Zell shield antibiotics

HCT-116 cells (DSMZ, Braunschweig, Germany) were grown in McCoy’s 5A medium (Biowest) containing 10% fetal bovine serum (FBS) (PAA Laboratories, Pasching, Austria). Caco-2 cells (ECACC, Salisbury, UK) were grown in MEM medium (Biowest, Nuaillé, France) containing 15% FBS (PAA Laboratories) and MEM non-essential amino acids (Biowest). Culture media were supplemented with 2 mM glutamine (Biowest) and Zell Shield antibiotics (Minerva Biolabs, Berlin, Germany). Cells were maintained in a humidified atmosphere at 37 °C and 5 °C CO₂.

Cells were treated while growing in adherence and then trypsinized and embedded in Matrigel (growth factor reduced, phenol red free; BD) on ice and seeded in 24-well plates (10–25 single cells/μL of Matrigel per well). The Matrigel was polymerized for 10–15 min at 37 °C and then 500 μL/well basal culture medium (advanced DMEM/F12) supplemented with Zell Shield antibiotics (Minerva Biolabs), 1× B27 and 1× N2 (Thermo Scientific, Waltham, MA, USA) was added. The number and size of organoids were analyzed by optical microscopy 1–2 weeks after seeding.
For wt or VilCre^ERT2Apc^fl/fl mouse organoids, crypts, fragments of epithelium, or single cells were seeded in 24-well plates (50 and 10 fragments per 10 μL of Matrigel per well, respectively). After 24 hours, organoids were treated with c-PTIO for 48 hours and proliferation was determined by analyzing the images with ImageJ software.

ASC52telo cells (ASC; ATCC, SCRC-4000) and normal human ASC (ATCC, PCS-500-011) were expanded in the dedicated medium (ATCC, Mesenchymal Stem Cell Basal Medium with Mesenchymal Stem Cell Growth Kit). For experiments, cells were switched to complete growth medium consisting of 89% MEM Alpha (ThermoFisher Scientific), 10% FBS Q (Biological Industries) and 1% ZellShield antibiotics (Minerva Biolabs). Human bone marrow stem cells (BMSC) were obtained from the iliac crest and dental pulp stem cells (DPSC) from the molar tooth of adult donors according to the approved institutional review board protocol no. 1072.6120.253.2017. BMSC and DPSC were expanded in complete growth medium and used for experiments at passages 4–7. Cell cultures were maintained in culture incubator at 37 °C, 5% CO₂ humidified atmosphere; culture media were exchanged every 2–3 days and cells were passaged using 0.25% trypsin/EDTA (ThermoFisher Scientific) before they reached full confluence. For experiments, ASC cells were seeded at the density of 1000/cm², BMSC and DPSC at the density of 10,000/cm².