I don't have the proper and well detailed protocol of Animal cell culture in cell culture lab. so please any one send me the pdf of well detailed protocol for animal cell culture?
For pdf click these link
https://www.atcc.org/~/media/PDFs/Culture%20Guides/AnimCellCulture_Guide.ashx
https://shop.roche.com/wcsstore/RASCatalogAssetStore/Articles/Culture%20_of_Animal_Cells-Basic_Techniques_TT.pdf
https://dmice.ohsu.edu/bd2k/skillscourse/files/CellCultureProtocol.pdf
https://www.vanderbilt.edu/viibre/CellCultureBasicsEU.pdf
And also more information
https://www.researchgate.net/publication/266455722_BASIC_TECHNIQUES_IN_ANIMAL_CELL_CULTURE
https://www.qiagen.com/us/resources/molecular-biology-methods/animal-cell-culture/
Best of luck, Bhaskara Rao Simma
dear Monyer , please see this link
https://www.vanderbilt.edu/viibre/CellCultureBasicsEU.pdf
Protocols and useful hints for the successful culture of animal cells
This section provides useful hints for culturing animal cells (i.e., cells derived from higher eukaryotes such as mammals, birds, and insects). It covers different types of animal cell cultures, considerations for cell culture, and cell culture protocols.
Animal cell cultures
Safety and handling considerations for animal cell culture
Cell culture conditions
Essential protocols for animal cell culture
Counting cells
Freezing and viability staining of cells
References
Animal cell cultures
Depending on their origin, animal cells grow either as an adherent monolayer or in suspension.
Adherent cells are anchorage-dependent and propagate as a monolayer attached to the cell culture vessel. This attachment is essential for proliferation — many adherent cell cultures will cease proliferating once they become confluent (i.e., when they completely cover the surface of cell culture vessel), and some will die if they are left in this confluent state for too long. Most cells derived from tissues are anchorage-dependent.
Suspension cells can survive and proliferate without being attached to a substratum. Hematopoietic cells (derived from blood, spleen, or bone marrow) as well as some transformed cell lines and cells derived from malignant tumors can be grown in suspension.
Primary cells, finite cultures, and continuous cell lines differ in their proliferative potential (see below). Different cell types vary greatly with respect to their growth behavior and nutritional requirements. Optimization of cell culture conditions is necessary to ensure that cells are healthy and in optimal condition for downstream applications.
Extensive information on culturing cells can be found in reference 1.
Primary cell cultures
Primary cell cultures come from the outgrowth of migrating cells from a piece of tissue or from tissue that is disaggregated by enzymatic, chemical, or mechanical methods. Primary cultures are formed from cells that survive the disaggregation process, attach to the cell culture vessel (or survive in suspension), and proliferate.
Primary cells are morphologically similar to the parent tissue. These cultures are capable of only a limited number of cell divisions, after which they enter a non-proliferative state called senescence and eventually die out. Adherent primary cells are particularly susceptible to contact inhibition, that is, they will stop growing when they have reached confluency. At lower cell densities, however, the normal phenotype can be maintained. Primary cell culture is generally more difficult than culture of continuous cell lines.
Primary cell cultures are sometimes preferred over continuous cell lines in experimental systems. Primary cells are considered by many researchers to be more physiologically similar to in vivo cells. In addition, cell lines cultured for extended periods of time can undergo phenotypic and genotypic changes that can lead to discrepancies when comparing results from different laboratories using the same cell line. Furthermore, many cell types are not available as continuous cell lines.
Finite cell cultures
Finite cell cultures are formed after the first subculturing (passaging) of a primary cell culture. These cultures will proliferate for a limited number of cell divisions, after which they will senesce. The proliferative potential of some human finite cell cultures can be extended by introduction of viral transforming genes (e.g., the SV40 transforming-antigen genes). The phenotype of these cultures is intermediate between finite cultures and continuous cultures. The cells will proliferate for an extended time, but usually the culture will eventually cease dividing, similar to senescent primary cells. Use of such cells is sometimes easier than use of primary cell cultures, especially for generation of stably transfected clones.
Continuous cell lines
Finite cell cultures will eventually either die out or acquire a stable, heritable mutation that gives rise to a continuous cell line that is capable of unlimited proliferative potential. This alteration is commonly known as in vitro transformation or immortalization and frequently correlates with tumorigenicity.
Rodent primary cell cultures form continuous cell lines relatively easily, either spontaneously or following exposure to a mutagenic agent. In contrast, human primary cell cultures rarely, if ever, become immortal in this way and require additional genetic manipulation to form a continuous cell line. However, cell cultures derived from human tumors are often immortal.
Continuous cell lines are generally easier to work with than primary or finite cell cultures. However, it should be remembered that these cells have undergone genetic alterations and their behavior in vitro may not represent the in vivo situation.
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Safety and handling considerations for animal cell culture
Legislation and regulatory guidelines
Before undertaking any work with human or animal tissue (e.g., to establish a primary cell culture), it is necessary to ensure that the nature of the work conforms to the appropriate medical-ethical and animal-experiment legislation and guidelines. It may be necessary to seek approval from the relevant regulatory authorities and/or individuals.
Safety considerations and biohazards
When working with potentially hazardous material, it is important to be aware of the possible risks associated with both the material and the experimental protocol. All cell cultures are considered a biohazard because of their potential to harbor an infectious agent (e.g., a virus).
The degree of hazard depends on the cells being used and the experimental protocol. Primary cell cultures in particular should be handled carefully as these cultures have a high risk of containing undetected viruses. Although commonly used cell lines are generally assumed to be free of infectious agents, care should still be exercised when working with these cell lines as it is possible that they contain infectious agents, such as latent viruses. Cell cultures used to study specific viruses should be assumed to have the same degree of hazard as the virus under study.
We recommend handling all material as potentially infectious to ensure the safest possible working environment. Work should be performed in an approved laminar flow hood using aseptic technique, and the creation of aerosols should be avoided (see Handling cell cultures). After the work is complete, all waste media and equipment (i.e., used flasks, pipets, etc.) should be disinfected by autoclaving or immersion in a suitable disinfectant according to institutional and regional guidelines.
Handling cell cultures
Adherence to good laboratory practice when working with cell cultures is essential for two reasons: first, to reduce the risk of exposure of the worker to any potentially infectious agent(s) in the cell culture, and second, to prevent contamination of the cell culture with microbial or other animal cells (see Aseptic technique and minimization of aerosols).
Aseptic technique and minimization of aerosols
Aseptic technique and the proper use of laboratory equipment are essential when working with cell cultures. Always use sterile equipment and reagents, and wash hands, reagent bottles, and work surfaces with a biocide or 70% ethanol before beginning work.
Creation of aerosols should be avoided — aerosols represent an inhalation hazard, and can potentially lead to cross-contamination between cultures. To avoid aerosols, use TD (to deliver) pipets, and not TC (to contain) pipets; use pipets plugged with cotton; do not mix liquids by rapidly pipetting up and down; do not use excessive force to expel material from pipets; and do not bubble air through liquids with a pipet. Avoid releasing the contents of a pipet from a height into the receiving vessel. Expel liquids as close as possible to the level of liquid of the receiving vessel, or allow the liquid to run down the sides of the vessel.
Proper use of equipment can also help minimize the risk of aerosols. For example, when using a centrifuge, ensure the vessel to be centrifuged is properly sealed, avoid drops of liquid near the top of the vessel, and use centrifuge buckets with caps and sealed centrifuge heads to prevent contamination by aerosols.
Laminar flow hoods
For the most efficient operation, laminar flow hoods should be located in an area of the laboratory where there is minimal disturbance to air currents. Avoid placing laminar flow hoods near doorways, air vents, or locations where there is high activity. Hoods are often placed in dedicated cell culture rooms.
Tips:
Keep laminar flow hoods clean, and avoid storing equipment inside the hood.
Before starting work, disinfect the work surface of the hood as well as the outside of any bottles (e.g., by wiping with 70% ethanol), and then place everything needed for the cell culture procedure in the hood.
Arrange equipment, pipets, waste containers, and reagent bottles so that used items are not placed near clean items, and avoid passing used items over clean items.
Place used items (e.g., pipets) in a container inside the hood, and disinfect or seal before removing from the hood.
Contamination
The presence of microorganisms can inhibit cell growth, kill cells, and lead to inconsistent results. Contamination of cell cultures can occur with both cell culture novices and experts.
Potential contamination routes are numerous. For example, cultures can be infected through poor handling, from contaminated media, reagents, and equipment (e.g., pipets), and from microorganisms present in incubators, refrigerators, and laminar flow hoods, as well as on the skin of the worker and in cultures coming from other laboratories.
Bacteria, yeasts, fungi, molds, mycoplasmas, and other cell cultures are common contaminants in animal cell culture. To safeguard against accidental cell culture loss by contamination, we recommend freezing aliquots of cultured cells to re-establish the culture if necessary (see Freezing and viability staining of cells).
Microbial contamination
The characteristic features of microbial contamination are presented in the table Characteristic features of microbial contamination. The presence of an infectious agent sometimes can be detected by turbidity and a sharp change in the pH of the medium (usually indicated by a change in the color of the medium), and/or cell culture death. However, for some infections, no turbidity is observed and adverse effects on the cells are not easily observed.
Cell cultures should be routinely evaluated for contamination. Mycoplasmal infections are one of the more common and difficult-to-detect infections; their detection and eradication are described in further detail below.
Characteristic features of microbial contamination
Characteristic
Bacteria
Yeast
Fungi
Change in pH
pH drop with most infections
pH change with heavy infections
pH changes sometimes
Cloudy medium: Under microscope (100–400x)
Shimmering in spaces between cells; rods or cocci may be observed
Round or ovoid particles that bud off smaller particles
Thin filamentous mycelia; sometimes clumps of spores
Mycoplasmal infection — detection
Mycoplasmas are small, slow-growing prokaryotes that lack a cell wall and commonly infect cell cultures. They are generally unaffected by the antibiotics commonly used against bacteria and fungi. Furthermore, as mycoplasma do not overgrow cell cultures and typically do not cause turbidity, they can go undetected for long periods of time and can easily spread to other cell cultures. The negative effects of mycoplasmal contamination include inhibition of metabolism and growth, as well as interference with nucleic acid synthesis and cell antigenicity. Acute infection causes total deterioration of the cell culture, sometimes with a few apparently resistant colonies that may, in fact, also be chronically infected. There are two main approaches to detect mycoplasma — Hoechst 33258 staining (1, 3) and mycoplasma-specific DNA probes. Alternatively, a PCR-based, mycoplasma-testing service is offered by the ATCC or other organizations on a fee-for-service basis.
Mycoplasmal infection — eradication
The best action to take with a culture containing chronic mycoplasmal infection is to discard it by either autoclaving or incineration. Only if the cell culture is absolutely irreplaceable should eradication be attempted. This process should be performed by experienced personnel in an isolated hood that is not used for cell culture, preferably in a separate room. Elimination of mycoplasma is commonly achieved by treatment with various commercially available antibiotics such as a quinolone derivative (Mycoplasma Removal Agent), ciprofolxacin (Ciprobay), enrofloxacin (Baytril), and a combination of tiamulin and minocycline (BM-Cyclin). Treatment procedures and appropriate antibiotic concentrations can be found in the suppliers’ instructions and in references 1 and 3.
Cross-contamination of cell lines
Cross-contamination of one cell culture with fast-growing cells from another culture (such as HeLa) presents a serious risk. To avoid cross-contamination, only use cell lines from a reputable cell bank; only work with one cell line at a time in the hood; use different pipets, bottles of reagents, and bottles of media for different cell lines; and check cells regularly for the correct morphological and growth characteristics.
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Cell culture conditions
Media and serum
The choice of cell culture medium is extremely important, and significantly affects the success of cell culture experiments. Different cell types have highly specific growth requirements, and the most suitable medium for each cell type must be determined experimentally. Common basal media include Eagle minimal essential medium (MEM), Dulbecco’s modified Eagle medium (DMEM), RPMI 1640, and Ham F10. These contain a mixture of amino acids, glucose, salts, vitamins, and other nutrients, and are available either as a powder or as a liquid from various commercial suppliers.
Basal media are usually supplemented just before use with serum, L glutamine, and antibiotics and/or fungicides to give complete medium (also called growth medium). Serum is a partially undefined material that contains growth and attachment factors, and may show considerable variation in the ability to support growth of particular cells. Fetal calf serum (FCS) is the most frequently used serum, but for some applications, less expensive sera such as horse or calf serum can be used. Different serum batches should be tested to find the best one for each cell type. L-glutamine is an unstable amino acid that, with time, converts to a form that cannot be used by cells, and should be added to medium just before use. Antibiotics and fungicides can be used as a supplement to aseptic technique to prevent microbial contamination. The working concentration of commonly used antibiotics and fungicides is provided in the tables Commonly used antibiotics for animal cell culture and Commonly used fungicides for animal cell culture. Some cell types, particularly primary cells, require additional supplements (e.g., collagen and fibronectin, hormones such as estrogen, and growth factors such as epidermal growth factor and nerve growth factor) to attach to the cell culture vessel and proliferate.
Media, serum, and supplements should be tested for sterility before use by incubation of a small aliquot at 37°C for 48 hours. If microbial growth has occurred after this incubation, the medium or supplement should be discarded.
Commonly used antibiotics for animal cell culture
Antibiotic
Working concentration
Effective against
Stability at 37°C
Penicillin
50–100 U/ml
Gram-positive bacteria
3 days
Streptomycin
50–100 µg/ml
Gram-negative bacteria
5 days
Kanamycin
100 µg/ml
Gram-positive and gram-negative bacteria; mycoplasma
5 days
Gentamycin
5–50 µg/ml
Gram-positive and gram-negative bacteria; mycoplasma
5 days
Adapted from reference 4.
Commonly used fungicides for animal cell culture
Antibiotic
Working concentration
Effective against
Stability at 37°C
Nystatin
100 U/ml
Yeasts and molds
3 days
Amphotericin B
0.25–2.5 µg/ml
Yeasts and molds
3 days
Adapted from reference 4.
Incubation conditions
The incubation conditions used to culture cells are also important. Cell cultures should be incubated in an incubator with a tightly regulated temperature (e.g., a water-jacketed incubator) and CO2 concentration. Most cell lines grow at 37°C and 5% CO2 with saturating humidity, but some cell types require incubation at lower temperatures and/or lower CO2concentrations.
Cell culture vessel
The choice of growth vessel can influence the growth of adherent cells. Sterile, disposable dishes and flasks that have been treated to allow attachment of animal cells to the growing surface are available commercially.
Cell banking
For some cell cultures, especially those that are valuable, it is common practice to maintain a two-tiered frozen cell bank: a master cell bank and a working cell bank. The working cell bank comprises cells from one of the master bank samples, which have been grown for several passages before storage. If future cell samples are needed, they are taken from the working cell bank. The master cell bank is used only when absolutely necessary. This ensures that a stock of cells with a low passage number is maintained, and avoids genetic variation within the cell culture.
Culture instability
The growth rate of cells that have been repeatedly subcultured may sometimes unexpectedly decrease, and the cytotoxicity of, for example, a transfection process may unexpectedly increase. This instability can result from variations in cell culture conditions, genomic variation, and selective overgrowth of constituents of the cell population. We recommend using cells with a low passage number (
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