Growth of CACO-2 Cells on 24-well Plates

CACO-2 cells from ATCC at passages 20-50 are used. CACO-2 cells are stored in cryovials in liquid nitrogen. Original cryovials from ATCC are handled as described in the ATCC procedure (see also Chen et al. 2002). This ensures constant cell source for many years once cells have been splitted, grown under the same conditions and than frozen in liquid nitrogen. 

The suspension is mixed carefully and than centrifuged at 150 g for 5 min. The cell pellet is resuspended after discarding freezing medium in cultivation medium and placed in an 175 cm2 cultivation flask containing 80 ml complete medium. Cells are cultivated at 37 °C, 95 % humidity and 10 % CO2. 

Medium change is performed every 3 days. After 10 days cells are splitted. 

Cell splitting starts with washing the attached cells with 20 ml PBS for 2 1 min. After removal of PBS trypsin/EDTA is added (20 ml) for 2 min. After removal of trypsin/EDTA 2 ml of trypsin/EDTA are added and incubated for 5 min at 37 °C with gently shaking the flasks. Trypsinization is stopped when cells start to detach by suspending the cells in 20 ml complete medium while trypsinization procedure stops. After a centrifugation step (to remove trypsin) at 150 g for 5 min cells are resuspended in complete medium and splitted to 3 flasks of 175 cm2 (2.4-2.6 x 10s cells per flask). 

After 7 days of cultivation with medium changes every 3 days (confluency 80-90 %) cells are trypsinated and prepared for use in 24-well plates. 

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