Part IV: Essential protocols for animal cell culture
Welcome to the next of our series of articles aimed at providing useful
hints for culturing animal cells. This article contains useful protocols
for animal cell culture. The series will continue in future issues of QIAGEN
News, followed by hints for successful transfection.
Maintaining cell cultures
Establishment and maintenance of animal cell cultures require standardized
approaches for media preparation, feeding, and passaging (or subculturing)
of the cells. Cultures should be examined regularly to check for signs of
contamination and to determine if the culture needs feeding or passaging.
The cell culture protocols below have been adapted from the following sources:
Culture of Animal Cells; a Manual of Basic Technique (1), Current Protocols
in Molecular Biology (2), and Cells: A Laboratory Manual (3). These protocols
are examples of methods for general cell culture, and have not been rigorously
validated and optimized by QIAGEN. There are many alternative protocols
in current use.
Potentially biohazardous materials (e.g., cells, culture
medium, etc.) should be sterilized before disposal, and disposed of according
to your institutions guidelines.
Heat a water bath to 37C, and warm the growth medium into
which the cells will be plated.
. Add prewarmed growth medium to an appropriately sized cell culture
Remove a vial of frozen cells from liquid nitrogen, and place
in the water bath until thawed.
Wear protective goggles and gloves when thawing vials
that have been stored in liquid nitrogen. Vials may explode when removed
from liquid nitrogen.
Proceed to step 4 as soon as the cells have thawed. Do
not allow the cells to warm up before transferring them into growth medium.
Wash the outside of the vial with 70% ethanol or another suitable
Slowly pipet the thawed cell suspension into the cell culture
vessel containing prewarmed growth medium. Swirl the vessel gently to mix
the cells with the medium.
Tip - Immediate removal of DMSO may sometimes be necessary, especially for
suspension cells, primary cells, and sensitive cell types. For such cell
types, pipet the thawed cell suspension into a sterile centrifuge tube containing
prewarmed medium, centrifuge at 200 x g for 2 min, aspirate the supernatant,
resuspend the cells in fresh growth medium, and then transfer to an appropriate
cell culture vessel.
Thoroughly mix the cells in the cell culture vessel to
ensure even distribution of the cells throughout the vessel.
Incubate cells overnight under their usual growth conditions.
The next day, replace the growth medium
Trypsinization is a technique that uses the proteolytic enzyme trypsin to
detach adherent cells from the surface of a cell culture vessel. This procedure
is performed whenever the cells need to be harvested (e.g., for passaging,
counting, or for nucleic acid isolation).
Aspirate the medium and discard.
. Wash cells with PBS or HBSS (see Table 1), aspirate, and discard.
Tip - The volume of PBS or HBSS should be approximately the same as the
volume of medium used for culturing the cells.
. Add enough warmed 1x trypsinEDTA solution (see Table 1)
to cover the monolayer, and rock the flask/dish 45 times to coat the
. Place the flask/dish in a CO2 incubator at 37C for 12
. Remove flask/dish from incubator and firmly tap the side of the
flask/dish with palm of hand to assist detachment.
Tip - If cells have not dislodged, return the flask/dish to the incubator
for a few more minutes.
Do not leave cells in 1x trypsinEDTA solution for
extended periods of time. Do not force the cells to detach before they are
ready to do so, or clumping may occur.
Tip - Overly confluent cultures, senescent cells, and some cell lines may
be difficult to trypsinize. While increasing the time of trypsin exposure
may help to dislodge resistant cells, some cell types are very sensitive
to trypsin and extended exposure may result in cell death. In addition,
some cell lines will resist this treatment and will produce cell clumps.
. Once dislodged, resuspend the cells in growth medium containing
Tip - Use medium containing the same percentage of serum as used for growing
the cells. The serum inactivates trypsin activity.
Gently pipet the cells up and down in a syringe with a needle
attached to disrupt cell clumps.
Tip - If pipetted too vigorously, the cells will become damaged. Ensure
that pipetting does not create foam.
Proceed as required (e.g., with passaging, freezing, nucleic acid
Table 1. Composition of solutions for animal cell culture protocols
* Store 1x trypsin-EDTA solution at 20C. Small aliquots can
be stored at 28C for 12 weeks. Work quickly when using
trypsin during cell culture, since trypsin degrades and enzymatic activity
declines at 37C.
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 a confluent state for too
long. Adherent cell cultures therefore need to be routinely passaged,
that is, once the cells are confluent, a fraction of the cells need to
be transferred to a new cell culture vessel. Suspension cells will exhaust
their culture medium very quickly once the cell density becomes too high,
so these cultures similarly require regular passaging.
IMPORTANT: Although regular passaging is necessary to maintain
animal cell cultures, the procedure is relatively stressful for adherent
cells as they must be trypsinized. We do not recommend passaging adherent
cell cultures more than once every 48 h.
1. Harvest the cells, either by trypsinization (adherent cell cultures)
or by centrifugation at 200 x g for 5 min (suspension cell cultures).
Resuspend the cells in an appropriate volume of prewarmed growth medium
Tip- The volume of medium used to resuspend the cells depends on the spl
ratio required (see step 2) and the size of the cell culture vessel. If
too small a volume is used, it may be difficult to accurately pipet the
desired volume to the new culture vessel. Conversely, if too large a volume
is used, the culture vessel may be too full following transfer of the
Tip - Removal of trypsin may sometimes be necessary following harvesting
of adherent cells, especially for primary and sensitive cell types. Centrifuge
the cells at 200 x g for 5 min, carefully aspirate the supernatant, and
resuspend the cells in an appropriate volume of prewarmed medium containing
2. Transfer an appropriate volume of the resuspended cells to a
fresh cell culture vessel containing prewarmed growth medium. Swirl the
vessel gently to mix the cells with the medium.
IMPORTANT: Thoroughly mix the cells in the cell culture vessel
to ensure even distribution of cells.
IMPORTANT: Some cell types will not survive if too few cells are
transferred. We do not recommend high split ratios for primary cells,
sensitive cell types, or senescent cultures.
Tip - For adherent cells, we recommend adding enough cells so that the
culture takes approximately one week to reach confluence again. This minimizes
the number of times the cells are trypsinized as well as the handling
time required to maintain the culture.
Tip - When determining how many cells to transfer to the new cell culture
vessel, it can be helpful to think in terms of how many cell divisions
will be required for the culture to reach confluence again. For example,
if half the cells are transferred, t
hen it will take the culture one cell
division to reach confluency again; if a quarter of the cells are transferred
then it will take 2 cell divisions, and so on. If a culture divides once
every 30 h or so, then in one week it will undergo approximately 5 cell
divisions. A split ratio of 1:32 (1:25) should therefore be appropriate
for the cells to reach confluency in about one week. In step 1, resuspend
the cells in 8 ml medium, and transfer 0.25 ml to the new cell culture
3. Incubate cells under their usual growth conditions.
1. Freshney, R.I. (1993)
Culture of Animal Cells: a
Manual of Basic Technique.
3rd ed. New York:
2. Ausubel, F.M. et al., eds.
(1991) Current Protocols
in Molecular Biology.
New York: Wiley
3. Spector, D., Goldman,
R.R., and Leinwand, L.A.,
eds. (1998) Cells: a
Laboratory Manual. Cold
Spring Harbor, NY: Cold
Spring Harbor Laboratory
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