Handling of Suspension cells

CLS Cell Lines Services

1. Thawing Suspension cells

a. Prewarm complete medium to room temperature (not 37°C) and transfer 8ml of it to a 15ml tube; transfer another 10ml to a T25 flask
b. Quickly thaw the cryovial at 37°C ( a small ice clump should still remain and the cryovial should still be cold)
c. Transfer the complete content of the cryovial to the 8ml of media and resuspend well
d. Spin the cell suspension at 300xg for 3min and discard the supernatant carefully
e. Take 5ml out of the T25 flask and resuspend the cell pellet in the 15ml tube. Take a small aliquot of cells (<300μl) to count the cells and determine the viability.
f. Transfer the rest of the cell suspension to the T25 flask, resuspend well with the remaining 5ml of media.
g. Incubate at 37°C for at least 24 hours

2. Subculture Suspension cells

a. The cells should not exceed a cell density of 1x10^6 cells/ml.
b. Carefully transfer the cell suspension to a 15ml tube and centrifuge at 300xg for 3min.
c. Aspirate the supernatant of the cells and resuspend the cells in 5 or 10ml of medium (depending on the cell pellet). Take a small aliquot to count the cells and determine the viability.
d. Seed the cells at a density of 1x10^5 cells/ml in either T75 flasks or T150 flasks. Transfer 15ml of fresh medium to each T75 flask (or 30ml for each T150 flask) and dispense the cell suspension equally to the T75/T150 flasks.

3. Freezing Suspension cells

a. Carefully transfer the cell suspension to a 15ml or 50ml tube and centrifuge at 300xg for 3min.
b. Aspirate the supernatant and resuspend the pellet in a defined volume of medium (e.g. 5ml, 10ml, 20ml; depending on the size of the cell pellet). Take a small aliquot to count the cells and determine the viability.
c. Depending on the cell count, determine the amount of cryovials.
d. Centrifuge the cell suspension at 300xg for 3min and resuspend the cell pellet in the calculated amount of CM-1 (freezing medium).
e. Store the cryovials at -20°C immediately for at least 40min. Transfer all cryovials to - 80°C overnight. Prolonged storage must be in liquid nitrogen (-196°C).