General Procedures for Cell Culture
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
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
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
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.
II. MATERIALS AND
- 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
- 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
- No eating, drinking, or smoking should be
- 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
- 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
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)
(Cat. No. 3055) and 75-cm2
3375) nonvented tissue culture flasks, and 25-cm2
No. 3056) and 75-cm2
(Cat. No. 3376) vented tissue
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.
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
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
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
- Low-speed biological centrifuge
- CO2 incubator
- Inverted microscope with phase-contrast
- 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
B. Subculturing Cells
- 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
- 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
- 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
- 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,
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
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
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
): Thaw stock solution
and aliquot into sterile 5-ml amounts and store at
100× penicillin/streptomycin solution
: Aliquot stock solution
into sterile 5-ml amounts and store at-20°C.
: 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
1% EDTA solution
: Add I g of EDTA to 100 ml of ultrapure
water and autoclave. Store sterile solution at
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.
: Prepare l× DMEM from 10× powder
according to the manufacturer's instructions, supplement
the medium with NaHCO3
(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
(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.
- 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
- 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,
- 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.
- 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
- Inactivate the trypsin by adding an equal
volume of serum-containing (complete) media to the
- Remove the cell suspension from the flask and
place in to a sterile container. Centrifuge typically at
1000 rpm for 5 min.
- 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,
2. Subculturing Suspension Cells
- 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
- 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.
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
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
: 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.
C. Cryopreservation of Cells
- 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).
- 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%.
- Transfer the cells to a sterile container and centrifuge
the cells at 1000rpm for 5 min to form a pellet.
- Discard the supernatant and resuspend the pellet
with fresh media.
- 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).
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
- 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
- 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
- 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
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
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
Protocol 3. Cryopreservation of Adherent and
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.
- 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
- Thaw the 2× freezing medium and leave at 4°C
- 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
- 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.
- 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).
- Remove the vials from the vapour phase of the
liquid nitrogen container and transfer them to the
liquid phase for storage (-196°C).
Protocol 4. Thawing of Cryopreserved Cells
- 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
- Alternative freeze medium (e.g., glycerol): check
the suitability on individual cell lines.
- 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).
- 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.
- Allow the contents to thaw until a small amount
of ice remains in the cryovial.
- 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.
- Centrifuge the resulting cell suspension at 1000
rpm for 5 min to remove the cryoprotectant. Remove
the supernatant and resuspend the pellet in fresh
- Assess the viability of the cells as described in the
article by Hoffman to ensure that the viability is above
- 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
- 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).
Angerman, D. (ed.) (1999). "Handbook of Compressed Gases:
Compressed Gas Association,"
4th Ed. Kluwer Academic, New
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
4th Ed. Wiley-Liss, New York.
Gey, G., Coffman, W. D., and Kubiech, M. T. (1952). Cancer Res
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
Ohno, T., Kurita, K., Abe, S., Eimori, N., and Ikawa, Y. (1988). A
simple freezing medium for serum-flee cultured cells. Cytotechnology
Richmond, J. Y., and McKinney, R. W. (eds.) (1999). "Biosafety in
Microbiological and Biomedical Laboratories." U.S. Department
of Health and Human Services, Public Health Service Centers for
Disease Control and Prevention and National Institutes of
Health. 4th Ed. U.S. Government Printing Office, Washington.
1999 Web edition:
http: / / www. cdc. gov / od / ohs/biosfty / bmbl4 / bmbl4toc, htm.
Shaw, A. J. (ed.) (1996). "Epithelial Cell Culture: A Practical Approach."
IRL Press, Oxford.