Rpmi 1640

RPMI 8226, U266B1, SKO-007, IM-9, Hs 602, RPMI 6666, Jiyoye, Daudi, HuT 78, H9, 293T, NK-92MI cell lines were obtained from Korean Cell Line Bank or American Type Culture Collection. According to the supplier’s recommendation, RPMI 8226, U266B1, and IM-9 cell lines were cultured in RPMI 1640 (Cytiva, Massachusetts, USA) supplemented 15% fetal bovine serum (FBS, Cytiva, Massachusetts, USA) with 1× Penicillin and Streptomycin (P/S, Thermo Fisher Scientific, Massachusetts, USA). SKO-007 cell line was cultured in the same medium with 0.02 mg/mL 6-thioguanine (Merck, Massachusetts, USA), 1 mM sodium pyruvate and 2 mM L-glutamine (Corning, New York, USA) added. Hs 602, Daudi, and 293 T-cell lines were cultured in RPMI 1640 supplemented 10% FBS with P/S. RPMI 6666, Jiyoye, and H9 cell lines were cultured in RPMI 1640 supplemented 20% FBS with P/S. HuT 78 cell line was cultured in Dulbecco's Modified Eagle Medium (DMEM) (Lonza, Switzerland) supplemented 10% FBS with P/S. NK-92MI cell lines were cultured in RPMI 1640 supplemented 25% FBS with P/S. Cells were cultured at 37°C, 5% CO₂ and confirmed to be Mycoplasma-negative with MycoAlert Detection Kit (Lonza, Switzerland).

iPSCs were differentiated into HEPs following previous protocols²² (link)^,23 with modification as follows: iPSCs were replated onto Matrigel-coated plates at 0.8 × 10⁵ cells/cm² in StemMACS iPSC-Brew XF medium (Miltenyi Biotec) with 10 μmol/L Y-27632 (Selleck Chemicals). After 1 day, the medium was changed to RPMI-1640 (Lonza) containing 50 ng/mL Activin A (PeproTech, Rocky Hill, NJ), 0.5 mg/mL bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO), B27 supplement (Gibco, Grand Island, NY), and 0.5 mmol/L sodium butylate (Sigma). The concentration of sodium butylate was reduced to 0.1 mmol/L for 4 days to induce definitive endoderm differentiation. For HB induction, the definitive endoderm was cultured in RPMI-1640 (Lonza) supplemented with 10 ng/mL HGF (PeproTech) and 10 ng/mL FGF4 (PeproTech), 0.5 mg/mL BSA (Sigma-Aldrich), and B27 supplement (Gibco) for 5 days. HB stage cells were then cultured in Hepatocyte Culture Medium (without epidermal growth factor (EGF) supplement; Lonza) with 10 ng/mL HGF, 10 ng/mL OSM (PeproTech), and 0.1 mmol/L dexamethasone (Dex) for final differentiation.

The two prostate cancer cell lines PC-3 and LNCaP American Type Culture Collection (ATCC, CRL-1435 and CRL-1740, USA) were used as a model to produce tdEVs. Both cell lines were cultured at 37°C and 5% CO₂ in RPMI-1640 with L-glutamine medium (Lonza, Cat. No.: 12–702 F) supplemented with 10% (v/v) foetal bovine serum (FBS), 10 units/mL penicillin and 10 μg/mL streptomycin. The initial cell density was 10,000 cells/cm² as recommended by the ATCC. Medium was refreshed every second day. At 80-90% confluence, cells were washed three times with phosphate-buffered solution (PBS) and cultured in FBS-free RPMI-1640 with L-glutamine medium (Lonza, Basel, Switzerland, Cat. No.: 12–702 F) supplemented with 1 unit/mL penicillin and 1 μg/mL streptomycin. After 2–3 days of cell culture, cell supernatant was collected in a 15 mL tube (Cellstar® tubes, Greiner Bio-one BV, Alphen a/d Rijn, The Netherlands) and centrifuged at 500–800 xg at room temperature for 10 min (Centrifuge 5804, Eppendorf, Hamburg, Germany). The pellet containing dead or apoptotic cells and large cell fragments was discarded. The collected supernatants containing PC-3 and LNCaP EVs were stored in aliquots at −80°C. Samples were thawed in a water bath at 37°C before use. EV size distribution and concentration were determined by both nanoparticle tracking analysis (NTA) (NanoSight NS500, UK) and flow cytometry (SI).