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 0.05 % 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 2-3 min. Observe cell detachment under an inverted microscope. As soon as the cells are detached, add Trypsin-Inhibitor (20 µl/cm²), resuspend the cells in growth medium (about 160 µl/cm²) and aspirate the cells by pipetting. Pass cell suspension through an EASY 100 µM cell strainer 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, transfer appropriate aliquots of the cell suspension to new culture vessels supplemented with growth medium (final volume of 240 µl/cm²). A split ratio of 1:4 – 1:6 twice a week is recommended (after cells have reached about 60-70 % confluence). Never allow the culture to become confluent! Cultivate cells at 37°C in a humidified atmosphere with 5% CO2.

Cryopreservation