A primary culture is grown to confluency in a 60-mm petri plate or 25-cm2
tissue culture flask containing 5 mL tissue culture medium. Cells are
trypsin treatment and then reseeded into secondary cultures. The process
removing cells from the primary culture and transferring them to
constitutes a passage, or subculture.
- Primary cultures of cells
- HBSS without Ca2+ and Mg2+ at 37°C
- Trypsin/EDTA solution
- Complete medium with serum with 10% to 15% (v/v) FBS
- Sterile Pasteur pipettes
- 37°C warming tray or incubator
- Tissue culture plasticware or glassware, including pipettes and 25-cm2 flasks or 60-mm petri plates, sterile
- Remove all medium from primary culture with a sterile Pasteur pipette.
the adhering cell monolayer once or twice with a small volume of
HBSS without Ca2+ and Mg2+ to remove any residual FBS that may
inhibit the action of trypsin.
Use a buffered salt solution that is Ca2+ and Mg2+ free to wash cells. Ca2+ and Mg2+ in the salt solution can cause cells to stick together.
this is the first medium change, rather than discarding medium that is
removed from primary culture, put it into a fresh dish or flask. The
contains unattached cells that may attach and grow, thereby providing a
- Add enough 37°C trypsin/EDTA solution to culture to cover adhering cell
- Place plate on a 37°C warming tray 1 to 2 min. Tap the bottom of the
the countertop to dislodge cells. Check culture with an inverted
sure that cells are rounded up and detached from the surface.
cells are not sufficiently detached, return plate to warming tray for an
additional minute or 2.
2 mL 37°C complete medium. Draw cell suspension into a Pasteur
pipette and rinse cell layer 2 or 3-times to dissociate cells and
remaining adherent cells. As soon as cells are detached, add serum or
medium containing serum to inhibit further trypsin activity that might
cultures are to be split 1/
rather than 1/
add sufficient medium
that 1 mL of cell suspension can be transferred into each fresh
- Add an equal volume of cell suspension to fresh plates or flasks that
Alternatively, cells can be counted using a hemocytometer or Coulter
diluted to the desired density so a specific number of cells can be
to each culture vessel. A final concentration of ~5 × 104 cells/mL
appropriate for most subcultures.
primary cultures and early subcultures, 60-mm petri plates or 25-cm2 flasks are generally used; larger vessels (e.g., 150-mm plates or 75-cm2 flasks) may be used for later subcultures.
Cultures should be labeled with date of subculture and passage number.
- Add 4 mL fresh medium to each new culture. Incubate in a humidified
37°C, 5% CO2 incubator.
If using 75 cm2 culture flasks, add 9 mL medium per flask.
Some labs now use incubators with 5% CO2 and 4% O2.
The low oxygen
concentration is thought to stimulate the in vivo environment of cells
enhance cell growth.
For some media, it is necessary to adjust the CO2 to a higher or lower level
to maintain the pH at 7.4.
- If necessary, feed subconfluent cultures after 3 or 4 days by removing old
medium and adding fresh 37°C medium.
- Passage secondary culture when it becomes confluent by repeating steps
1 to 7, and continue to passage as necessary.