Establishment of Patient-Derived Tumoroids

Patient-derived tumoroids were established as described by Jeppesen et al. [39 (link)] with minor modifications. In short, freshly resected tumour samples of 0.01–1 g were divided into 1 mm³ pieces and digested for 20 min at 37⁰C with 1 mg/ml collagenase type II (Thermo Fisher Scientific) diluted in STEM medium. Subsequently, the tissue suspension was sequentially filtered through a 70 μm cell strainer (VWR, Soeborg, Denmark) and a 30 μm pre-separation filter (MACS, Miltenyi Biotec, Bergisch Gladbach, Germany). Tissue retained by the 70 μm filter was collected and redigested for 10 min at 37⁰C and passed through the filters again. This step was repeated until all tissue could pass through the 70 μm filter. Retained tumour fragments (30–70 μm) were seeded in STEM cell medium in petri dishes coated with 1.5% agarose (Sigma-Aldrich) and cultured at 37⁰C in a 5% CO₂ humidified incubator. After 3–5 days the tissue fractions which had densified into rounded tumoroids with smooth surfaces could be collected for further culture. Tumoroids established from colonic tumours were denoted C after the patient number and tumoroids from liver metastases were denoted L.