1. 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 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. Aspirated into a 10cm petri dish containing 10ml of culture medium and gently shaken back and forth to make the cells in the petri dish evenly distributed.
●4. Label the cell type and date, the name of the cultivator, etc., and place it in a CO2 incubator for cultivation. After the cells adhere to the wall, change the medium.
●5. Change the medium every 3 days.
2. Passaging
●1. Passage when the cell coverage in the culture dish reaches 80%-90%.
●2. Aspirate the original medium.
●3. Add appropriate trypsin (just enough to 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. Use a pipette to suspend the cells by pipetting.
●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 medium, and blow up the cells.
●8. Transfer the cells to several dishes depending on the cell type. Generally, there are 5 cancer cells and 3 normal cells. Continue to cultivate.
3. Cryopreservation Digest the cells and centrifuge (ibid.). Suspend the cells with the prepared freezing solution, and distribute them into sterilized cryovials, let them stand for a few minutes, and indicate the cell type and cryopreservation date. 4°C for 30min, -20°C for 30min, -80°C overnight, and then placed in liquid nitrogen for storage. Preparation of cryopreservation solution: 70% complete medium + 20% FBS + 10% DMSO. DMSO should be slowly added dropwise, shaking while dripping.
