General Procedures for Cell Culture

I. INTRODUCTION
A. Background
Mammalian cell culture emerged as a valuable research tool in the 1950s when the first cell line, HeLa, was successfully cultured from a human cervical cancer (Gey et al., 1952). However, it is only since the mid-1980s that reproducible and reliable largescale culture of mammalian cells has been achieved. The development of cell culture led to new experimental approaches to cellular physiology in which isolated, functionally differentiated cells could be maintained in culture under conditions that allowed direct manipulations of the environment and measurement of the resulting changes in the function of a single cell type. Today many aspects of research and development involve the use of animal cells as in vitro model systems, substrates for viruses, and in the production of diagnostic and therapeutic products in the pharmaceutical industry.

The process of initiating a culture from cells, tissues, or organs taken directly from an animal and cultured either as an explant culture or following dissociation into a single cell suspension by enzyme digestion is known as primary culture. Certain primary cultures may be passaged for a finite number of population doublings before senescence occurs, but usually the number of doublings is limited in adult-derived or differentiated cell types. However, these cells are still invaluable as they retain many of the differentiated characteristics of the cell in vivo. After a number of subcultures a cell line will either die out, referred to as a finite cell line (and is usually diploid), or a population of cells can transform to become a continuous cell line. Lines of transformed cells can also be obtained from normal primary cell cultures by infecting them with oncogenic viruses or treating them with carcinogenic chemicals. It is often very difficult to obtain a normal human cell line from a culture of normal tissue. In contrast, neoplasms from humans have been generated into many cell lines. It appears that the possession of a cancerous phenotype allows the easier adaptation to cell culture, which may be due in part to the fact that cancer cells are aneuploid. Transformed cell lines have the advantage of almost limitless availability; however, they often retain very little of the original in vivo characteristics.

Cell cultures in vitro take one or two forms, either growing in suspension (as single cells or small clumps) or as an adherent monolayer attached to the surface of the tissue culture flask. It is necessary that cell culture medium is produced so that it mimics the physiological conditions within tissues. In vitro growth of cell lines requires a sterile environment in which all the nutrients for cellular metabolism can be provided in a readily accessible form at the optimal pH and temperature for growth. Media formulations vary in complexity and have been developed to support a wide variety of cell types, including Eagle's minimum essential medium (MEM), Dulbecco's modified Eagle's medium (DMEM), RPMI 1640, and Ham's F12. Cell culture media essentially consist of a number of factors required for the growth of the cells, including amino acids (essential and nonessential), lipids (essential fatty acids, glycerides, etc.), trace elements, vitamins, and cofactors. Carbohydrates such as glucose or fructose are usually added as an energy source. Other essential components include inorganic salts, which provide buffering capacity and osmotic balance (260-320 mOsm/kg) to counteract the effects of carbon dioxide and lactic acid produced during cellular metabolism. The pH of medium should ideally be between 7.2 and 7.4 (however, fibroblasts prefer a pH range of 7.4-7.7 whereas transformed cells prefer a pH range of 7.0-7.4). When phenol red is included in the medium, the medium turns purple above pH 7.6 and yellow below pH 7.0. Buffering of culture media is usually provided by sodium bicarbonate and cells are usually maintained in vented tissue culture flasks in an atmosphere of 5% CO₂. Synthetic buffers such as HEPES can also help maintain correct pH levels in closed, nonvented tissue culture flasks, although it may be toxic to some cell types. Serum, which is a complex mixture of albumins, growth promoters, and growth inhibitors, may also be incorporated into the growth medium at concentrations from 5 to 20%, although certain production processes and experimental procedures require the use of serum-free conditions. The majority of cell lines require the addition of serum to defined culture medium to stimulate growth and cell division but can be subject to significant biological variation. The most common source is bovine and this may be of adult, newborn, or foetal origin.

A number of other books give more detailed and comprehensive treatment of procedures for mammalian cell culture and the reader is recommended to refer to these books for additional information (Doyle et al., 1998; Freshney, 1992, 2000; Shaw, 1996).

B. General Safety when Working with Mammalian Cells in Culture
In general, because of the potential risks that may be associated with material of biological origin, standard and specific laboratory regulations should always be adhered to.

• Antibodies, sera, and cells (particularly but not exclusively those of human and nonhuman primate origin) may pose a potential threat of infection or other biological hazard (e.g., prion disease).
• Many animal cells contain C-type particles, which may be retrovirus related. All such materials may harbour pathogens and should be handled as potentially infectious material in accordance with local guidelines.
• Laboratory coats are essential. The Howie-type coat is the only recommended coat for biological work. Coats should be used only in the culture area and should be laundered frequently.
• Protective glasses should be worn at all times while contact lenses should never be worn in the laboratory area.
• No eating, drinking, or smoking should be permitted.
• No mouth pipetting of any solutions.
• Operators must make sure that any cuts, especially on the hands, are covered. Wearing gloves is strongly recommended, particularly for manipulations involving cells and biological material. Gloves should be of a standard appropriate to the risk of the agent being handled. Nitrile gloves may provide superior protection with lower allergy potential than traditional latex gloves.
• Thorough washing of hands before and after cell work with appropriate laboratory soap is essential.
• Immunisation against hepatitis B may be recommended if working with primary human material. In certain countries, where tuberculosis (TB) vaccination is not a standard (or staff are employed from such countries and where the material being handled may have a TB hazard) BCG vaccination may also be recommended (Richmond and McKinney, 1999). However, this should be at the discretion of the individual operator, and regular follow-up and paperwork, including titre estimation, are necessary to have an effective vaccination policy.
• To comply with current safety regulations, a cell culture laboratory should be fully ventilated, preferably with high efficiency particulate air (HEPA) filters on the inlets, and equipped with HEPA-filtered workstations where the airflow is directed away from the operator. A class II (type A) downflow recirculating laminar flow biological safety cabinet will provide a safe working environment for standard hazard material and should be checked yearly (or as recommended by manufacturer) for containment, airflow velocity, and efficiency. A horizontal flow cabinet should never be used, as it can, even in the absence of viruses, possibly increase exposure to allergens.
• In addition to standard alcohol-based disinfection for routine work and introduction of consumables, primary equipment and work areas should be regularly disinfected with laboratory disinfectants, e.g., Virkon, Tego, or equivalent. Manufacturer guidelines should be followed as some disinfectants may corrode materials.

II. MATERIALS AND INSTRUMENTATION
A. Materials The following are from Sigma Aldrich: DMEM (Cat. No. D5648), Ham's F12 (Cat. No. N2650), RPMI (Cat. No. R6504), NaHCO₃ (Cat. No. S5761), HEPES (Cat. No. H4034), dimethyl sulphoxide (DMSO, Cat. No. D5879), foetal calf serum (FCS, Cat. No. F7524), EDTA (Cat. No. E5134), soybean trypsin inhibitor (Cat. No. T6522), haemocytometers (Neubauer improved chamber) (Cat. No. Z35, 962-9), and replacement coverslips (Cat. No. Z37, 535-7).

The following are from Invitrogen (GIBCO brand): 200mM (100x) L-glutamine (Cat. No. 25030-024), penicillin/ streptomycin (5000IU/500µg/ml) (Cat. No. 15070-063), 2.5% trypsin (10×) (Cat. No. 15090-046), and Trypan blue (Cat. No. 15250-061).

The following are from Corning (Costar brand): 10- ml (Cat. No. 4101CS) and 25-ml (Cat. No. 4251) pipettes, 25-cm² (Cat. No. 3055) and 75-cm² (Cat. No. 3375) nonvented tissue culture flasks, and 25-cm² (Cat. No. 3056) and 75-cm² (Cat. No. 3376) vented tissue culture flasks.

The following are from Greiner: 30-ml (Cat. No. 2011-51) and 50-ml (Cat. No. 210161G) sterile containers/ centrifuge tubes, cryovials (Cat. No. 122278G), and autoclave bags (Cat. No. Bag1).

The following are from Millipore: 0.22-µm low protein-binding filters for small volumes (Cat. No. SLGVR25KS) and 0.22-µm filters for large volumes (Cat. No. SPGPM10RJ).

Phosphate-buffered saline (PBS) (Cat. No. BR14A) is from Oxoid. Laboratory disinfectants such as Virkon (Cat. No. CL900.05) and Tego (Cat. No. 2000) are from Antec and Goldschmidt, respectively.

B. Instrumentation
Automatic pipette aids (Accu-jet, Model No. Z33,386-7) are from Sigma Aldrich, the inverted microscope with phase-contrast optics (Model No. DM 1L) is from Leica Microsystems, and the centrifuge is from Eppendorf (Model No. 5810). The 37°C incubator (Model No. 310) is from Thermo Forma. The laminar flow cabinet (Model No. NU-425-600) is from Nuaire. Other standard laboratory apparatus includes refrigerators, -20°C and -80°C freezers, and a liquid nitrogen freezer. Clean autoclaves should be available for solutions, glassware, and other items that require sterilisation by moist heat. A separate waste autoclave should be available for general biological laboratory waste, including plastics and waste media. Dishwashers should also be available to ensure thorough cleaning of all glassware used in cell culture procedures.

III. PROTOCOLS
A. Good Practice and Safety Considerations
Equipment in the designated area for cell culture should be kept to the minimum required for the job. There should be proper entry facilities and internal surfaces must be easy to clean and dust free. When setting up a cell culture laboratory the following equipment is essential:

• Class II downflow recirculating laminar flow cabinet
• Low-speed biological centrifuge
• CO₂ incubator
• Inverted microscope with phase-contrast capabilities
• Refrigeration and freezing facilities
• Cell storage (liquid nitrogen) facilities

Prevention of contamination by bacteria, fungi (especially yeast), mycoplasma, or viruses is absolutely necessary in cell culture. Good laboratory practice requires that the following standard procedures are followed:

• Cell culture should be performed in a designated area that is easy to clean and free from clutter. Equipment used should broadly be designated for that purpose to prevent potential chemical or biological contamination by or due to other laboratory processes and operation. Ensure that all equipment are cleaned and serviced regularly.
• Cell culture by definition involves the handling of biological material. As such, all biological material can potentially harbour infective agents. Therefore, routine precautions to prevent infection should be exercised. Waste media and items coming into contact with biological material should be disinfected; autoclaving is probably the most broadly useful method. Where material of primary origin is in use and in the absence of specific legal guidelines, validation of the inactivation of biological material is vital.
• Although the majority of common culture lines are characterised as biosafety level 1, the Centers for Disease Control and Prevention (CDC) in the United States suggest that handling procedures be of biosafety level 2 standard, where material is of mixed origin, including some primary material. Biosafety level 2 or 2+ will be mandatory unless prior knowledge indicates the need for even higher standards of safe handling (Richmond and McKinney, 1999).
• It is important that cell lines are obtained from a reputable source, preferably the laboratory of origin or an established cell repository (e.g., ATCC or ECACC). Cells from all sources should be handled in quarantine until all quality control checks are completed, particularly microbial (and especially Mycoplasma) contamination should be checked.
• Only sterile, wrapped items (i.e., pipettes, culture flasks) should enter the room, and discarded media and waste should be removed each day. All used materials should be disposed of safely, efficiently, and routinely in accordance with local regulatory requirements. Keep cardboard packaging to a minimum in all cell culture areas.
• Stock cultures of two cell lines should never be worked on at the same time in a laminar flow cabinet. When working with different cell lines, a thorough cleaning of surfaces with a suitable disinfectant is required and a minimum of 15 min between handling different cell lines is essential to prevent cross contamination. Use of pipettes, medium/waste bottles, and so on for more than one cell line is another possible source of cross contamination and must be avoided.
• When setting up a large frozen stock line, aliquots should be thawed to test for viability, growth, and absence of contamination (including Mycoplasma). They should also be characterised and authenticated by some appropriate criteria (e.g., DNA fingerprinting and cytogenetic analysis), both for comparison to the parent cell line and as a standard for comparison of future stocks.
• It is advisable not to use antibiotics continuously in culture medium as this will inevitably lead to the appearance of antibiotic-resistant strains.
• Quality control all of the reagents used in tissue culture to ensure all materials used provide reproducible growth characteristics and are free from contamination prior to use with cells.
• Ensure laboratory coats are changed regularly.
• A disinfectant, e.g., 70% isopropyl alcohol [or alternatives such as 70% industrial methylated spirits (IMS) or ethanol] should be used liberally to disinfectant work surfaces, bottles, plastics, and so on.
• Cell culture is often combined with other methodologies, including chemical and drug methodologies. These may necessitate additional consideration of the chemical/toxicological hazards involved (e.g., use of cytotoxic laminar flow cabinets, cytoguards).

B. Subculturing Cells
In order to maintain cell cultures in optimum conditions it is essential to keep cells in the log phase of growth as far as it is practicable. There are two general types of culture method appropriate for adherent and suspension cells.

1. Subculturing Adherent Cells
Adherent cells (usually of epithelial, endothelial, and fibroblastic origin) will continue to grow in vitro until either they have covered the surface available for growth or they have depleted the nutrients in the surrounding medium. Cells kept for prolonged periods in the stationary phase of growth will lose plating efficiency and may become senescent, lose viability and other characteristics, or even die. The frequency of subculture is dependent on a number of factors, including inoculation density, growth rate, plating efficiency, and saturation density. These factors will vary between cell lines.

Protocol 1. Subculturing of Adherent Cells
The following procedure is described for the adherent cell line A549 (human lung adenocarcinoma, purchased from the ATCC) of epithelial origin, which can be subcultured indefinitely. These cells can be grown in HEPES-buffered medium and can therefore be routinely grown in nonvented tissue culture flasks in a normal 37°C incubator.

Media and Solutions
100× L-glutamine stock (200mM): Thaw stock solution and aliquot into sterile 5-ml amounts and store at -20°C.

100× penicillin/streptomycin solution: Aliquot stock solution into sterile 5-ml amounts and store at-20°C.

FCS: Thaw stock solution and aliquot into sterile 20- to 50-ml amounts and store at-20°C.

l×phosphate-buffered saline (PBS): Add I tablet to 500ml of ultrapure water, according to manufacturer's instructions, and autoclave. Store sterile solution at 4°C.

1% EDTA solution: Add I g of EDTA to 100 ml of ultrapure water and autoclave. Store sterile solution at 4°C.

0.25% trypsin/EDTA solution: Thaw the 2.5% (10×) stock trypsin solution. To 440ml of sterile PBS solution, add 10ml of sterile 1% EDTA and 50ml of 2.5% trypsin stock solution. Mix resultant solution by gentle inversion and aliquot into sterile 20ml amounts. Store aliquots at-20°C.

l× DMEM: Prepare l× DMEM from 10× powder according to the manufacturer's instructions, supplement the medium with NaHCO₃ and HEPES (adjust pH to 7.2-7.4), and filter sterilise using a 0.22-µm filter capable of handling large volumes or, alternatively, purchase premade l× medium from a relevant supplier. Store basal media at 4°C.

1× Ham's F12: Prepare l× Ham's F12 from 10× powder according to the manufacturer's instructions, supplement the medium with NaHCO₃ and HEPES (adjust pH to 7.2-7.4), and filter sterilise using a 0.22-µm filter capable of handling large volumes or, alternatively, purchase premade l× medium from a relevant supplier. Store basal media at 4°C.

Complete DMEM/Ham's F12 1:1 medium: To prepare 100ml of complete medium, mix 47ml of l× DMEM with 47ml of l× Ham's F12 and supplement with 5 ml of FCS and 1 ml of 100× L-glutamine. One milliliter of antibiotic solution (e.g., 100× penicillin/ streptomycin solution) may also be supplemented to the medium if absolutely necessary.

Procedure

1. Examine the condition of the cells using an inverted microscope with phase-contrast capabilities. Ensure that the cells are healthy and subconfluent (i.e., in the exponential phase of growth) and free of contamination.
2. Sanitise the laminar flow cabinet by wiping the surface of the working area with 70% IMS (or equivalent alternative). Wipe the surfaces of any materials prior to starting work, including gloves, media bottles, and pipettes.
3. Remove the spent growth medium from the flask using a pipette and wash the monolayer with a sufficient volume of prewarmed trypsin/EDTA solution (0.25% trypsin/EDTA solution in PBS) to ensure the removal of all media from the flask.
4. Add an appropriate volume of the 0.25% trypsin/EDTA solution to the flask (2ml for a 25-cm² flask, 4 ml for a 75-cm² flask, 7-8 ml for a 175-cm² flask) and incubate at 37°C to allow the cells to detach from the inside surface of the flask (the length of time depends on the cell line, but usually this will occur within 2-10 min). Examine the cells with an inverted microscope to ensure all the cells are detached and in suspension. Gently tap the flask with the palm of the hand a couple of times to release any remaining detached cells.
5. Inactivate the trypsin by adding an equal volume of serum-containing (complete) media to the flask.
6. Remove the cell suspension from the flask and place in to a sterile container. Centrifuge typically at 1000 rpm for 5 min.
7. Pour off the supernatant from the container and resuspend the pellet in complete medium. Perform a viable cell count (see article by Hoffman) and reseed a flask with an aliquot of cells at the required density. The size of culture flask used depends on the number of cells required. An appropriate volume of complete medium is added to the flask (5-7 ml for a 25-cm² flask, 10-15 ml for a 75-cm² flask, or 20-30ml for a 175-cm² flask). A cell count may not always be necessary if the cell line has a known split ratio (refer to ATCC or ECACC data sheets for the required seeding density). Label each flask with cell line name, passage number, and date.

Notes

• It is recommended to prepare fresh complete medium 1 week in advance of use, which gives time for a 5-day sterility check.
• L-Glutamine is more labile in cell culture solution than any other amino acid and the rate of degradation is dependent on storage temperatures, age of product, and pH. It is usually added in excess to culture medium, as it can be a limiting factor during cell growth. However, the degradation of L-glutamine causes a buildup of ammonia, which can have a detrimental effect on some culture suspensions. It is important to be very cautious when exceeding the formulated level of r-glutamine originally in the medium. Its degradation occurs at a faster rate at 37°C compared to 4°C; therefore it is recommended to keep medium containing L-glutamine at 4°C.
• To maintain serum-free conditions for specific cultures, trypsin may be neutralised with soybean trypsin inhibitor where an equal volume of inhibitor at a concentration of 1 mg/ml is added to the trypsinised cells. Cells can then be processed as described in steps 6 and 7 described earlier. For more details, refer to the article by Gayme and Gruber.
• An alternative to proteases is cell-scraping methods.
• Although most cells will detach in the presence of trypsin alone, EDTA is added to enhance the activity of the enzyme.
• Cells should only be exposed to trypsin long enough to detach the cells. Prolonged exposure can damage surface receptors on cells.

2. Subculturing Suspension Cells
Once established in culture, many tumour cell lines do not produce attachment factors and remain in suspension either as single cells or as clumps of cells. Examples of this cell type are lymphoblasts and cells of haematopoietic origin. Lymphoblastoids and many other tumour cell lines do not require a surface on which to grow and therefore do not require proteases such as trypsin for subculturing. Nonadherent cells may be subcultured by a number of methods, including subculture by direct dilution and subculture by sedimentation followed by dilution. The disadvantage of subculturing suspension cells by dilution is that during growth, cells produce metabolic by-products, which become toxic if allowed to accumulate in the medium. Initially, sufficient waste metabolites will be removed by diluting cell cultures, allowing growth to continue but the viability of the cells will gradually decline after a few subcultures. Surviving cells may also undergo selection pressures, resulting in altered characteristics. Subculture by sedimentation overcomes this problem of toxic metabolites but one must be careful not to overdilute the cells, which will increase the lag phase of the cells and may prevent them from dividing and also removes any growth factors produced by the cells.

Protocol 2. Subculture of Suspension Cells by Sedimentation
The following procedure is described for the human haematopoietic cell line HL60 (purchased from the ATCC), which can be subcultured indefinitely in suspension culture. HL60 cells cannot be grown in medium containing HEPES as a buffering agent. These cells need to be grown in vented tissue culture flasks in an atmosphere of 5% CO₂. HEPES is known to be toxic to some cell lines, so this needs to be checked before culturing the cell line of interest.

Media and Solutions
l× RPMI: Prepare 1x RPMI from 10× powder according to the manufacturer's instructions and supplement the medium with NaHCO₃ (adjust pH to 7.2-7.4) and filter sterilise using a 0.22µm filter capable of handling large volumes or, alternatively, purchase premade l× medium from a relevant supplier. Store basal media at 4°C.

Complete RPMI medium: To prepare 100ml of complete medium, aliquot 89ml of l× RPMI and supplement with 10 ml of FCS and 1 ml of 100× L-glutamine. One milliliter of antibiotic solution (e.g., 100× penicillin/ streptomycin) may also be supplemented to the medium if absolutely necessary.

Procedure

1. Examine the cultures microscopically for signs of cell deterioration and high-density growth. Cells in the exponential phase of growth will appear bright, round, and refractile, whereas dying cultures show cell lysis and shrunken cells. Hybridomas may be sticky and adhere loosely to the surface of the flask. These may require a gentle tap to the flask to detach the cells into suspension. Examination of the colour of the media may also indicate the growth stage of the cells (pH indicator in media).
2. Remove a volume of the cells for a viable cell count (refer to the article by Hoffman for details). The viability of suspension cell cultures should not be allowed to fall below 90%.
3. Transfer the cells to a sterile container and centrifuge the cells at 1000rpm for 5 min to form a pellet.
4. Discard the supernatant and resuspend the pellet with fresh media.
5. Set up the required number of vented tissue culture flasks and add an appropriate volume of prewarmed growth medium to the flasks. Add the appropriate number of cells to the flasks and incubate the flasks at 37°C in a CO₂ incubator. Refer to data sheets for recommended seeding densities of the cell line of interest (a typical growing density range for many suspension cells is 10⁵-10⁶ cells/ml).

C. Cryopreservation of Cells
Cell lines can change properties over time such as growth rate, antigen expression, and isoenzyme profile. It is therefore essential to set up a large frozen stock of each cell line, i.e., a working cell bank, and to work only within a defined number of cell doublings (or passages at a defined dilution/split ratio). A 10- passage period is satisfactory for many cell lines to maintain characteristic profiles.

1. Good Practice and Safety Considerations
Liquid nitrogen is the main refrigerant used for the long-term storage of biological material on a laboratory scale. The nitrogen boils above-196°C (-320°F) and thus the liquid is intensely cold and can pose several forms of safety hazard (Angerman, 1999).

• Cold of this magnitude will almost instantly burn skin and tissue. Such burns will also be produced by contact with material that has been exposed to liquid nitrogen. In some cases, items may actually adhere to skin and tissue that they contact, exacerbating burn damage.
• Liquid nitrogen can only be transported and stored in appropriately insulated containers. Most materials become extremely brittle in contact with such temperatures. For small quantities, special polystyrene containers can be used for transport. For larger quantities, purpose-built vacuum-insulated dewars are vital.
• The constant boiling of liquid nitrogen, particularly the instantaneous boiling that can occur if objects at room temperature are encountered, necessitates that all exposed areas of skin are covered. Hands, in particular, will need specific thermal-insulating gloves designed for low temperatures and these must never be used if damp or wet. The face should also be protected by a full face visor. This becomes particularly important when cryovials are being removed from liquid phase storage. The liquid gas can enter such vials during storage. The gas phase of nitrogen occupies approximately 700-fold the space of the liquid. Hence boiling of the liquid in a closed vial generates unbearable pressure that can result in explosive projections.
• The huge expansion of liquid nitrogen also causes boiling of the liquid to displace air if ventilation is insufficient. As nitrogen is an asphyxiant gas, careful consideration must be given to storage and transport. Ventilation must be adequate at all times to prevent buildup of the boiled gas produced under normal storage and the amount of gas that could be immediately produced in the event of a vessel rupture. Storage of large liquid nitrogen volumes needs specialist attention, including automatic oxygen level monitoring in the area. There have unfortunately been notable cases of death by asphyxiation where storage and monitoring have not been appropriate.

DMSO, which is often used in cryopreservation, must be handled with care. The chemical is flammable, although high temperatures are required to generate a risk of explosion. DMSO readily permeates some types of gloves and the skin. In itself DMSO is not hugely toxic to the body; however, DMSO may carry other chemicals and allergens through the skin barrier (Freshney, 2000).

2. Cryopreservation of Adherent and Suspension Cells
It is impractical to maintain cell lines in culture indefinitely as cell cultures will undergo genetic drift with continuous passage and risk losing their differentiated characteristics. It therefore becomes necessary to store cell stocks for future use and as a backup in case of contamination or alterations in the characteristics of the cells. There should be a limit placed on the number of passages any cell line can undergo before being discarded and replaced with new cells from the cryopreserved stock (e.g., a limit of 10 passages). Nearly all cell lines can be cryopreserved indefinitely in liquid nitrogen at-196°C. The cryopreservation process is based on slow freeze and fast thaw, together with a high protein concentration (foetal calf serum) and the presence of a cryopreservative agent that increases membrane permeability (e.g., DMSO or glycerol).

Cryopreservatives may be classed as being penetrative or nonpenetrative. Penetrative cryopreservatives, such as DMSO and glycerol, protect the cells against freezing damage caused by intracellular ice crystals and osmotic effects. Nonpenetrative cryopreservatives, such as serum, protect the cells from damage by extracellular ice crystals. Cells should only be cryopreserved when in the exponential phase of growth to increase the chances of good recovery. It is important to check each batch of cryopreserved vials for viability, sterility, and maintenance of specific cell characteristics.

Protocol 3. Cryopreservation of Adherent and Suspension Cells
Media and Solutions
2× freezing medium (20% DMSO): To prepare 20ml of freezing medium, add 4ml of DMSO to 16ml of FCS. Filter sterilise the solution using a 0.22-µm low-protein-binding filter. Aliquot the final solution into 5-ml amounts and store at-20°C.

Procedure

1. Check cultures using an inverted microscope to assess the degree of cellular growth and to ensure that the cells are free of contamination. Adherent or suspension cells are harvested for cryopreservation in the exponential phase of growth (refer to Protocols 1 and 2) and are counted as described in the article by Hoffman. Ideally, the cell viability should be greater than 90% in order to achieve a good recovery after freezing.
2. Thaw the 2× freezing medium and leave at 4°C until required.
3. Resuspend the cells in a suitable volume of serum. Slowly, in a dropwise manner, add an equal volume of the cold 20% DMSO/serum solution (or alternative freezing medium) to the cell suspension while at the same time gently swirling the cell suspension to allow the cells to adapt to the presence of DMSO. Note that DMSO is toxic to cells if added too quickly. The final concentration of DMSO should be 10%.
4. Place a total volume of 1 ml of this suspension, which ideally should contain between 5 × 10⁶ and 1 × 10⁷ cells, into each cryovial. Ensure that each cryovial is clearly marked with the cell line name, passage number, and date of freezing.
5. Place these vials in the vapour phase of a liquid nitrogen container, which is equivalent to a temperature of -80°C for a minimum of 3 h (or overnight).
6. Remove the vials from the vapour phase of the liquid nitrogen container and transfer them to the liquid phase for storage (-196°C).

Notes

• DMSO is a good universal cryoprotectant and most cell lines can be frozen down in a final concentration of 5-10% DMSO. However, it may not be suitable for every cell line
  1. Alternative freeze medium (e.g., glycerol): check the suitability on individual cell lines.
  2. It may have differentiation effects on certain cell lines (Freshney, 2000).
  
  
  
• Programmable freezers: the European Collection of Animal Cell Cultures (ECACC) recommends a cooling rate of-3°C per minute for most cell types.
• With serum-free conditions, nonpenetrative cryoprotectants such as methylcellulose and polyvinylpyrollidone (Merten et al., 1995; Ohno et al., 1988) can be used as alternatives to serum to maintain serum-free conditions (Keenan et al., 1998).

Protocol 4. Thawing of Cryopreserved Cells

1. Aliquot a volume of 9 ml of medium to a sterile container. Remove the cryopreserved cells from the liquid nitrogen and thaw at 37°C. It is important to thaw the cryopreserved cells rapidly to ensure minimal damage to the cells by the thawing process and the cryopreservation agent DMSO, which is toxic above 4°C.
2. Allow the contents to thaw until a small amount of ice remains in the cryovial.
3. Wipe the outside of the cryovial with a tissue moistened with 70% IMS (or alternative) and transfer the cell suspension to the previously aliquoted media.
4. Centrifuge the resulting cell suspension at 1000 rpm for 5 min to remove the cryoprotectant. Remove the supernatant and resuspend the pellet in fresh culture medium.
5. Assess the viability of the cells as described in the article by Hoffman to ensure that the viability is above 90%.
6. Add cells to an appropriately sized tissue culture flask (usually a 25- or 75-cm² flask) with a suitable volume of growth medium. Allow adherent cells to attach overnight and refeed the next day to remove any dead or floating cells. It is advisable to start cultures at between 30 and 50% of their final maximum cell density as this allows the cells rapidly to condition the medium and enter the exponential phase of growth.

IV. COMMENTS

• The procedures described in this article are applicable to many different continuous cell lines. However, some specialised cell types require special conditions for growth and differentiation (e.g., defined media, growth factors, coated flasks/plates) and are described elsewhere within this volume.
• A number of other basic cell culture procedures are also applicable to this article but are described elsewhere. These include cell counting (Hoffman) proliferation assays (Riss and Moravec), cell line authentication (Hay), and testing of cell cultures for microbial and viral contamination, including Mycoplasma (Hay and Ikonomi).

References
Angerman, D. (ed.) (1999). "Handbook of Compressed Gases: Compressed Gas Association," 4th Ed. Kluwer Academic, New York.

Doyle, A., Griffiths, J. B., and Newell, D. G. (eds.) (1998). "Cell and Tissue Culture: Laboratory Procedures." Wiley, New York.

Freshney, R. I. (ed.) (1992). "Animal Cell Culture, a Practical Approach," 2nd Ed. IRL Press, Oxford.

Freshney, R. I. (ed.) (2000). "Culture of Animal Cells: A Manual of Basic Techniques," 4th Ed. Wiley-Liss, New York.

Gey, G., Coffman, W. D., and Kubiech, M. T. (1952). Cancer Res. 12, 264.

Keenan, J., Meleady, E, and Clynes, M. (1998). Serum-free media. In "Animal Cell Culture Techniques" (M. Clynes, ed.), pp. 54-66. Springer-Verlag, New York.

Merten, O. W., Peters, S., and Couve, E. (1995). A simple serum free freezing medium for serum free cultured cells. Biologicals 23, 186-189.

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