How are cultured cells frozen?

Date:2022-06-09 10:33:09 Thursday

Summary:Adjust the centrifuge to 800 rpm for 5 minutes. The water bath was adjusted to a constant temperature of 37 degrees. Take the cell complete medium, DMSO, trypsin, etc., and put it in a water bath to preheat. Disinfect your hands and bench first. Tak......

Adjust the centrifuge to 800 rpm for 5 minutes. The water bath was adjusted to a constant temperature of 37 degrees. Take the cell complete medium, DMSO, trypsin, etc., and put it in a water bath to preheat.

Disinfect your hands and bench first. Take about 10ml of complete Cell spreader culture medium into a 15ml centrifuge tube.

Prepare the cell cryopreservation solution: The cryopreservation solution should be prepared in advance and kept at room temperature for future use to prevent the heat generated by the temporary preparation from damaging the cells. Among them, increasing the proportion of serum in the cryopreservation solution is very beneficial for preserving some fragile stem cells and some more precious cells.

After adding the components of the freezing solution according to the proportion, gently pipette and mix well, and leave it at room temperature for use.

Remove the cells from the cell incubator, sterilize the bottle, and discard the original medium of the cells. Add the digestion solution per 25cm2/ml trypsin/EDTA digestion solution and observe it under a microscope. After all the cells become round, they are immediately placed into the ultra-clean bench, and 2ml of complete cell culture medium is added to terminate the digestion immediately.

Use a sterile pipette tip to gently pipette the surface of the cells, pay attention to pipetting the entire culture surface, and put all the cell suspension into a clean 15 ml centrifuge tube by pipetting left and right, then up and down.

Balance centrifugation: balance the ends with a balance, centrifuge at 800 rpm for 5 minutes at room temperature.

Cell counts were performed using the Roche CASY-DT Rapid Cell Counting and Viability Analyzer. Add 10ml of CASY-ton to the tube marked as viable, add 100μl of cell suspension, invert and mix three times for cell counting. The total number of viable cells per milliliter of suspension can be seen.

Take out the cryovial, and indicate the cell name, passage, and date.

After centrifugation, discard the supernatant with a sterile pipette, do not aspirate the cell pellet at the bottom. Mix the cell pellet with the freezing solution thoroughly, and adjust the total number of cells to 5 × 10? per ml according to the counting result.

Divide the cell cryopreservation suspension into cell cryopreservation tubes, generally a two-ml cryopreservation tube is suitable for 1 to 1.5 ml of cell cryopreservation suspension.

After the seal is tightly sealed, perform programmed cooling and cryopreservation:

First -4°C for 30 minutes, 20°C for 30 minutes, -80°C overnight, and finally stored in liquid nitrogen.

Another practical cooling method is to wrap the cryopreservation tube tightly with a medical cotton gauze at least two centimeters thick, tie it tightly, and put it directly into a -70 ℃ refrigerator. Or it is more convenient to directly use a programmed cooling box.