Freezing cells for liquid nitrogen storage

Aim:

To make 6 x 1mL aliquot cell stocks starting with a T75 flask semi-confluent with cells. (1mL aliquots will include 10% DMSO.

Perform all work in the sterile laminar flow cell cabinet. Make sure cryo-tubes are clearly labelled.

Steps

1. For 6 aliquots mix 2600mL of cell media with 650mL DMSO, then add 0.5mL to 6 empty sterile cryo-tubes. Special cryo-tubes are required to ensure the freezing presure does not pop the lid off. Each tube will consist of 20% DMSO. (Approximately 250uL of mix should be left over as excess - waste).

2. Starting with a semi-confluent T75 remove media and wash 2 times with room temperature PBS. (6mL PBS for a T75).

3. Add 0.05% Trypsin & incubate for approximately 5 minutes at 37°C. (1.5mL Trypsin for a T75).

Trypsin is a mixture of proteases widely used for cell dissociation.

4. Dilute Trypsin by adding media. (6mL for a T75 = >1:4 Trypsin:media).

5. Transfer to 15mL tube, centrifuge at 1000 rpm for 5 minutes & pour off media. Resuspend gently in 3.5mL media.

6. Transfer 0.5mL of cells to each cryo-tube for a final volume of 1mL with 10% DMSO. Work quickly as cells do not like liquid DMSO. DMSO helps protect cells from freezing. Approximately 500uL of cells / media should be left over as excess. This can be added to a new T25 or T75 flask or discarded.

7. Freeze cells at -70 to -80°C. Ideal to use a Mr. Frosty Freezing Container from ThermoFisher Scientific (or similar) with new isopropanol added after every 3 – 5 freezes. This ensures cells freeze more gradually limiting damage.

8. Once freezing is complete, transfer cells to liquid nitrogen for long term storage.