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 once with PBS.Remove PBS completely.Then, add Trypsin-EDTA solution (20 µl/cm²), make sure that all cells have been in contact with Trypsin-EDTA and incubate the culture flask at 37°C for approximately 2 – 3 min.Observe cell detachment under an inverted microscope. As soon as all cells are detached, add growth medium (about 160 µl/cm²) and aspirate cells by pipetting.Determine the viable cell number and add appropriate aliquots of the cell suspension to new Gelatin coated culture vessels filled with growth medium (final volume of 240 µl/cm²).A seeding density of 1200 cells/cm² is recommended.Cells should be split twice a week when having reached about 30 – 40 % confluence. Never allow the culture to become confluent!Cultivate cells at 37°C in a humidified atmosphere with 5% CO2.

Cryopreservation