In vitro propagation

Passaging of cells

For detachment of the cells remove and discard the culture medium and wash the cells once with PBS (about 160 µl/cm²). Remove PBS completely.

Then, add Trypsin-EDTA solution (20 µl/cm²), make sure that all cells have been in contact with this solution and incubate the culture flask at 37°C for approximately 2 – 5 min. Observe the cell detachment under an inverted microscope. As soon as all cells are detached (if necessary, agitate the cells by gently hitting the flask), add Trypsin-Inhibitor (20 µl/cm²). Resuspend the cells in growth medium (about 160 µl/cm²) and centrifuge at 170 g for 5 min. Discard the supernatant, resuspend the cell pellet in the remaining droplet and add growth medium (about 160 µl/cm²). Then, add appropriate aliquots of the cell suspension to new culture vessels supplemented with growth medium (final volume of 240 µl/cm²). A split ratio of 1:2 to 1:3 twice a week is recommended (after having reached about 95 % confluence). Cells should be passaged when near confluent. Cultivate cells at 37°C in a humidified atmosphere with 5% CO2.

Cryopreservation