Ham s f12

Human bladder cancer cell lines UMUC3, J82, RT4 and immortalized urothelial cell line SV-HUC-1 were purchased from the American Type Culture Collection (Manassas, VA, USA), and bladder cancer cell line 5637 was kindly provided from the Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University. UMUC3 was cultured in Dulbecco's modified Eagle's medium (DMEM; Wako, Osaka, Japan), and 5637 and RT4 were cultured in RPMI 1640 medium (Wako). J82 was cultured in Eagle's minimum essential medium (EMEM; Wako), and SV-HUC-1 was cultured in Ham's F-12 (Wako), with 10% exosome-depleted fetal bovine serum (Thermo Fisher Scientific) and 5 μg/ml gentamaicin (MSD, NJ, USA) and 60 μg/ml tylosin (Sigma-Aldrich, St. Louis, USA), and incubated at 37°C in a humidified atmosphere containing 5% CO₂. Cells were grown to confluence for 72 h. Conditioned media were pooled and centrifuged to obtain EVs according to the same protocol as the urine samples.

CHO-K1 and CHO (Chr.21) containing human chromosome 21^{10 (link)} were cultured in Ham’s F12 (Wako, Osaka, Japan) containing 10% fetal bovine serum (FBS)(Biowest, Vieux Bourg, France), 1% penicillin /streptomycin (Wako) and 800µg/mL G418 (Promega, Madison, USA). HT1080 and B16F10 cells were cultured in Dulbecco′s Modified Eagle’s Medium (DMEM) (Wako) containing 10% FBS.

The loggerhead sea turtles were maintained as a closed colony at the Port of Nagoya Public Aquarium, Minato-ku, Nagoya, Japan. During the labelling of the sea turtles with identification tags (at approximately 1–2 years of age; see Fig. 1 of the reference), we obtained small (3 mm × 3 mm) dermal-tissue biopsies from the flipper-like fins^{39 (link)}. All biopsy processes were authorized by a veterinary doctor affiliated with the Port of Nagoya Public Aquarium. The experimental protocol and procedure were approved by the committee of animal handling at the Port of Nagoya Public Aquarium. The biopsy samples were immediately immersed in DMEM/F12 medium (Life Technologies, Carlsbad, CA, USA; product code, 10565-042) containing 10% FCS, and 1× antibiotics (Nacalai Tesque, Kyoto, Japan; product code, 02892-54). For optimization of the cell culture conditions, we used four different media: DMEM (Nacalai Tesque; product code, 845935), DMEM/F12 (Life Technologies), Ham’s F12 (Wako Chemicals, Osaka, Japan; product code, 8708335) and RPMI 1640 (Wako Chemicals; product code, 18902025). Each medium contained 10% fetal calf serum and 1× antibiotics.

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).

HTR-8/SVneo, a trophoblast-derived cell line, was kindly supplied by CH Graham^{38 (link)} and BeWo, a choriocarcinoma-derived cell line, was obtained from American type culture collection. HTR-8/SVneo cells were cultured in RPMI 1640 (Wako, Osaka) supplemented with 10% FBS, and BeWo cells in Ham’s F12 (Wako) supplemented with 10% FBS.