Cryopreservation of Mammalian Cells

Cryopreservation is the technique of freezing cells at very low temperatures at

which the biological material remains genetically stable and metabolically inert,
while minimizing ice crystal formation. In general, when a tissue is subjected to
low temperatures, ice crystals will eventually form which will pierce the cell
membrane leading to the death of the cell. The goal of cryopreservation is to
replace some of the water with other compounds that will not form large crystals
when frozen. The most commonly used replacement is dimethyl sulfoxide
(DMSO). DMSO aids in cryopreservation by making the cell membrane more
permeable, dehydrating the cell, which will minimize ice crystal formation inside
the cell. DMSO also prevents the formation of ice lattice, which will prevent large
ice crystals. Cells are suspended in a media with DMSO and placed in a liquid
nitrogen freezer. As the medium begins to freeze, the salt concentration outside
the cells will become greater than that in the cells causing water to leave the
cells. The cryopreservative both protects the cells from mechanical and physical
stress and reduces the water content within the cells, thus minimizing the
formation of cell-lysing ice crystals.
Generally, the optimum cell density to freeze per 1ml of cell suspension depends
on the type of cell. Mammalian cells are usually frozen between 1 X 106 cells per
ml to 1 X 107 cells per ml.
The following cryopreservation media are generally used:
Adherent Cell Types
90% Fresh complete media
10% DMSO
Suspension Cell Types
45% Fresh complete media
45% Spent (used) media
10% DMSO

Cryopreservation of Adherent Cell Cultures

The following procedure will be performed utilizing aseptic technique, as
described in Aseptic Techniques Used for Mammalian Cell Culture
1. Expand culture to allow for adequate cell density for the desired volume to
freeze.
a. Optimum conditions include cells currently in the log phase of the
growth cycle (approximately 80% confluency)
2. Follow Dissociation of Adherent Cells.
3. Centrifuge cells at approximately 1200 rpm for 7 minutes, allowing cells to
form a pellet.
4. Determine the amount of freezing media to prepare by calculating the total
number of vials to be frozen.
a. Total cell concentration/desired freezing density = number of
possible 1mL aliquots at desired density
5. Label the appropriate number of vials with the following:
a. cell type
b. passage
c. dilution
d. your initials
e. date
f. any other information you deem necessary
6. Remove supernatant by aspiration from the centrifuged tube.
7. Resuspend cell pellet in 500L of the freezing media using a Pipetteman
to unclump the cells, then add remaining freezing media, pipetting up and
down 5 times to ensure a single-cell suspension
8. Dispense into the labeled Nalgene cryogenic vials.
a. 1ml/vial
9. Immediately transfer the vials to a -20C freezer for one hour.
10. Transfer the vials to a -80C freezer for 24 hours. Alternatively, a
Styrofoam insulated box with lid containing dry ice may be used.
11. After 24 hours at -80C, cells may be transferred to liquid nitrogen storage
by following Liquid Nitrogen SOP section Depositing Cell Vials within the
Liquid Nitrogen Dewar.

Cryopreservation of Suspension Cell Cultures

The following procedure will be performed utilizing aseptic technique, as
described in Aseptic Techniques Used for Mammalian Cell Culture
1. Expand culture to allow for adequate cell density for the desired volume to
freeze.
a. Optimum conditions include cells currently in the log phase of the
growth cycle (approximately 3-4 days after splitting)
2. Combine the cell suspensions into a single sterile vessel. From this single
suspension, count the cells to determine cell viability and total cell
concentration.
3. Centrifuge cells at approximately 1200 rpm for 7 minutes, allowing cells to
form a pellet.
4. Determine the amount of freezing media to prepare by calculating the total
number of vials to be frozen.
a. Total cell concentration/desired freezing density = number of
possible 1mL aliquots at desired density
5. Label the appropriate number of vials with the following:
a. cell type
b. passage
c. dilution
d. your initials
e. date
f. any other information you deem necessary
6. When centrifugation is complete, transfer the calculated amount of spent
media to a sterile container and set aside. Aspirate the remaining
supernatant.
7. Complete the freezing media by adding appropriate volumes of fresh
media and DMSO to the spent media. Cap and invert gently three times.
8. Resuspend cell pellet in 500l of the freezing media using a Pipetman to
un-clump the cells, then add the remaining freezing media, pipetting up
and down 5 times to ensure a single-cell suspension.
9. Dispense into the labeled Nalgene cryogenic vials.
a. 1ml/vial
10. Immediately transfer the vials to a -20C freezer for one hour
11. Transfer the vials to a -80C freezer for 24 hours. Alternatively, a
Styrofoam insulated box with lid containing dry ice may be used
12. After 24 hours at -80C, cells may be transferred to liquid nitrogen storage
by following Liquid Nitrogen SOP section Depositing Cell Vials within the
Liquid Nitrogen Dewar.