Amphotericin b suspension

The cells were grown overnight in DMEM (HeLa S3), Ham’s F-12 (LoVo), McCoy’s 5A (HCT116), or RPMI-1640 (DLD-1) medium supplemented with 10% (HeLa, HCT116, and DLD-1 cells) or 20% (LoVo cells) fetal bovine serum (FBS), 100× Penicillin–Streptomycin solution (Wako Pure Chemical Industries), and Amphotericin B suspension (Wako Pure Chemical Industries), at 37 °C in a humidified 5% CO₂ incubator. The cells were seeded into the wells of a TF1205M micro slide glass (Matsunami Glass Industries) or into a 96-well plate, grown to approximately 90% confluence, and transfected with the plasmids (300 ng per well) using Lipofectamine 2000 (Thermo Fisher Scientific, 0.5 μl per well), according to the manufacturer’s instructions. After 24 h, the cells were analyzed with the Olympus IX71 fluorescence microscopy system (pBSII EGFP C·G and C/A) or with the Keyence BZ-9000 microscope (pBSII EGFP-tdTomamo C·G and C/A), and the fluorescence intensities were quantified using the ImageJ 1.50i software (National Institutes of Health) or the BZII-analyzer software (Keyence).

Bilateral uterine horns were isolated from C57BL/6 J mice at approximately 10 weeks of age. The tissues were minced into 2–3 mm pieces, washed several times with cold PBS, and dissociated with 2 U/mL dispase II and 1 mg/mL collagenase P (Roche Diagnostics K.K., Tokyo, Japan) for 30 min at 37 °C. Dissociated cells were subjected to the Matrigel bilayer organoid culture (MBOC) protocol [22 (link)]. Briefly, cells were resuspended in the medium supplemented with L-glutamine solution (Wako, Osaka, Japan), penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO), amphotericin B suspension (Wako), 50 ng/mL EGF (Peprotech, Rocky Hill, NJ), 250 ng/mL R-spondin1 (R&D, Minneapolis, MN), 100 ng/mL Noggin (Peprotech), 10 μM Y27632 (Wako, Osaka, Japan), 1 μM Jagged-1 (AnaSpec, Fremont, CA), and 2.5 μM CHIR-99021 (Cayman Chemical, Ann Arbor, MI), and plated on 65 μL of solidified Matrigel (BD Biosciences, Franklin Lakes, NJ) per well in a 12-well plate. After overnight incubation at 37 °C, floating or dead cells were removed, leaving only viable cells attached onto Matrigel, which were covered with 70 μL of Matrigel and the medium. Passage was conducted every 5–10 days at 1:2 to 1:3 dilution. In each passage, organoids were dissociated using Accutase (Innovative Cell Technologies, San Diego, CA) for 5 min at 37 °C and vigorous pipetting, followed by seeding on Matrigel.