3. Centrifuge the cells at 500 Â g for 5 min at room temperature.
4. Discard the supernatant and resuspend the pellet with the
cryopreservation medium at a density of 2 Â 10
6 cells/mL.
5. Quickly dispense 1 mL aliquots of the suspension into
each cryogenic vial.
6. Transfer the cryogenic vials into the slow cooling cryocontainer and store at À80
C freezer overnight.
7. The next day, transfer the vials to a liquid nitrogen tank for
long-term storage.
3.2 Culture of NK-92
Cell Line
3.2.1 Thawing
1. Warm the NK-92 cell culture medium to room temperature.
2. Quickly thaw the cryopreserved NK-92 cells in a 37
C water
bath until only a small ice crystal remains.
3. Wipe the outside of the cryovial with 70% ethanol.
4. Gently pipette the cell suspension and transfer to a 15-mL
conical tube containing 9 mL of α-MEM basal medium.
5. Centrifuge the cells at 500 Â g for 5 min at room temperature.
6. Discard the supernatant and resuspend the pellet in 5 mL of
culture medium.
7. Transfer the cell suspension into a 25-cm
2 cell culture flask and
incubate at 37
C, 5% CO 2 .
3.2.2 Passaging
Passaging is performed when the cells reach 80% confluence, usually every 3–4 days (see Note 2).
1. Gently resuspend the cells using a serological pipette and transfer the cell suspension to a 15-mL conical tube.
2. Aliquot a small amount of the cell suspension into a 1.5-mL
Eppendorf tube for cell counting.
3. Centrifuge the cells at 500 Â g for 5 min at room temperature.
4. During centrifugation, perform cell counting using trypan blue
exclusion assay to determine the cell number and viability.
5. After centrifugation, discard the supernatant and resuspend the
cell pellet with 1 mL of culture medium.
6. Transfer the cells to a 25-cm
2 cell culture flask containing 5 mL
of culture medium at a seeding density of 2 Â 10
5 viable cells/
mL.
7. Incubate at 37
C, 5% CO 2 .
8. Change the medium and adjust the cell density every 3–4 days.
3.2.3 Cryopreservation
1. Prepare the NK-92 cell cryopreservation medium and chill at
4
C.
2. Gently resuspend the cells and transfer the suspension into a
15-mL conical tube.
220
Nontaphat Thongsin and Methichit Wattanapanitch
4. Discard the supernatant and resuspend the pellet with the
cryopreservation medium at a density of 2 Â 10
6 cells/mL.
5. Quickly dispense 1 mL aliquots of the suspension into
each cryogenic vial.
6. Transfer the cryogenic vials into the slow cooling cryocontainer and store at À80
C freezer overnight.
7. The next day, transfer the vials to a liquid nitrogen tank for
long-term storage.
3.2 Culture of NK-92
Cell Line
3.2.1 Thawing
1. Warm the NK-92 cell culture medium to room temperature.
2. Quickly thaw the cryopreserved NK-92 cells in a 37
C water
bath until only a small ice crystal remains.
3. Wipe the outside of the cryovial with 70% ethanol.
4. Gently pipette the cell suspension and transfer to a 15-mL
conical tube containing 9 mL of α-MEM basal medium.
5. Centrifuge the cells at 500 Â g for 5 min at room temperature.
6. Discard the supernatant and resuspend the pellet in 5 mL of
culture medium.
7. Transfer the cell suspension into a 25-cm
2 cell culture flask and
incubate at 37
C, 5% CO 2 .
3.2.2 Passaging
Passaging is performed when the cells reach 80% confluence, usually every 3–4 days (see Note 2).
1. Gently resuspend the cells using a serological pipette and transfer the cell suspension to a 15-mL conical tube.
2. Aliquot a small amount of the cell suspension into a 1.5-mL
Eppendorf tube for cell counting.
3. Centrifuge the cells at 500 Â g for 5 min at room temperature.
4. During centrifugation, perform cell counting using trypan blue
exclusion assay to determine the cell number and viability.
5. After centrifugation, discard the supernatant and resuspend the
cell pellet with 1 mL of culture medium.
6. Transfer the cells to a 25-cm
2 cell culture flask containing 5 mL
of culture medium at a seeding density of 2 Â 10
5 viable cells/
mL.
7. Incubate at 37
C, 5% CO 2 .
8. Change the medium and adjust the cell density every 3–4 days.
3.2.3 Cryopreservation
1. Prepare the NK-92 cell cryopreservation medium and chill at
4
C.
2. Gently resuspend the cells and transfer the suspension into a
15-mL conical tube.
220
Nontaphat Thongsin and Methichit Wattanapanitch