1. Harvesting: Digest well-grown cells into single cells or cell clumps (the method depends on cell type). Quench digestion by neutralization or dilution. Collect cells into a centrifuge tube and take an aliquot for counting to calculate the total cell number.

2. Centrifugation: Remove the supernatant after centrifugation.

3. Resuspension: Add pre-chilled (2–8°C) PluriCryo cryopreservation medium according to the calculated cell count. Pipette gently to homogenize and aliquot the cell suspension into cryovials. The recommended cell density for cryopreservation is generally in the range of 1×106 to 1×107 cells/mL.

4. Place the cryovials directly into a -80°C freezer. Transfer to liquid nitrogen for long-term storage after 24 hours.

1. Pre-warm the water bath and medium required for cell thawing in advance.

2. Retrieve the cryovial from liquid nitrogen, place it on dry ice, and transport it to the cell culture room.

* It is recommended to leave the cryovial on dry ice for 10–20 minutes before thawing to prevent explosion caused by sudden heating of residual liquid nitrogen inside. If dry ice is unavailable, loosen and re-tighten the cryovial cap inside the biosafety cabinet prior to thawing to release internal pressure.

3. Hold the cryovial with forceps and immerse the lower half of the tube in a 37°C water bath. Gently swirl until the contents thaw with only a small ice crystal remaining. Spray the tube surface with 75% ethanol and transfer it into the biosafety cabinet.

4. Aspirate an appropriate volume of medium using a 1 mL pipette tip and add it dropwise into the cryovial. Transfer the cell suspension to a 15 mL centrifuge tube, add medium at 5–10 times the volume of the cryopreservation medium, mix well, centrifuge and discard the supernatant.

5. Resuspend the cell pellet with culture medium, mix and perform cell counting, then seed cells at the desired ratio.