Endogro ls complete culture medium

Human epidermoid carcinoma (A431) cells and murine macrophages (RAW 264.7) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Lonza, Walkersville, MD, USA) supplemented with 10% fetal bovine serum (FBS) (Bodinco, Alkmaar, the Netherlands), 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM l-glutamine (all from Lonza). Human umbilical vein endothelial cells (HUVECs) were isolated as described in [25 (link)] and maintained in EndoGro-LS complete culture medium (Merck Millipore, Billerica, MA, USA). HUVECs were grown in Primaria cell culture flasks (Corning Life Sciences, Tewksbury, MA, USA). Human perihilar cholangiocarcinoma (SK-ChA-1) cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 culture medium (Lonza) supplemented with 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 2 mM l-glutamine, and 143 µM β-mercaptoethanol. All cells were maintained at standard culture conditions (37 °C, 5% CO₂, 95% air, humidified atmosphere).

The uptake of the EVs was evaluated in the human monocyte cell line (THP-1 cells, ATCC) and human endothelial cells (HMEC cells, ATCC). The THP-1 cells were cultured in RPMI-1640 medium, supplemented with 10% FBS, while the HMEC cells were cultured in EndoGRO-LS complete culture medium (Merck Millipore, Burlington, MA, USA). MSC-EVs were labeled with DiR (2.5 µM per 1.00 × 10¹⁰ particles) and incubated for 1 h in PBS (DiR-EVs). DiR dye (2.5 μM) was diluted in PBS alone and used as control (DiR-PBS). The free, unbound dye was eliminated from the preparations, by applying 3 serial centrifugations at 4 °C at 2000× g, using the 100 kDa Amicon filters (Merck Millipore). The EV-DiR and PBS-DiR were then collected from the filter (around 150 μL). The same staining protocol was used for the in vivo experiments. The THP-1 and HMEC cell lines were seeded in 8-well chamber slides (2.00 × 10⁴ cells/well and 1.00 × 10⁴ cells/well, respectively) and incubated for 24 h. After that, DiR-PBS or DiR-EVs (1.00 × 10⁹ particles/well) were added and incubated for 24 h. Then, cells were fixed in PFA 4% for 10 min and washed and stained with phalloidin-FITC for 20 min. After washing, Vectashield with DAPI was added. Images were taken using a Leica microscope at 40×.