and peptides that are used in organisms as signaling compounds. These chemical signals are similar to hormones and neurotransmitters and are used to allow one cell to communicate with another. The cytokine family consists mainly of smaller water-soluble proteins and glycoproteins (proteins with an added sugar chain) with a mass of between 8 and 30 kDa. While hormones are released from specific organs into the blood and neurotransmitters are released by nerves, cytokines are released by many types of cells. They are particularly important in both innate and adaptive immune responses

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Due to their central role in the immune system, cytokines are involved in a variety of immunological, inflammatory and infectious diseases. However, not all their functions are limited to the immune system, as they are also involved in several developmental processes during embryogenesis.

Cytokines are produced by a wide variety of cell types (both haemopoietic and non-haemopoietic) and can have effects on both nearby cells or throughout the organism. Sometimes these effects are strongly dependent on the presence of other chemicals and cytokines.

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EffectsEach cytokine binds to a specific cell-surface receptor. Subsequent cascades of intracellular signalling then alter cell functions. This may include the upregulation and/or downregulation of several genes and their transcription factors, in turn resulting in the production of other cytokines, an increase in the number of surface receptors for other molecules, or the suppression of their own effect by feedback inhibition.The effect of a particular cytokine on a given cell depends on the cytokine, its extracellular abundance, the presence/abundance of the complementary receptor on the cell surface, and downstream signals activated by receptor binding; these last two factors can vary by cell type. Interestingly, cytokines are characterized by considerable "redundancy", in that many cytokines appear to share similar functions.

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Generalization of functions is not possible with cytokines; nonetheless, their actions may be comfortably grouped as:autocrine, if the cytokine acts on the cell that secretes it paracrine, if the action is restricted to the immediate vicinity of a cytokine's secretion endocrine, if the cytokine diffuses to distant regions of the body (carried by blood or plasma) to affect different tissues. NomenclatureCytokines have been variously named as lymphokines, interleukins and chemokines, based on their presumed function, cell of secretion or target of action

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Manipulation of cultured cellsManipulation of cultured cellsAs cells generally continue to divide in culture, they generally grow to fill As cells generally continue to divide in culture, they generally grow to fill

the available area or volume. This can generate several issues:the available area or volume. This can generate several issues:Nutrient depletion in the growth media Nutrient depletion in the growth media

Accumulation of Accumulation of apoptotic//necrotic (dead) cells. (dead) cells. Cell-to-cell contact can stimulate cell cycle arrest, causing cells to stop Cell-to-cell contact can stimulate cell cycle arrest, causing cells to stop

dividing known as dividing known as contact inhibition. . Cell-to-cell contact can stimulate promiscuous and unwanted Cell-to-cell contact can stimulate promiscuous and unwanted

cellular differentiation. . These issues can be dealt with using tissue culture methods that rely on These issues can be dealt with using tissue culture methods that rely on

sterile techniquesterile technique. These methods aim to avoid contamination with . These methods aim to avoid contamination with bacteria or yeast that will compete with mammalian cells for nutrients bacteria or yeast that will compete with mammalian cells for nutrients and/or cause cell infection and cell death. Manipulations are typically and/or cause cell infection and cell death. Manipulations are typically

carried out in a carried out in a biosafety hood or or laminar flow cabinet to exclude to exclude contaminating micro-organisms. contaminating micro-organisms. Antibiotics can also be added to the can also be added to the

growth media.growth media.Amongst the common manipulations carried out on culture cells are Amongst the common manipulations carried out on culture cells are

media changes, passaging cells, and transfecting cellsmedia changes, passaging cells, and transfecting cells

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Media changesThe 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

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Passaging cellsPassaging or splitting cells involves transferring a small number of cells into a new vessel. Cells can be cultured for a longer time if they are split regularly, as it avoids the senescence associated with prolonged high cell density. Suspension cultures are easily passaged with a small amount of culture containing a few cells diluted in a larger volume of fresh media. For adherent cultures, cells first need to be detached; this was historically done with a mixture of trypsin-EDTA, however other enzyme mixes are now available for this purpose. A small number of detached cells can then be used to seed a new culture.

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Transfection and transductionAnother common method for manipulating cells involves the introduction of foreign DNA by transfection. This is often performed to cause cells to express a protein of interest. More recently, the transfection of RNAi constructs have been realised as a convenient mechanism for suppressing the expression of a particular gene/protein.DNA can also be inserted into cells using viruses, in methods referred to as transduction, infection or transformation. Viruses, as parasitic agents, are well suited to introducing DNA into cells, as this is a part of their normal course of reproduction.

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Transfection describes the introduction of foreign material into eukaryotic cells. Transfection typically involves opening transient pores or 'holes' in the cell plasma membrane, to allow the uptake of material. Genetic material (such as supercoiled plasmid DNA or siRNA constructs), or even proteins such as antibodies, may be transfected. Transfection is frequently carried out by mixing a cationic lipid with the material to produce liposomes, which fuse with the cell plasma membrane and deposit their cargo inside.

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The term transfection is most often used in reference to mammalian cells, while the term transformation is used for the same process in bacteria and, occasionally, plants. The original meaning of transfection was 'infection by transformation', i.e. introduction of DNA (or RNA) from a eukaryote virus or bacteriophage into cells, resulting in an infection. Because the term transformation had another sense in eukaryotic cell biology (a genetic change allowing long-term propagation in culture, or acquisition of properties typical of cancer cells), the term transfection acquired, for animal cells, its present meaning of a change in cell properties caused by introduction of DNA.

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MethodsThere are various methods of introducing foreign DNA into a eukaryote cell. One of the cheapest (and least reliable) is transfection by calcium phosphate, originally discovered by S. Bacchetti and F. L. Graham in 1977. HEPES-buffered saline solution (HeBS) containing phosphate ions is combined with a calcium chloride solution containing the DNA to be transfected. When the two are combined, a fine precipitate of calcium phosphate will form, binding the DNA to be transfected on its surface. The suspension of the precipitate is then added to the cells to be transfected (usually a cell culture grown in a monolayer). By a process not entirely understood, the cells take up some of the precipitate, and with it, the DNA. Other methods of transfection include electroporation, heat shock, magnetofection, nucleofection and proprietary transfection reagents such as Lipofectamine, Fugene.

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Other methods use highly branched organic compounds, so-called dendrimers, to bind the DNA and get it into the cell. A very efficient method is the inclusion of the DNA to be transfected in liposomes, i.e. small, membrane-bounded bodies that are in some ways similar to the structure of a cell and can actually fuse with the cell membrane, releasing the DNA into the cell. For eukaryotic cells, lipid-cation based transfection is more typically used, because the cells are more sensitive.Another method is DEAE-Dextran transfection, in which a mutant plasmid and Luciferase reporter are used. Dextran is positively charged. A cell performs endocytosis to take in the positively charged Dextran, which is attracted to the DNA (negatively charged).

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A direct approach to transfection is the gene gun, where the DNA is coupled to a nanoparticle of an inert solid (commonly gold) which is then "shot" directly into the target cell's nucleus. DNA can also be introduced into cells using viruses as a carrier. In such cases, the technique is called viral transduction, and, the cells are said to be transduced.Stable and transient transfectionFor most applications of transfection, it is sufficient if the transfected gene is only transiently expressed. Since the DNA introduced in the transfection process is usually not inserted into the nuclear genome, the foreign DNA is lost at the later stage when the cells undergo mitosis. If it is desired that the transfected gene actually remains in the genome of the cell and its daughter cells, a stable transfection must occur.

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To accomplish this, another gene is co-transfected, which gives the cell some selection advantage, such as resistance towards a certain toxin. Some (very few) of the transfected cells will, by chance, have inserted the foreign genetic material into their genome. If the toxin, towards which the co-transfected gene offers resistance, is then added to the cell culture, only those few cells with the foreign genes inserted into their genome will be able to proliferate, while other cells will die. After applying this selection pressure for some time, only the cells with a stable transfection remain and can be cultivated further.A common agent for stable transfection is Geneticin, also known as G418, which is a toxin that can be neutralized by the product of the neomycin resistant gene.

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Transformation .In molecular biology, Transformation is the genetic alteration of a cell resulting from the uptake and expression of foreign genetic material (DNA). Separate terms are used for genetic alterations resulting from introduction of DNA by plasmid-encoded conjugation or by viruses (transduction). Transformation of animal cells is usually called transfection.The term transformation is also used more generally to describe mechanisms of DNA and RNA transfer in molecular biology (i.e. not only the genetic consequences). For example the production of transgenic plants such as transgenic maize requires the insertion of new genetic information into the maize genome using an appropriate mechanism for DNA transfer; the process is commonly referred to as transformation.

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RNA molecules may also be transferred into cells using similar methods, but this does not normally produce heritable change and so is not true transformation.BacteriaIn bacteria, transformation refers to a stable genetic change brought about by taking up naked DNA (DNA without associated cells or proteins), and competence refers to the state of being able to take up exogenous DNA from the environment. Two different Natural competenceSome bacteria (around 1% of all species) are naturally capable of taking up DNA under laboratory conditions; many more may be able to take it up in their natural environments . Such species carry sets of genes specifying machinery for bringing DNA across the cell's membrane or membranes

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Artificial competenceArtificial competence is not encoded in the cell's genes. Instead it is induced by laboratory procedures in which cells are passively made permeable to DNA, using conditions that do not normally occur in nature. These procedures are comparatively easy and simple, and are widely used to introduce plasmids and genetically engineered bacteria. Artificially competent cells of standard bacterial strains may also be purchased frozen, ready to use.Chilling cells in the presence of divalent cations such as Ca2+ (in CaCl2) prepares the cell walls to become permeable to plasmid DNA. Cells are incubated on ice with the DNA and then briefly heat shocked (42oC for 30-120 seconds), which causes the DNA to enter the cell. This method works very well for circular plasmid DNAs. An excellent preparation of competent cells will give ~108 colonies per μg of plasmid. A poor preparation will be about 104/μg or less. Good non-commercial preps should give 105 to 106 transformants per microgram of plasmid.

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The method does not work for linear molecules such as fragments of chromosomal DNA, probably because exonuclease enzymes in the cell rapidly degrade linear DNAs. Cells that are naturally competent are usually transformed more efficiently with linear DNA than with plasmids.Electroporation is another way to make holes in bacterial (and other) cells, by briefly shocking them with an electric field of 10-20kV/cm. Provided plasmid DNA is present when the shock is applied, it can enter the cell through these holes. Natural membrane-repair mechanisms will rapidly close these holes after the shock.

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Electroporation: make transient holes in cell membranes using electric shock; this allows DNA to enter as described above for Bacteria. Viral transformation (transduction): Packages the desired genetic material into a suitable plant virus and is allow this modified virus to infect the plant. If the genetic material is DNA, it can recombine with the chromosomes to produce transformant cells. However genomes of most plant viruses consist of single stranded RNA which replicates in the cytoplasm of infected cell. For such genomes this method is a form of transfection and not a real transformation, since the inserted genes never reach the nucleus of the cell and do not integrate into the host genome. The progeny of the infected plants is virus free and also free of the inserted gene.

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Transfection of mammalian cells by electrophoration1. Grow cells to ~ 70-80 % confluence2. Trypsinize and resuspend them in complete medium ( 10 7 cells/ml)3. Combine DNA (40ug) with resistance (2ug) plasmid (neo or Hyg) and mix.4. Electrophorate in a gene pulser@950uF and o.22KV/cm(t=20-30ms)5. Perform duplicate electrophorations.6. Plate cells in six 10 cm plates as follows 1. 2000ul 5x10 5 2.1200ul 3x10 5 3. 800ul 2x 10 5 4. 400ul 1x 10 5 5. 200ul 5x 10 4 6. 100ul 2.5x 10 4

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LIPOFACTAMINE:1. Wash the cell once with serum free medium.2. Seed the cell (2-3 x 106/well) in six well plate in 0.8

ml serum free medium.3. Prepare the solutions in 12x 75 mm sterile tubes;

Solution A: For each tarnsfection dilute 1-3 ug of DNA into 100ul serum free medium (OPTI-MEM).

Solution B: For each Transfection dilute 2-25 of LIPOFECTAMINE reagent into 100 ul of serum free medium.

4. Combine the two solutions, mix gently and incubate 2 hr at 37.C .

5. Following incubation add 4 ml complete medium to each well and incubate at incubator and add DNA mixture gently and incubate 24-72 hr

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Basic Techniques - The "Do's and Don'ts" of Cell Culture

Given below are a few of the essential "do’s and don'ts" of cell culture. Some of these are mandatory e.g. use of personal protective equipment (PPE). Many of them are common sense and apply to all laboratory areas. However some of them are specific to tissue culture.

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The Do’s 1. Use personal protective equipment, (laboratory

coat/gown, gloves and eye protection) at all times. In addition, thermally insulated gloves, full-face visor and splash-proof apron should be worn when handling liquid nitrogen.

2. Always use disposable caps to cover hair. 3. Wear dedicated PPE for tissue culture facility and

keep separate from PPE worn in the general laboratory environment. The use of different colored gowns or laboratory coats makes this easier to enforce.

4. Keep all work surfaces free of clutter.

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5.Correctly label reagents including flasks, medium and ampules with contents and date of preparation.

6.Only handle one cell line at a time. This common-sense point will reduce the possibility of cross contamination by mislabeling etc. It will also reduce the spread of bacteria and mycoplasma by the generation of aerosols across numerous opened media bottles and flasks in the cabinet.

7.Clean the work surfaces with a suitable disinfectant (e.g. 70% ethanol) between operations and allow a minimum of 15 minutes between handling different cell lines.

8.Wherever possible maintain separate bottles of media for each cell line in cultivation.

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9. Examine cultures and media daily for evidence of gross bacterial or fungal contamination. This includes medium that has been purchased commercially.

10. Quality Control all media and reagents prior to use. 11. Keep cardboard packaging to a minimum in all cell

culture areas. 12. Ensure that incubators, cabinet, centrifuges and

microscopes are cleaned and serviced at regular intervals.

13. Test cells for mycoplasma on a regular basis.

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The Don’ts 1. Do not continuously use antibiotics in culture

medium as this will inevitably lead to the appearance of antibiotic resistant strains and may render a cell line useless for commercial purposes.

2. Don’t allow waste to accumulate particularly within the microbiological safety cabinet or in the incubators.

3. Don't have too many people in the lab at any one time.

4. Don't handle cells from unauthenticated sources in the main cell culture suite. They should be handled in quarantine until quality control checks are complete.

5. Avoid keeping cell lines continually in culture without returning to frozen stock

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The Don’ts 1. Avoid cell culture becoming fully confluent.

Always sub-culture at 70-80% confluency or as advised on cell culture data sheet.

2. Do not allow media to go out of date. Shelf life is only 6 weeks at +4ºC once glutamine and serum is added.

3. Avoid water baths from becoming dirty . 4. Don’t allow essential equipment to become

out of calibration. Ensure microbiological safety cabinets are tested regularly.

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Aseptic Technique and Good Cell Culture Practice

• AimTo ensure all cell culture procedures are performed to a standard that will prevent contamination from bacteria, fungi and mycoplasma and cross contamination with other cell lines.

• Materials Chloros / Presept solution (2.5g/l) 1% formaldehyde based disinfectant e.g.Virkon,Tegador 70% ethanol in water .• Equipment Personal protective equipment (sterile gloves, laboratory coat,

safety visor) Microbiological safety cabinet at appropriate containment level

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Procedure 1. Sanitize the cabinet using 70% ethanol before commencing

work. 2. Sanitize gloves by washing them in 70% ethanol and allowing

to air dry for 30 seconds before commencing work. 3. Put all materials and equipment into the cabinet prior to

starting work after sanitizing the exterior surfaces with 70% ethanol.

4. Whilst working do not contaminate gloves by touching anything outside the cabinet (especially face and hair). If gloves become contaminated re-sanitize with 70% ethanol as above before proceeding.

5. Discard gloves after handling contaminated cultures and at the end of all cell culture procedures.

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Procedure 6. Equipment in the cabinet or that which will be taken into the

cabinet during cell culture procedures (media bottles, pipette tip boxes, pipette aids) should be wiped with tissue soaked with 70% ethanol prior to use.

7. Movement within and immediately outside the cabinet must not be rapid. Slow movement will allow the air within the cabinet to circulate properly.

8. Speech, sneezing and coughing must be directed away from the cabinet so as not to disrupt the airflow.

9. After completing work disinfect all equipment and material before removing from the cabinet. Spray the work surfaces inside the cabinet with 70% ethanol and wipe dry with tissue. Dispose of tissue by autoclaving.

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Procedure 10. Cell culture discard in chloros must be kept in the

cabinet for a minimum of two hours (preferably overnight) prior to discarding down the sink with copious amounts of water.

11. Periodically clean the cabinet surfaces with a disinfectant such as Presept,Tegador or Virkon or fumigate the cabinet according to the manufacturers instructions. However you must ensure that it is safe to fumigate your own laboratory environment due to the generation of gaseous formaldehyde, consult your on-site Health and Safety Advisor.

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Resuscitation of Frozen Cell Lines

• AimMany cultures obtained from a culture collection, such as ATCC, will arrive frozen and in order to use them the cells must be thawed and put into culture. It is vital to thaw cells correctly in order to maintain the viability of the culture and enable the culture to recover more quickly. Some cryoprotectants, such as DMSO , are toxic above 4ºC therefore it is essential that cultures are thawed quickly and diluted in culture medium to minimize the toxic effects.

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Resuscitation of Frozen Cell Lines

• Materials Media– pre-warmed to the appropriate temperature (refer to the ECACC Cell

Line Data Sheet for the correct medium and size of flask to resuscitation into.) 70% ethanol in water DMSO • Equipment Personal protective equipment (sterile gloves, Laboratory coat, safety visor) Waterbath set to appropriate temperature Microbiological safety cabinet at appropriate containment level CO2 incubator Pre labeled flasks Marker Pen Pipettes Ampule Rack Tissue

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Procedure 1. Read Technical data sheet to establish specific requirements

for your cell line. 2. Prepare the flasks as appropriate (information on technical

data sheet). Label with cell line name, passage number and date.

3. Collect ampule of cells from liquid nitrogen storage wearing appropriate protective equipment and transfer to laboratory in a sealed container.

4. Still wearing protective clothing, remove ampule from container and place in a water bath at an appropriate temperature for your cell line e.g. 37ºC for mammalian cells. Submerge only the lower half of the ampule. Allow to thaw until a small amount of ice remains in the vial - usually 1-2 minutes. Transfer to class II safety cabinet.

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Procedure5. Wipe the outside of the ampule with a tissue

moistened (not excessively) with 70% alcohol hold tissue over ampule to loosen lid.

6. Slowly, dropwise, pipette cells into pre-warmed growth medium to dilute out the DMSO (flasks prepared in Step 2).

7. Incubate at the appropriate temperature for species and appropriate concentration of CO2 in atmosphere.

8. Examine cells microscopically (phase contrast) after 24 hours and sub-culture as necessary

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Key Points 1. Most text books recommend washing the thawed cells in

media to remove the cryoprotectant. This is only necessary if the cryoprotectant is known to have an adverse effect on the cells. In such cases the cells should be washed in media before being added to their final culture flasks.

2. Do not use an incubator to thaw cell cultures since the rate of thawing achieved is too slow resulting in a loss of viability.

3. For some cultures it is necessary to subculture before

confluence is reached in order to maintain their characteristics e.g. the contact inhibition of NIH 3T3 cells is lost if they are allowed to reach confluence repeatedly

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Subculture of Adherent Cell Lines

• AimAdherent cell lines will grow in vitro until they have covered the surface area available or the medium is depleted of nutrients. At this point the cell lines should be sub-cultured in order to prevent the culture dying. To subculture the cells they need to be brought into suspension. The degree of adhesion varies from cell line to cell line but in the majority of cases proteases, e.g. trypsin, are used to release the cells from the flask. However, this may not be appropriate for some lines where exposure to proteases is harmful or where the enzymes used remove membrane markers/receptors of interest. In these cases cells should be brought into suspension into a small volume of medium mechanically with the aid of cell scrapers

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Subculture of Adherent Cell Lines

• Materials Media– pre-warmed to 37ºC (refer to the ECACC Cell Line Data Sheet for the correct

medium) 70% ethanol in water PBS without Ca2+/Mg2+

0.25% trypsin/EDTA in HBSS, without Ca2+/Mg2+

Trypsin Soybean trypsin Inhibitor• Equipment Personal protective equipment (sterile gloves, Laboratory coat, safety visor) Waterbath set to appropriate temperature Microbiological safety cabinet at appropriate containment level CO2 incubator Pre-labeled flasks Marker Pen Pipettes Ampule Rack Tissue

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Procedure 1. View cultures using an inverted microscope to assess the

degree of confluency and confirm the absence of bacterial and fungal contaminants.

2. Remove spent medium. 3. Wash the cell monolayer with PBS without Ca2+/Mg2+ using a

volume equivalent to half the volume of culture medium. Repeat this wash step if the cells are known to adhere strongly.

4. Pipette trypsin/EDTA onto the washed cell monolayer using 1ml per 25cm2 of surface area. Rotate flask to cover the monolayer with trypsin. Decant the excess trypsin.

5. Return flask to the incubator and leave for 2-10 minutes.

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Procedure6. Examine the cells using an inverted microscope to ensure that

all the cells are detached and floating. The side of the flasks may be gently tapped to release any remaining attached cells.

7. Resuspend the cells in a small volume of fresh serum-containing medium to inactivate the trypsin. Remove 100-200uL and perform a cell count .

8. Transfer the required number of cells to a new labeled flask containing pre-warmed medium (refer to Cell Line Data Sheet for the required seeding density).

9. Incubate as appropriate for the cell line. 10. Repeat this process as demanded by the growth

characteristics of the cell line.

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Key Points 1. Some cultures whilst growing as attached lines adhere only

lightly to the flask, thus it is important to ensure that the culture medium is retained and the flasks are handled with care to prevent the cells detaching prematurely.

2. Although most cells will detach in the presence of trypsin alone the EDTA is added to enhance the activity of the enzyme.

3. Trypsin is inactivated in the presence of serum. Therefore, it is essential to remove all traces of serum from the culture medium by washing the monolayer of cells with PBS without Ca2+/Mg2.

4. Cells should only be exposed to trypsin/EDTA long enough to detach cells. Prolonged exposure could damage surface receptors.

5. Trypsin should be neutralized with serum prior to seeding cells into new flasks otherwise cells will not attach

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Key Points6. Trypsin may also be neutralized by

the addition of soybean trypsin inhibitor , where an equal volume of inhibitor at a concentration of 1mg/ml is added to the trypsinised cells. The cells are then centrifuged, resuspended in fresh culture medium and counted . This is especially necessary for serum-free cell culture.

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Subculture of Semi-Adherent Cell Lines

• AimSome cultures grow as a mixed population (e.g. B95-8 ) where a proportion of cells do not attach to the tissue culture flask and remain in suspension. Therefore to maintain this heterogeneity both the attached cells and the cells in suspension must be subcultured.

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Procedure 1. View cultures using an inverted phase contrast microscope to

assess the degree of confluency and confirm the absence of bacterial and fungal contaminants. Give the flask a gentle knock first, this may dislodge the cells from the flask and remove the need for a trypsinisation step with the subsequent loss of some cells due to the washings.

2. Decant spent medium into a sterile centrifuge tube and retain. 3. Wash any remaining attached cells with PBS without

Ca2+/Mg2+ using 1-2ml for each 25cm2 of surface area. Retain the washings.

4. Pipette trypsin/EDTA onto the washed cell monolayer using 1ml per 25cm2 of surface area. Rotate flask to cover the monolayer with trypsin. Decant the excess trypsin.

5. Return flask to incubator and leave for 2-10 minutes

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Procedure6. Examine the cells using an inverted microscope to ensure that

all the cells are detached and floating. The side of the flasks may be gently tapped to release any remaining attached cells.

7. Transfer the cells into the centrifuge tube containing the retained spent medium and cells.

8. Centrifuge the remaining cell suspension at 150g for 5 minutes. Also centrifuge the washings from Number 3 above if they contain significant numbers of cells.

9. Decant the supernatants and resuspend the cell pellets in a small volume (10-20mls) of fresh culture medium. Pool the cell suspensions. Count the cells.

10. Pipette the required number of cells to a new labeled flask and dilute to the required volume using fresh medium (refer to ECACC Cell Line Data Sheet for the required seeding density).

11. Repeat this process every 2-3 days as necessary.

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Key Points 1. Although most cells will detach in the presence of trypsin alone

the inclusion of EDTA is used to enhance the activity of the enzyme.

2. Trypsin is inactivated in the presence of serum. Therefore, it is essential to remove all traces of serum from the culture medium by washing the monolayer of cells with PBS without Ca2+/Mg2+. Repeated warming to 37ºC also inactivates trypsin.

3. Cells should only be exposed to trypsin/EDTA long enough to detach cells. Prolonged exposure could damage surface receptors. In general a shorter time of exposure to trypsin is required for semi adherent cell lines.

4. Trypsin should be neutralized with serum prior to seeding cells into new flasks otherwise cells will not attach.

5. Trypsin may also be neutralized by the addition of Soybean trypsin Inhibitor , where an equal volume of inhibitor at a concentration of 1mg/ml is added to the trypsinised cells. The cells are then centrifuged, resuspended in fresh culture medium and counted as above.

6. If a CO2 incubator is not available gas the flasks for 1-2 minutes with 5% CO2 in 95% air filtered through a 0.25m filter. 60

Subculture of Suspension Cell Lines

• AimIn general terms cultures derived from blood (e.g. lymphocytes) grow in suspension. Cells may grow as single cells or in clumps (e.g. EBV transformed lymphoblastoid cell lines). For these types of lines subculture by dilution is relatively easy. But for lines that grow in clumps it may be necessary to bring the cells into a single cell suspension by centrifugation and resuspension by pipetting in a smaller volume before counting.

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Procedure 1. View cultures using an inverted phase contrast

microscope. Cells growing in exponential growth phase should be bright, round and refractile. Hybridomas may be very sticky and require a gentle knock to the flask to detach the cells. EBV transformed cells can grow in very large clumps that are very difficult to count and the center of the large clumps may be non-viable.

2. Do not centrifuge to subculture unless the pH of the medium is acidic (phenol red = yellow) which indicates the cells have overgrown and may not recover. If this is so, centrifuge at 150g for 5 minutes, re-seed at a slightly higher cell density and add 10- 20% of conditioned medium (supernatant) to the fresh media.

3. Take a small sample of the cells from the cell suspension (100-200uL ). Calculate cells/ml and re-seed the desired number of cells into freshly prepared flasks without centrifugation just by diluting the cells. The data sheet will give the recommended seeding densities.

4. Repeat this every 2-3 days. 62

Key Points

1. If the cell line is a hybridoma or other cell line that produces a substance (e.g. recombinant protein or growth factor) of interest retain the spent media for analysis.

63

Cell Quantification • Aim

For the majority of manipulations using cell cultures, such as transfections, cell fusion techniques, cryopreservation and subculture routines it is necessary to quantify the number of cells prior to use. Using a consistent number of cells will maintain optimum growth and also help to standardize procedures using cell cultures. This in turn gives results with better reproducibility.

• Materials Media– pre-warmed to appropriate temperature (refer

to the ECACC Cell Line Data Sheet for the correct medium and temperature)

70% ethanol in water 0.4% Trypan Blue Solution Trypsin/EDTA

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Cell Quantification• Equipment Personal protective equipment (sterile gloves,

laboratory coat, safety visor) Waterbath set to appropriate temperature Microbiological safety cabinet at appropriate

containment level Centrifuge CO2 incubator Haemocytometer , Improved Neubauer, Camlab

CCH.AC1) Inverted phase contrast microscope Pre-labeled flasks

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Procedure 1. Bring adherent and semi adherent cells into

suspension using trypsin/EDTA and resuspend in a volume of fresh medium at least equivalent to the volume of trypsin. For cells that grow in clumps centrifuge and resuspend in a small volume and gently pipette to break up clumps.

2. Under sterile conditions remove 100-200uL of cell suspension.

3. Add an equal volume of Trypan Blue (dilution factor =2) and mix by gentle pipetting.

4. Clean the haemocytometer. 5. Moisten the coverslip with water or exhaled breath.

Slide the cover-slip over the chamber back and forth using slight pressure until Newton’s refraction rings appear (Newton’s refraction rings are seen as rainbow-like rings under the cover-slip).

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Procedure6. Fill both sides of the chamber (approx. 5-10uL) with

cell suspension and view under a light microscope using x20 magnification.

7. Count the number of viable (seen as bright cells) and non-viable cells (stained blue) . Ideally >100 cells should be counted in order to increase the accuracy of the cell count . Note the number of squares counted to obtain your count of >100.

8. Calculate the concentration of viable and non-viable cells and the percentage of viable cells using the equations below.

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A is the mean number of viable cells counted, i.e. Total viable cells counted divided by Number of squares

B is the mean number of non-viable cell counted, i.e. Total non-viable cells counted divided by Number of squares

C is the dilution factor and D is the correction factor (this is provided by the

haemocytometer manufacturer). • Concentration of viable cells (cells/ml) = A x C x D

Concentration of non-viable cells (cells/ml) = B x C x DTotal number of viable cells = concentration of viable cells x volumeTotal number of cells = number of viable + number of dead cellsPercentage Viability = (No of viable cells x 100) divided by Total No of cells

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Key Points 1. Trypan Blue is toxic and is a potential carcinogen.

Protective clothing, gloves and face/eye protection should be worn. Do not breathe the vapor.

2. The central area of the counting chamber is 1mm2.This area is subdivided into 25 smaller squares (1/25mm2). Each of these is surrounded by triple lines and is then further divided into 16 (1/400mm2).The depth of the chamber is 0.1mm.

3. The correction factor of 104 converts 0.1mm3 to 1ml (0.1mm3 = 1mm2 x 0.1mm)

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Key Points4. There are several sources of inaccuracy:

o The presence of air bubbles and debris in the chamber.

o Overfilling the chamber such that sample runs into the channels or the other chamber

o Incomplete filling of the chamber. o Cells not evenly distributed throughout the chamber. o Too few cells to count. This can be overcome by

centrifuging the cells, resuspending in a smaller volume and recounting.

o Too many cells to count. This can be overcome by using a higher dilution factor in trypan blue e.g. 1:1070

Cryopreservation of Cell Lines

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• Materials • Freeze medium (commonly 70% basal

medium, 20% FCS, 10% DMSO or glycerol, check data sheets for details).

• 70% ethanol in water • PBS without Ca2+ Mg2+• 0.25% trypsin/EDTA in HBSS, without

Ca2+/Mg2+• DMSO • Trypsin/EDTA

Cryopreservation of Cell Lines

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Cryopreservation of Cell Lines• Equipment • Personal protective equipment (sterile gloves,

Laboratory coat) • Full-face protective mask/visor • Waterbath set to 37ºC • Microbiological safety cabinet at appropriate

containment level • Centrifuge • Haemocytometer , Improved Neubauer – Camlab

CCH.AC1) • Pre labeled ampules/cryotubes • Cell Freezing Device

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Procedure 1. View cultures using an inverted microscope to assess

the degree of cell density and confirm the absence of bacterial and fungal contaminants.

2. Bring adherent and semi adherent cells into suspension using trypsin/EDTA as above (Protocol 3 and 4 – Subculture of adherent/attached and semi-adherent cell lines) and re-suspend in a volume of fresh medium at least equivalent to the volume of trypsin. Suspension cell lines can be used directly.

3. Remove a small aliquot of cells (100-200uL) and perform a cell count (Protocol 6 – Cell Quantification). Ideally the cell viability should be in excess of 90% in order to achieve a good recovery after freezing.

4. Centrifuge the remaining culture at 150g for 5 minutes

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Procedure1. View cultures using an inverted microscope to

assess the degree of cell density and confirm the absence of bacterial and fungal contaminants.

2. Bring adherent and semi adherent cells into suspension using trypsin/EDTA as above (Protocol 3 and 4 – Subculture of adherent/attached and semi-adherent cell lines) and re-suspend in a volume of fresh medium at least equivalent to the volume of trypsin. Suspension cell lines can be used directly.

3. Remove a small aliquot of cells (100-200uL) and perform a cell count (Protocol 6 – Cell Quantification). Ideally the cell viability should be in excess of 90% in order to achieve a good recovery after freezing.

4. Centrifuge the remaining culture at 150g for 5 minutes 75

Procedure5. Re-suspend cells at a concentration of 2-4x106

cells per ml in freeze medium. 6. Pipette 1ml aliquots of cells into cyroprotective

ampules that have been labeled with the cell line name, passage number, cell concentration and date.

7. Place ampules inside a passive freezer e.g. Nalgene Mr. Frosty . Fill freezer with isopropyl alcohol and place at –80ºC overnight.

8. Frozen ampules should be transferred to the vapor phase of a liquid nitrogen storage vessel and the locations recorded

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Key Points • Culture cell line in the absence of antibiotics for 2

passages prior to testing. • Bring attached cells into suspension with the use of

a cell scraper. Suspension cell lines may be tested directly.

• Inoculate 2 x Thioglycollate Medium (TGM) and 2 x Tryptone Soya broth (TSB) with 1.5ml test sample.

• Inoculate 2 (TGM) and 2 (TSB) with 0.1ml C.albicans (containing 100 colony forming units, cfu).

• Inoculate 2 (TGM) and 2 (TSB) with 0.1ml B. subtilis (containing 100cfu).

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Key Points5 An alternative to the Mr. Frosty system is the Taylor Wharton

passive freezer where ampules are held in liquid nitrogen vapor in the neck of Dewar. The system allows the ampules to be gradually lowered thereby reducing the temperature. Rate controlled freezers are also available and are particularly useful if large numbers of ampules are frozen on a regular basis.

6. As a last resort if no other devices are available ampules may be placed inside a well insulated box (such as a polystyrene box with sides that are at least 1cm thick) and placed at –80ºC overnight. It is important to ensure that the box remains upright throughout the freezing process. Once frozen, ampules should be transferred to the vapor phase of a liquid nitrogen storage vessel and the locations recorded.

7. If using a freezing method involving a -80ºC freezer it is important to have an allocated section for cell line freezing so that samples are not inadvertently removed. If this happens at a crucial part of the freezing process then viability and recovery rates will be adversely affected.

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Testing for Bacteria and Fungi • Aim

In cases of gross contamination the naked eye may identify the presence of bacteria and fungi. However, it is necessary to detect low-level infections by incubation of cell cultures and/or their products in microbiological broth. Equally these sterility tests can be used to confirm the absence of bacteria and fungi from the preparation which is important when preparing cell banks or cell culture products.

• Materials • Soybean Casein Digest (Tryptone Soya Broth,TSB) (15ml

aliquots) TSB Powder • Fluid Thioglycollate Medium (20ml aliquots) (TGM) • Bacillus subtilis NCTC* • Candida albicans NCTC* • Clostridium sporogenes NCTC*

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Procedure 1. Culture cell line in the absence of antibiotics for 2

passages prior to testing. 2. Bring attached cells into suspension with the use of

a cell scraper. Suspension cell lines may be tested directly.

3. Inoculate 2 x Thioglycollate Medium (TGM) and 2 x Tryptone Soya broth (TSB) ( with 1.5ml test sample.

4. Inoculate 2 (TGM) and 2 (TSB) with 0.1ml C.albicans (containing 100 colony forming units, cfu).

5. Inoculate 2 (TGM) and 2 (TSB) with 0.1ml B. subtilis (containing 100cfu).

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Procedure6. Inoculate 1 TGM with 0.1ml C. sporogenes

(containing 100cfu). 7. Leave 2 (TGM) and 2 (TSB) un-inoculated as

negative controls. 8. Incubate broths as follows:

o For TSB, incubate one broth of each pair at 32ºC the other at 22ºC for 14 days

o For TGM, incubate one broth of each pair at 32ºC the other at 22ºC for 14 days

o For the TGM inoculated with C.sporogenes incubate at 32ºC for 14 days

9. Examine Test and Control broths for turbidity after 14 days.

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• Criteria for a Valid ResultAll positive control broths show evidence of bacteria and fungi within 14 days of incubation and the negative control broths show no evidence of bacteria and fungi.

• Criteria for a Positive ResultTest broths containing bacteria or fungi show turbidity.

• Criteria for a Negative ResultTest broths should be clear and show no evidence of turbidity

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• Notes • The positive controls should be handled in a

laboratory remote from the main tissue culture laboratory.

• Control organisms (Bacillus subtilis, Clostridium sporogenes and Candida albicans) are also available from the National Collection of Type Cultures (NCTC), UK*.

• This test procedure should be carried out in a microbiology laboratory away from the cell culture laboratory

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Detection of Mycoplasma by Culture

• AimDetection of mycoplasma by culture is the reference method of detection and has a theoretical level of detection of 1 colony-forming unit (cfu). However there are some strains of mycoplasma that are non-cultivable (certain strains of Mycoplasma hyorhinis). The method is suitable for the detection of mycoplasma in both cell cultures and cell culture reagents and results are obtained within 4 weeks. Mycoplasma colonies observed on agar plates have a ‘fried egg’ appearance

• Materials • 70% ethanol in water • Mycoplasma Pig Agar plates (in 5cm petri dishes) • Mycoplasma Pig Agar broths (in 1.8ml aliquots) • M. orale NCTC • M. pneumoniae NCTC

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• Equipment • Personal protective equipment (sterile gloves,

laboratory coat, safety visor) • Waterbath set to 37ºC • Microbiological safety cabinet at appropriate

containment level • CO2 Incubator set at 32ºC • Gas Jar (Gallenkamp) • Gas Pak Anaerobic System (Gallenkamp) • Gas Pak Catalyst (Gallenkamp) • Gas Pak Anaerobic Indicator (Gallenkamp

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Procedure • Inoculate 2 agar plates with 0.1ml of test sample. • Inoculate 1 agar plate with 100cfu M. pneumoniae. • Inoculate 1 agar plate with 100cfu M. orale. • Leave 1 agar plate un-inoculated as a negative control. • Inoculate 1 broth with 0.2 ml of test sample. • Inoculate 1 broth with 100cfu M. pneumoniae. • Inoculate 1 broth with 100cfu M. orale. • Leave 1 agar plate un-inoculated as a negative control. • Incubate agar plates anaerobically for 14 days at 37ºC

using a gas jar with anaerobic gas pak and catalyst. • Incubate broths aerobically for 14 days at 37ºC. • Between 3 and 7 days and 10 and 14 days incubation,

subculture 0.1 ml of test broth onto an agar plate and incubate plate anaerobically as above.

• Observe agar plates after 14 days incubation at x300 magnification using an inverted microscope for the presence of mycoplasma colonies 86

• Criteria for a Valid ResultAll positive control agar plates and broths show evidence of mycoplasma by typical colony formation on agar plates and usually a color change in broths.All negative control agar plates and broths show no evidence of mycoplasma.

• Criteria for a Positive ResultTest agar plates infected with mycoplasma show typical colony formation.

• Criteria for a Negative ResultThe test agar plates show no evidence of mycoplasma.

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• Notes • Mycoplasma colonies have a typical colony formation

commonly described as “fried egg” due to the opaque granular central zone of growth penetrating the agar surrounded by a flat translucent peripheral zone on the surface. However in many cases only the control zone will be visible.

• Positive controls may be included at a concentration to give 100 colony-forming units. These controls should obviously be handled in a laboratory remote from the main tissue culture laboratory.

• Control organisms (M. pneumoniae, and M. orale) are available from National Collection of Type Cultures (UK).

• Mycoplasma pneumoniae is a potential pathogen and must be handled in a class II microbiological safety cabinet operating to ACDP Category 2 Conditions.

• This test procedure should be carried out in a microbiology laboratory away from the cell culture laboratory.

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Testing for Mycoplasma by Indirect DNA Stain (Hoechst 33258 stain)

• AimDNA staining methods such as Hoechst staining techniques are quick with results available within 24 hours, which compares favorably with 4 weeks for detection by culture. However the staining of cultures directly with a DNA stain, results in a much-reduced sensitivity (~106cfu/ml). This may be improved by co-culturing the test cell line in the presence of an indicator cell line such as Vero (Prod.No. 84113001-1v1). This enrichment step results in a sensitivity of 104 cfu/ml of culture. This step also improves sensitivity by increasing the surface area upon which mycoplasma can adhere. Like detection by culture, DNA staining methods are suitable for the detection of mycoplasma from cell cultures or cell culture reagents.

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• Materials • Media– pre-warmed to 37ºC (refer to Cell Line Data

Sheet for the correct medium) • 70% ethanol in water • Methanol • Acetic Acid Glacial • Hoechst 33258 stain solution • Vero cells (Prod. No. 84113001-1v1) • Mountant (Autoclave 22.2ml 0.2M citric acid with

27.8ml 0.2M disodium phosphate. Add 50ml glycerol. Filter sterilize and store at 4ºC)

• Mycoplasma hyorhinis NCTC* 10112

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• Equipment • Personal protective equipment (sterile gloves,

laboratory coat, safety visor) • Waterbath set to 37ºC • Microbiological safety cabinet of appropriate

containment level • Centrifuge • CO2 Incubator set at 37ºC • Microscope (uv Epi-Fluorescent.) • 35mm plastic tissue culture dishes • Multidish 24 well • Cell scraper • Microscope slides and 22mm cover slips • Aluminum foil

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• Procedure Equipment • For each sample and control sterilize 2 cover slips in a

hot oven at 180ºC for 2 hours or by immersing in 70% ethanol and flaming in a blue Bunsen flame until the ethanol has evaporated. Also sterilize 2 cover slips to use as a negative control.

• Place the cover slips in 35mm culture dishes (1 per dish). • Store until needed. • To prepare the Vero indicator cells add 2x104 cells in

2ml of antibiotic-free growth medium to each tissue culture dish.

• Incubate at 37ºC in 5% CO2 for 2 – 24 hrs to allow the cells to adhere to the cover slips.

• Bring attached test cell lines into suspension using a cell scraper. Suspension cell lines may be tested directly.

• Remove 1ml of culture supernatant from duplicate dishes and add 1ml of test sample to each. Inoculate 2 dishes with 100cfu M. hyorhinis and 2 with 100cfu M. orale.. org.uk

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• Leave duplicate tissue culture dishes un-inoculated as negative controls.

• Incubate dishes at 37ºC in 5% CO2 for 1-3 days. • After 1 day observe one dish from each pair for bacterial or

fungal infection. If contaminated discard immediately. Leave the remaining dish of each pair for a further 2 days.

• Fix cells to cover-slip by adding a minimum of 2ml of freshly prepared fixative (1:3 glacial acetic acid: absolute methanol) to the tissue culture dish and leave for 3 to 5 minutes.

• Decant used fixative to toxic waste bottle. Add another 2ml aliquot of fixative to cover-slip and leave for a further 3 to 5 min. Decant used fixative to toxic waste.

• Air dry cover-slip by resting it against the tissue culture dish for 30-120 min.

• Replace cover-slip in dish and add a minimum of 2ml Hoechst stain . Leave for 5 minutes shielded from direct light by aluminum foil .

• Decant used and unused stain to toxic waste. • Add 1 drop of mountant to a pre-labeled microscope slide and

place cover-slip (cell side down) onto slide. • Keep slide covered with aluminum foil , allowing it to set for at

least 15 min at 37ºC or for 30 min at room temperature. • Observe slide under uv Epi-Fluorescence at x1000 93

• Criteria for a Valid ResultNegative controls show no evidence of mycoplasma infectionPositive controls show evidence of mycoplasma infectionVero cells clearly seen as fluorescing nuclei.

• Criteria for a Positive ResultSamples infected with mycoplasma are seen as fluorescing nuclei plus extra-nuclear fluorescence of mycoplasma DNA (small cocci or filaments).

• Criteria for a Negative ResultUninfected samples are seen as fluorescing nuclei against a dark background. There should be no evidence of mycoplasma.

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• Notes • DNA stains such as Hoechst stain bind specifically to DNA.

In all cultures cell nuclei will fluoresce. Uncontaminated cultures will show only fluorescent nuclei whereas mycoplasma positive cultures contain small cocci or filaments which may or may not be adsorbed onto the cells.

• Hoechst stain is toxic and should be handled and discarded with care.

• Culture dishes should be placed in a sealed box or cultured in large petri dishes to reduce evaporation.

• Positives should obviously be handled in a laboratory remote from the main tissue culture laboratory.

• Control organisms (M. hyorhinis) are available from the National Collection of Type Cultures (UK).

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• In some instances results may be difficult to interpret for the following reasons:

– Bacterial/yeast/fungal contamination – Too much debris in the background – Broken nuclei as cells are all dead – Too few or no live cells

• Although this procedure recommends the setting up of positive controls, this may not necessarily be feasible nor desirable in a cell culture facility with limited resources. If positive controls are to be set up they should be done so in a separate laboratory from the main tissue culture facility. If this is not possible then positive slides can be purchased from ECACC. If positive controls are not being used then it is strongly recommended that you get an independent testing laboratory to periodically test your cell lines.

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• For animal cell culture the level of risk is dependent upon the cell line to be used and is based on whether the cell line is likely to cause harm to humans. The different classifications are given below:

• Low risk Non human/non primate continuous cell lines and

some well characterized human diploid lines of finite lifespan (e.g. MRC-5).

• Medium risk Poorly characterized mammalian cell lines.• High risk Cell lines derived from human/primate tissue or

blood. Cell lines with endogenous pathogens (the precise

categorization is dependent upon the pathogen) – refer to ACDP guidelines, 1985, for details.

Cell lines used following experimental infection where the categorization is dependent upon the infecting agent - refer to ACDP guidelines, 1985, for details*. 97

Waste Disposal • Any employer has a ‘duty of care’ to dispose of all

biological waste safely in accordance with national legislative requirements. Given below is a list of ways in which tissue culture waste can be decontaminated and disposed of safely. One of the most important aspects of the management of all laboratory-generated waste is to dispose of waste regularly and not to allow the amounts to build up. The best approach is ‘little and often’. Different forms of waste require different treatment.

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• Tissue culture waste (culture medium) - Inactivate overnight in a solution of hypochlorite (10,000ppm) prior to disposal to drain with an excess of water

• Contaminated pipettes should be placed in hypochlorite solution (2500ppm) overnight before disposal by autoclaving and incineration

• Solid waste such as flasks, centrifuge tubes, contaminated gloves, tissues etc. should be placed inside heavy duty sacks for contaminated waste and autoclaved prior to incineration. These bags are available from Bibby Sterilin and Greiner.

• If at all possible waste should be incinerated rather than autoclaved

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Sourcing of Cell Lines • Large numbers of cell lines look identical. Cell lines

with very different origins and biological characteristics typically cannot be separated on grounds of morphology or culture characteristics. Infection or contamination of a cell line with an adventitious virus or mycoplasma may significantly change the characteristics of the cells but again such contamination will be inapparent. Cell lines will also change with time in culture, and to add to all these natural hazards it is all too easy to mis-label or cross-contaminate different cell lines in a busy cell culture laboratory

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Main Types of Cell Culture • Primary Cultures• Primary cultures are derived directly from excised,

normal animal tissue and cultured either as an explant culture or following dissociation into a single cell suspension by enzyme digestion. Such cultures are initially heterogeneous but later become dominated by fibroblasts. The preparation of primary cultures is labor intensive and they can be maintained in vitro only for a limited period of time. During their relatively limited life span primary cells usually retain many of the differentiated characteristics of the cell in vivo.

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• Continuous Cultures• Continuous cultures are comprised of a single cell type

that can be serially propagated in culture either for a limited number of cell divisions (approximately thirty) or otherwise indefinitely. Cell lines of a finite life are usually diploid and maintain some degree of differentiation. The fact that such cell lines senesce after approximately thirty cycles of division means it is essential to establish a system of Master and Working banks in order to maintain such lines for long periods.

• Continuous cell lines that can be propagated indefinitely generally have this ability because they have been transformed into tumor cells. Tumor cell lines are often derived from actual clinical tumors, but transformation may also be induced using viral oncogenes or by chemical treatments. Transformed cell lines present the advantage of almost limitless availability, but the disadvantage of having retained very little of the original in vivo characteristics.

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Commonly used cell lines of each culture type

• Attached Cell Lines • Name Species and tissue of origin Morphology• MRC-5 Human lung Fibroblast• HELA Human cervix Epithelial• VERO African Green Monkey Kidney Epithelial• NIH 3T3 Mouse embryo Fibroblast• L929 Mouse connective tissue Fibroblast• CHO Chinese Hamster Ovary Fibroblast• BHK-21 Syrian Hamster Kidney Fibroblast• HEK 293 Human Kidney Epithelial• HEPG2 Human Liver Epithelial• BAE-1 Bovine aorta Endothelial

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• Suspension Cell Lines • Name Species and tissue of origin Morphology• NSO Mouse myeloma

Lymphoblastoid-like• U937 Human Hystiocytic Lymphoma Lymphoblastoid• Namalwa Human Lymphoma

Lymphoblastoid• HL60 Human Leukaemia Lymphoblastoid-like• WEHI 231 Mouse B-cell Lymphoma Lymphoblastoid• YAC 1 Mouse Lymphoma

Lymphoblastoid• U 266B1 Human Myeloma

Lymphoblastoid• SH-SY5Y Human neuroblastoma Neuroblast

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