Thawing Frozen Cells and Cell Freezing
We can obtain a large number of cells with the same characteristics through cell culture, so that they can be used for subsequent cell research. The critical start of cell culture is to freeze and resuscitate cells. So how to thaw cells and how to freeze down cells correctly, the following content will be explained in detail.
Thawing Frozen Cells
- First, preheat the water bath to 37°C, remember to wear clean disposable PE gloves and glasses, and prepare sterile centrifuge tubes.
- Then the frozen cells were taken out from the liquid nitrogen tank and immediately placed in a water bath preheated to 37°C. The cells can be taken out of the liquid nitrogen tank and put into PE gloves, quickly submerged in a water bath, and gently shaken the cryotube to accelerate thawing. The entire duration should be controlled at about 1 minute.
- Add 2 times the cell volume of the cell culture medium into the prepared sterile centrifuge tube, and slowly add the cell suspension into the centrifuge tube with a pipette in a sterile environment.
- Next, centrifuge according to the actual cell type and volume, and remove the supernatant after centrifugation.
- Remove the supernatant from centrifugation, add fresh medium, and slowly resuspend the cells using a pipette.
- Then transfer it to the most suitable sterile culture container, supplement the culture medium to an appropriate level, and put it into an incubator suitable for the culture environment for cultivation.
- After 24 hours, observe the state of the cells, replace with fresh cell culture medium, continue culturing, and observe the growth.
- If the cells grow well and the cell density is high, the cells can be subcultured in time.
DMSO in the cryopreservation solution at room temperature has great damage to the cells, so we need to thaw the cryopreservation solution within a minute or two. Shaking the cryovial can speed up the thawing, and the frozen cells that are revived in time have a high survival rate and good growth and shape.
The cell experiment cycle is long, so it is common to freeze a part of the cells for later use, and the next step is about the steps of freezing the cells.
- First of all, you need to prepare clean and sterile centrifuge tubes and freezer tubes, and prepare for experiments in a sterile environment.
- Cells were collected into centrifuge tubes according to conventional methods.
- After centrifugation, the supernatant can be discarded using a sterile pipette.
- Add an appropriate amount of serum-free cell freezing solution to the cell pellet, mix them well, and make a cell suspension at the recommended cell freezing density.
- Aliquot the cell mixture in the centrifuge tube and add it to a cryopreservation tube and label it.
- After sealing tightly, you can start to cool down and freeze. It can be slow-frozen and manually cooled according to the temperature gradient cooling method, or it can be placed in a programmed cooling box.
- The method of manual cooling is to place frozen cells in an environment of 4°C for 30 minutes, then cool down to -20°C for 1 hour, and finally drop to -80°C for 24 hours. Finally, it can be transferred to a liquid nitrogen tank for storage.
- The programmed cooling box is a special freezing container that can quickly cool the cells to -80°C, and can also be moved to liquid nitrogen for storage after overnight.
The above is about thawing frozen cells and how to freeze down cells, we hope it can be helpful to you. If you need test-related centrifuge tubes or cell culture vessels, Hawach product is your very wise choice.