Cell Recovery
Recovery Medium: RPMI 1640+20% FBS(Gibco)+1% P.S
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.
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: RPMI 1640+10% FBS(Gibco)+1% P.S+0.5 μg/mL Puromycin
For the first 1 to 2 passages post-resuscitation, use the recovery medium. Once the cells have stabilized, switch to a growth medium.
a) After the first resuscitation, the cells can be subcultured after approximately 2–3 days. After 1 to 2 passages, the culture medium can be changed to a growth medium supplemented with antibiotics. If passage is not possible within 3 days, it is recommended to supplement with recovery medium as appropriate and place the flask in a horizontal position.
b) When the cell density reaches 8E5 cells/mL, perform a 1:3 split, and continue subculturing every 3–4 days. Do not allow the cell density to exceed 1E6 cells/mL. It is recommended to use T25 flasks for passaging and culture.
c) These cells are suspension cells, and it is recommended to use the "half-medium change" method to maintain optimal cell conditions during passaging.
d) During passaging, you can directly add fresh growth medium to the culture flask, gently pipette to resuspend the cells, and then transfer the cell suspension to a new T-25 flask for continued culture.
Subcultivation Ratio: Maintain cultures at a cell concentraion between 2.5E5 and 8E5 viable cells/mL.
Medium Renewal: Every 3 to 4 days
Notes
a) It is recommended to increase the serum concentration to 20% during cell resuscitation.
b) Fetal bovine serum (FBS) should be heat-inactivated at 56°C for 30 minutes, which can deactivate complement and some viruses without significantly affecting the activity of most growth factors and cytokines.
