Cell Recovery
Recovery Medium: NK-92 cell-specific medium supplemented with 100 U/mL IL-2
To insure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If upon arrival, continued storage of the frozen culture is necessary, it should be stored in liquid nitrogen vapor phase and not at -70°C. Storage at -70°C will result in loss of viability.
a) Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water. Thawing should be rapid (approximately 2 - 3 minutes).
b) Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.
c) Transfer the vial contents to a centrifuge tube containing 5.0 mL complete culture medium and spin at approximately 176 x g for 5 minutes. Discard supernatant.
d) Resuspend cell pellet with the recommended recovery medium. And dispense into appropriate culture dishes.
e) Incubate the culture at 37°C in a suitable incubator. A 5% CO₂ in air atmosphere is recommended if using the medium described on this product sheet.
Cell Freezing
Freezing Medium: 90% FBS+10% DMSO
a) Centrifuge at 176 x g for 3 minutes to collect cells.
b) Resuspend the cells in pre-cooled freezing medium and adjust the cell density to 5E6 cells/mL.
c) Aliquot 1 mL into each vial.
d) Place the vial in a controlled-rate freezing container and store at -80°C for at least 1 day, then transfer to liquid nitrogen as soon as possible.
Cell passage
Growth medium: NK-92 cell-specific medium supplemented with 100 U/mL IL-2
For the first 1 to 2 passages post-resuscitation, use the recovery medium. Once the cells have stabilized, switch to a growth medium.
a) Cell recovery after thawing is challenging, and initial cell growth is slow (approximately one week), with a subset of cells undergoing lysis, leading to a large number of dead cells and cellular debris. The culture condition usually improves after two weeks. It is recommended to centrifuge the culture at 176 × g for 5 minutes every 1–2 weeks to remove part of the cellular debris and particles by discarding the supernatant and replacing it with fresh recovery medium. Upon initial recovery, if the cells are not passaged within 48 hours, it is advisable to supplement the culture with additional recovery medium.
b) When the cell density reaches 1.5–2E6 cells/mL, passage the cells at a 1:3 ratio, and continue subculturing every 2–3 days. Care should be taken not to allow the density to exceed 2E6 cells/mL. Passage cultures are recommended to be maintained in T25 flasks.
c) Except for the first passage post-thaw (which requires centrifugation to collect cells), subsequent media changes or subculturing are preferably carried out using a half-medium change approach: allow the cells to settle by gravity, aspirate half of the spent medium, then add an equal volume of fresh medium to the flask. Gently pipette to resuspend the cells evenly or split them into two new T25 flasks at a 1:2 ratio, and then add fresh complete medium to a final volume of 8–10 mL per flask.
Subcultivation Ratio: Maintain cultures at a cell concentraion between 3E5 and 1E6 viable cells/mL.
Medium Renewal: Every 2 to 3 days
Notes
a) This cell line is highly sensitive to cell density. Please ensure that cell density is maintained within an appropriate range during cultivation and passaging.
b) Cell growth is dependent on IL-2; in the absence of IL-2 in the culture medium, the cells are prone to rapid cell death. IL-2 is stable at -5 to -20°C and should be protected from repeated freeze-thaw cycles. Since IL-2 is prone to degradation, freshly prepared complete medium should be used within one week.
c) Cells grow in clusters (cell aggregation). The presence of numerous cell debris and granules during the logarithmic growth phase is normal. When cell density becomes high or the culture medium turns yellow, partial or complete medium replacement should be performed promptly. During medium change, disperse the cell clusters as much as possible.
Cell Recovery
Recovery Medium: NK-92 cell-specific medium supplemented with 100 U/mL IL-2
To insure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If upon arrival, continued storage of the frozen culture is necessary, it should be stored in liquid nitrogen vapor phase and not at -70°C. Storage at -70°C will result in loss of viability.
a) Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water. Thawing should be rapid (approximately 2 - 3 minutes).
b) Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.
c) Transfer the vial contents to a centrifuge tube containing 5.0 mL complete culture medium and spin at approximately 176 x g for 5 minutes. Discard supernatant.
d) Resuspend cell pellet with the recommended recovery medium. And dispense into appropriate culture dishes.
e) Incubate the culture at 37°C in a suitable incubator. A 5% CO₂ in air atmosphere is recommended if using the medium described on this product sheet.
Cell Freezing
Freezing Medium: 90% FBS+10% DMSO
a) Centrifuge at 176 x g for 3 minutes to collect cells.
b) Resuspend the cells in pre-cooled freezing medium and adjust the cell density to 5E6 cells/mL.
c) Aliquot 1 mL into each vial.
d) Place the vial in a controlled-rate freezing container and store at -80°C for at least 1 day, then transfer to liquid nitrogen as soon as possible.
Cell passage
Growth medium: NK-92 cell-specific medium supplemented with 100 U/mL IL-2
For the first 1 to 2 passages post-resuscitation, use the recovery medium. Once the cells have stabilized, switch to a growth medium.
a) Cell recovery after thawing is challenging, and initial cell growth is slow (approximately one week), with a subset of cells undergoing lysis, leading to a large number of dead cells and cellular debris. The culture condition usually improves after two weeks. It is recommended to centrifuge the culture at 176 × g for 5 minutes every 1–2 weeks to remove part of the cellular debris and particles by discarding the supernatant and replacing it with fresh recovery medium. Upon initial recovery, if the cells are not passaged within 48 hours, it is advisable to supplement the culture with additional recovery medium.
b) When the cell density reaches 1.5–2E6 cells/mL, passage the cells at a 1:3 ratio, and continue subculturing every 2–3 days. Care should be taken not to allow the density to exceed 2E6 cells/mL. Passage cultures are recommended to be maintained in T25 flasks.
c) Except for the first passage post-thaw (which requires centrifugation to collect cells), subsequent media changes or subculturing are preferably carried out using a half-medium change approach: allow the cells to settle by gravity, aspirate half of the spent medium, then add an equal volume of fresh medium to the flask. Gently pipette to resuspend the cells evenly or split them into two new T25 flasks at a 1:2 ratio, and then add fresh complete medium to a final volume of 8–10 mL per flask.
Subcultivation Ratio: Maintain cultures at a cell concentraion between 3E5 and 1E6 viable cells/mL.
Medium Renewal: Every 2 to 3 days
Notes
a) This cell line is highly sensitive to cell density. Please ensure that cell density is maintained within an appropriate range during cultivation and passaging.
b) Cell growth is dependent on IL-2; in the absence of IL-2 in the culture medium, the cells are prone to rapid cell death. IL-2 is stable at -5 to -20°C and should be protected from repeated freeze-thaw cycles. Since IL-2 is prone to degradation, freshly prepared complete medium should be used within one week.
c) Cells grow in clusters (cell aggregation). The presence of numerous cell debris and granules during the logarithmic growth phase is normal. When cell density becomes high or the culture medium turns yellow, partial or complete medium replacement should be performed promptly. During medium change, disperse the cell clusters as much as possible.
