E199 medium

Fresh peritoneal tissue was obtained directly from the operating room, with informed consent from the patient, and transferred immediately to the laboratory in E199 medium (Biochrom AG, Berlin, Germany). The tissue was then incubated for 15 min in phosphate-buffered saline (PBS)-containing penicillin/streptomycin and amphotericin (Sigma–Aldrich Chemie, St. Louis, MO, USA). Extraperitoneal fat was removed, and 7 × 7 mm tissue pieces were inserted between two stainless steel rings and cultured, with the mesothelial cell surface directed upwards, in E199 medium containing penicillin/streptomycin, L-glutamine (Biochrom AG, Berlin, Germany), FCS (Thermo Fisher Scientific GmbH, Waltham, MA, USA), hydrocortisone (Sigma–Aldrich Chemie, St. Louis, MO, USA), fibroblast growth factor (PeproTech GmbH, Hamburg, Germany), and heparin (Biochrom AG, Berlin, Germany), as described previously [12 (link)].

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.

Fresh peritoneal tissue was obtained directly from the operating room, with informed consent from the patients, and transferred immediately to the laboratory in E199 medium (Biochrom AG, Berlin, Germany). The tissue samples were then incubated for 15 min in phosphate-buffered saline (PBS) containing penicillin/streptomycin and amphotericin B (Sigma-Aldrich Chemie, St. Louis, MO, USA). Extraperitoneal fat was removed, and 7 × 7 mm tissue pieces were inserted between two stainless steel rings, which are available in various sizes, and thus enable large variations in the setting of these experiments. Moreover, stainless steel rings are more stable than plastic rings. In this setup, the ring system allows for tissue orientation with the mesothelial cells pointing upwards, thereby better reflecting the peritoneal cavity in the in vivo setting. The peritoneum was cultured in E199 medium containing penicillin/streptomycin, L-glutamine (Biochrom AG), FCS (Thermo Fisher Scientific), hydrocortisone (Sigma-Aldrich Chemie), fibroblast growth factor (PeproTech GmbH, Hamburg, Germany), and heparin (Biochrom AG), as described previously [12 (link)].