U bottom low attachment plates

CRC cells were seeded in a clinically relevant 1:1 ratio with CCD18co fibroblasts and 5% ECRF24 endothelial cells in 96-well U-bottom low attachment plates (650970, Greiner Bio-One, Frickenhausen, Germany) [31 (link),32 (link)]. The 3Dcc culture media contained a mixture of DMEM-RPMI-EMEM (1:1:1) supplemented with 2.5% Matrigel^® (354254, Corning, Bedford, MA, USA). The 3D-CCs were treated with drugs 48 h post-seeding.

LS174T and SW620 human CRC, as well as CCD18co and CCD841 non-malignant cells were obtained from ATCC with a corresponding authentication certificate. Human immortalized endothelial cells ECRF24 cells (via immortalization procedures with amphitropic replicant-deficient retrovirus [58 (link)]). LS174T and SW620 FOLFOXIRI resistant cell lines were generated in our laboratory through a chronic treatment [14 (link), 35 (link)]. The cells were cultured in a humidified incubator at 37 °C and 5% CO₂ in culture medium supplemented with 10% fetal bovine serum (S1810-500, Biowest, Nuaillé, France) and 1% penicillin/streptomycin (4-01F00-H, BioConcept, Allschwil, Switzerland). Cells were regularly tested for mycoplasma contamination using the MycoAlert kit (LT07-218, Lonza, Rockland, ME, USA), with controlled passage.
3D co-cultures were created in 96-well U-bottom low attachment plates (650970, Greiner Bio-One, Frickenhausen, Germany) with CRC cells or CCD841 cells seeded 1:1 with CCD18co cells and 5% ECRF24 cells [24 (link)]. Culture media consisted of a mixture of DMEM, RPMI and EMEM (1:1:1) supplemented with 2.5% Matrigel® (354254, Corning, Bedford, MA, USA). The 3Dcc were treated 48 h post-seeding (Day2).