Isolation and Characterization of Colorectal Cancer Stem Cells

Human CRC cell lines Colo205, SW480, HT29, SW620, LS174T, DLD1, Caco-2 were purchased from ATCC and cultured in RPMI-1640 medium. No mycoplasma contamination was detected. Human CCSCs were isolated and cultured as described previously (Bu et al., 2013 (link)). Briefly, CCSCs were isolated from patient tumors by FACS based on markers CD44, CD133 and ALDH1 and functionally validated by serial sphere formation, tumor initiation, and self-renewal assays. For this study, original frozen stocks for the first passage were used. The CCSCs have not been authenticated by STR profiling. No mycoplasma contamination was detected. CCSCs were cultured as spheres in ultralow-attachment flasks (Corning, Tewksgury, MA) in DMEM/F12 (Invitrogen, Pittsburgh, PA), supplemented with nonessential amino acids (Fisher, Pittsburgh, PA), sodium pyruvate (Fisher), Penicillin-streptomycin (Fisher), N2 supplement (Invitrogen), B27 supplement (Invitrogen), 4 μg/mL heparin (Sigma, Mendota Heights, MN), 40 ng/mL epidermal growth factor (Invitrogen), and 20 ng/mL basic fibroblast growth factor (Invitrogen) at 37°C and 5% CO₂.
To measure tumor sphere formation, single CCSCs were plated in 24-well ultra-low attachment plates (Corning) at 1,000 cells per well. Tumor spheres were counted after 2 weeks in culture by an inverted microscope (Olympus).