In vitro propagation

Passaging of cells

The new culture flasks have to be pre-coated with porcine gelatin. Therefore, the culture flasks are treated with gelatin solution (80 μl/cm2) at 37°C for at least 4 hours (up to one week). Before introducing cells, remove excess of gelatin solution. Use the pre-coated flasks immediately for seeding of cells, the surface must not dry out.

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

Then, add 0.025 % 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. Thereafter, resuspend the cells in growth medium (about 160 µl/cm²) and aspirate the cells by pipetting, centrifuge at 170 g for 5 min. Centrifuge at 170 g for 5 min. Then, discard the supernatant, resuspend the cell pellet in the remaining droplet and add growth medium (about 160 µl/cm²). Transfer appropriate aliquots of the cell suspension to gelatin-coated culture vessels supplemented with growth medium (final volume of 240 µl/cm²). A split ratio of 1:4 twice a week is recommended (after cells have reached about 80-90 % confluence). Cultivate cells at 37°C in a humidified atmosphere with 5% CO2.

Cryopreservation