Bovine plasma fibronectin

Primary human brain microvascular endothelial cells (HBMEC) used in the study were purchased from ScienCell Research laboratories (Carlsbad, CA, USA). HBMEC were isolated from human brain and cryopreserved at passage one. HBMEC were characterized by immunofluorescence with antibodies specific to vWF/Factor VIII and CD31 (PECAM). Cells were cultured on bovine plasma fibronectin (ScienCell)-coated dishes in endothelial cell medium (ECM). Specifically, 500 mL of basal ECM medium was supplemented with 25 mL of exosome-depleted fetal bovine serum (Exo-FBS; System Biosciences, Mountain View, CA, USA), 5 mL of endothelial cell growth supplement (ECGS, ScienCell), and 5 mL of penicillin/streptomycin solution (P/S, ScienCell). We initiated two separate cultures on 100 mm cell culture dishes to reduce the number of passages and subcultured the cells twice at the 1:4 ratio. This resulted in 32 confluent cultures, with the average cell number at the end of experiment of 9.065 × 10⁷ cells/dish. Sixteen confluent cultures were used for EV surface proteomics, and 16 for EV total proteomics. The treatment groups were: 1) Control exposed to vehicle, 2) Aβ alone, 3) HIV alone, 4) HIV plus Aβ, with four samples/group.

Both hBMSCs and HUVECs were purchased from Sciencell (Carlsbad, CA, United States). The cells were seeded at 5,000 cells/cm² in culture bottles treated with bovine plasma fibronectin (Sciencell). ECM culture medium (Sciencell) containing 5% fetal bovine serum (Gibco, Grand Island, NY, United States), 1% penicillin mixture, and 1% endothelial cell growth factor (Sciencell) was used for HUVECs. hBMSCs were cultured in mesenchymal stem cell medium (Sciencell) containing 5% fetal bovine serum, 1% penicillin mixture, and 1% mesenchymal stem cell growth factor. The cells were placed in a constant temperature incubator with 5% CO₂ at 37°C to subculture. The third to fifth passages of cells were used in the experiments.

Three human gastric cancer cell lines (MKN-28, MKN-45, and MKN-74), a human colorectal cancer cell line (SW480), and HHSECs (human hepatic sinusoidal endothelial cells) were used in this study. MKN-28 and its transfectants, MKN-45 and MKN-74, were maintained in Dulbecco’s modified Eagle medium (DMEM; 05919, Nissui Pharmaceuticals, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS; 1370978, Gibco, Gaithersburg, MD, USA), P.S. (100 Units/mL penicillin and 100 µg/mL streptomycin, Gibco), and L-glutamine (30 mg/mL, Nacalai Tesque, Kyoto, Japan). SW480 cells were cultured in Roswell Park Memorial Institute 1640 medium (13485; Gibco) supplemented with 10% FBS. HHSECs were maintained in endothelial cell medium (1001; ScienCell) in bovine plasma fibronectin (10%; 8284; ScienCell)-coated dishes. All cell lines were cultured at 37 °C in an atmosphere of 95% air and 5% CO₂.