Establishing Colorectal Cancer Spheroids

Human colorectal cancer cell lines HCT-116 and HT-29 were selected because of their reported sensitivity towards Artesunate [61 (link),62 (link)] and their spheroid formation ability [63 (link),64 (link),65 (link)]. Cell lines were purchased from ATCC (HCT-116, ATCC CCL-247; HT-29, ATCC HTB-38) and cultured according to the manufacturer’s instructions. Cell line-derived cancer spheroids were prepared as described previously [63 (link)]. Briefly, monolayer cultures with a confluency of at least 90% were detached with 0.05% trypsin/0.53 mM EDTA (Pan Biotech, Aidenbach, Germany). Cell number and viability were determined using the trypan blue exclusion assay. Cell suspensions with a viability of at least 90% were used for spheroid formation. 5 × 10⁴ vital cells were seeded in each well of a low adhesive 96-well microtiter plate and incubated for 48 h under standard culture conditions (37 °C, 5% CO₂). A single cancer spheroid was obtained in each well. Cancer cell lines were used for a maximum of 10 passages. Mycoplasma assays were performed quarterly.