Ccd 18co atcc crl 1459

The myofibroblasts-like cell line CCD-18Co (ATCC^® CRL-1459) was purchased from the ATCC (American Type Culture Collection; Rockville, MD, USA). This cell line was grown and maintained following the instructions given by the manufacturer. Unless otherwise stated, EMEM (pH 7.2–7.4) was supplemented with 10% FBS (v/v), antibiotics (100 U mL⁻¹ penicillin and 100 mg mL⁻¹ streptomycin), 1.5 g/L sodium bicarbonate, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, and 2 mM L-glutamine. The cells were seeded at 6000 cells/cm² in T75-flasks and incubated under optimal grown conditions (i.e., 37 °C, 5% CO₂/95% air atmosphere, and constant humidity) for several days. Once ≥80% confluence was reached, the cells were subcultured. The passages and population doubling levels used for the experiments were 15–16 and 33–36, respectively.

The HT29 and WiDr human colorectal adenocarcinoma cell lines, and the CCD-18Co (ATCC^® CRL-1459^™) human fibroblast cell line isolated from normal colon tissue (both from American Type Culture Collection, Manassas, VA, USA) were cultured in 75 cm² flasks (TPP AG, Trasadingen, Switzerland) in RPMI-1640 (Euroclone, Milano, Italy) supplemented with 10% (v/v) fetal calf serum (FCS) (Euroclone, Milano, Italy), 100 U/mL penicillin G, 40 µg/mL gentamycin sulfate, and 2.5 µg/mL amphotericin B (all from Sigma-Aldrich, Milano, Italy) at 37 °C in a humidified 5% CO₂ atmosphere. Cells were harvested twice a week with 0.25% porcine trypsin solution (Sigma-Aldrich, Milano, Italy).