The first recovery:

1. Take the cryovial out of the liquid nitrogen, immediately put it into a 37°C water bath, and shake it slightly. After the liquid has melted (about 1-1.5 minutes), take it out and spray some alcohol and put it on the ultra-clean workbench.
2. Aspirate the above cell suspension into a 15ml centrifuge tube filled with 10ml of medium (wash the cryopreservation tube with medium to wash off all the cells adhering to the wall), and centrifuge at 1000 rpm for 5 minutes.
3. Pour off the supernatant and add 1ml of medium to suspend the cells. Aspirate into a 10cm Square media bottle containing 10ml of medium and gently shake it back and forth to distribute cells evenly in the Square media bottle.
4. Label the cell type and date, the name of the cultivator, etc., and put it in a CO2 incubator for cultivation. After the cells adhere to the wall, replace the Square media bottle.
5. Change the medium every 3 days.

The second passaging step:

1. Passage when the cells in the Square media bottle reach 80%-90% coverage.
2. Aspirate the original medium.
3. Add appropriate trypsin (as long as it can cover the cells) and digest for 1-2 minutes.
4. After the cells are rounded, add an equal volume of serum-containing medium to terminate the digestion.
5. Suspend the cells by pipetting them.
6. Aspirate the cells into a 15ml centrifuge tube and centrifuge at 1000 rpm for 5 minutes.
7. Pour off the supernatant, add 1-2ml of medium, and blow up the cells.
8. Pass the cells into several Square media bottles according to the cell type. Generally, there are 5 cancer cells and 3 normal cells. Continue to cultivate.