Residual tissue from patients with gastrointestinal tumors without peritoneal metastasis undergoing major elective surgery was collected after informed consent. Fresh peritoneal tissue was directly obtained from the surgery room and immediately transferred to the laboratory in E199 medium (Biochrom AG, Berlin, Germany). Afterward, the tissue was incubated for 15 min in PBS containing penicillin/streptomycin and amphotericin (Sigma-Aldrich Chemie, St. Louis, Missouri, US). Extraperitoneal fat was carefully removed with a scalpel, and small tissue pieces were inserted between two stainless steel rings and cultured with the mesothelial cell surface pointing upward. The tissue was cultured in E199 medium containing penicillin/streptomycin, L-glutamine (Biochrom AG, Berlin, Germany), FCS (Thermo Fisher Scientific GmbH, Waltham, Massachusetts, US), hydrocortisone (Sigma-Aldrich Chemie, St. Louis, Missouri, US), FGF (PeproTech GmbH, Hamburg, Germany), and heparin (Biochrom AG, Berlin, Germany) as described previously [16 (link)]. For fresh-frozen sections, the tissue was carefully dislodged from the rings, embedded in Tissue Freezing Medium (Leica Microsystems, Wetzlar, Germany) and immediately frozen in liquid nitrogen. For FFPE sections, the tissue was fixed with 4% PFA (VWR International, Radnor, Pennsylvania, US) and embedded in paraffin.
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