Cell cryopreservation considerations - Database & Sql Blog Articles

LC03-3.3 SOP8 TVS Static Protection 3.3V

When the cells are cooled below zero, the following changes can occur: dehydration of the organelles, an increase in the concentration of soluble substances in the cells, and formation of ice crystals within the cells.

If it is slowly frozen, the cells can be gradually dehydrated, and large ice crystals will not be produced in the cells; on the contrary, the crystals will be large, and large crystals will cause damage and rupture of the cell membrane and the organelle. The recovery process should be fast-melting, in order to prevent small ice crystals from forming large ice crystals, that is, recrystallization of ice crystals.

Slow freezing program

1. Standard procedure: using cell cryostat

When the temperature is above -25 °C, 1~2 °C/min;

When the temperature reaches below -25 °C, 5~10 °C/min;

When the temperature reaches -100 ° C, it can be quickly put into liquid nitrogen.

2. Simple procedure: Put the cryotube (the nozzle is facing up) into the gauze bag, and the gauze bag is tied with a rope. The gauze bag is fixed to the liquid nitrogen tank can by the wire rope, and the temperature drops by 1~2 per minute. The speed of °C dropped to the surface of liquid nitrogen overnight in 40 min, and it was injected into liquid nitrogen in the next morning.

3. Traditional procedure: The cryotube is placed at 4 °C for 10 minutes → -20 °C for 30 minutes → -80 °C for 16 to 18 hours (or overnight) → Long-term storage of liquid nitrogen tank.

Cell cryopreservation method

Pre-formulated cryopreservation solution

(1) 10% DMSO + cell growth solution (20% serum + basal medium)

2. Take the logarithmic growth phase cells, after trypsinization, add appropriate amount of frozen solution, and pipette to make a cell suspension (1×10 ⁶ ~ 5 × 10 ⁶ cells/ml).

3. Add 1 ml of cells to the cryotube and seal and label the frozen cell name and freezing date. Liquid nitrogen is stored for a long time.

Resuscitation method for preserving cells

Quick thaw

After the frozen cells were taken out from the liquid nitrogen, they were immediately placed in a 37 ° C water bath, and the freezing tube was gently shaken to completely melt in 1 minute (not more than 3 minutes).

2. The thawed cells can be directly inoculated directly into a cell culture flask containing the fully grown culture medium, and the old culture solution is replaced with fresh complete culture solution to remove DMSO 24 hours later.

3. If the cells are particularly sensitive to cryoprotectants, the thawed cells should be centrifuged to remove the cryoprotectant and then inoculated into a flask containing the fully grown medium.