T175 flask

Primary hMSCs were isolated from bone marrow obtained from healthy donors, Galway University Hospital, Galway, Ireland. All procedures followed were in accordance with ethical standards of the responsible local committees for human experimentation (institutional and national) and the Helsinki Declaration of 1975, as revised in 2000, and informed consent was obtained from all marrow donors included in the study. Isolated hMSCs were expanded in a growth medium containing α-minimal essential medium (α-MEM, Life technologies, Carlsbad, CA, USA), 10% foetal bovine serum (FBS, Sigma, St. Louis, MO, USA) and 1% penicillin/streptomycin (P/S, Life technologies) supplemented with 5 ng/mL fibroblast growth factor-2 (FGF-2, Peprotech, Rocky Hill, NJ, USA) and cultured at 37 °C, 5% CO₂. The medium was refreshed 3 times weekly and cells were sub-cultured or frozen at 90% confluence in T175 flasks (Sarstedt, Newton, NC, USA). The immortalized human monocytes THP1 were cultured in an RPMI (Sigma) growth medium with 10% FBS and 1% P/S, and incubated at 37 °C, 5% CO₂. The medium was refreshed 3 times weekly and the cells were sub-cultured or frozen at 90% confluence in T175 flasks (Sarstedt).

MSCs were thawed and expanded in chemically defined complete medium (DMEM) supplemented with 10% FCS (Sigma Aldrich, F7524), 1% Pen/Strep and 1% amphotericin B until 80–90% confluency, passaged with trypsin (Gibco, 25300-096) and seeded at a defined density of 4 × 10⁶ cells per T175 flask (Sarstedt, Nümbrecht, Germany, 833912002). All cells used were at passage 2, cell morphology was assessed daily, and viability was measured using Trypan blue dye exclusion in conjunction with an automated cell counter (ThermoFisher Countess 3). After 24 h, cells were washed twice with 10 mL filtered (0.22 µm filter (Sarstedt, 831822)) PBS +/+ and then cultured in 22 mL serum-free DMEM (plus 1% Pen/Strep, 1% amphotericin). After 48 h and doubling of the cell number, which was estimated from preliminary studies with a population doubling level (PDL) of 3 (PDL = 2 + 3.322 (log 8 × 10⁶–log 4 × 10⁶)), the conditioned medium was collected and transferred into 50 mL Falcon tubes (Sarstedt, 64547254) and precentrifuged at 3000× g for 20 min at 4 °C to remove undesired cell debris. The obtained supernatant (SN) was carefully collected without disturbing the cell debris pellet and immediately processed.

BV2, an immortalized murine microglial cell line, was cultured in growing medium containing Dulbecco’s modified Eagle medium (DMEM) (Gibco™GlutaMAX™, Thermo Fisher Scientific) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin (Thermo Fisher Scientific) in 5% CO₂ in air at 37 °C in a humidified incubator. Cells were re-cultured every 2 days starting at a concentration of 2 × 10⁵ cells/ml in T75 flask (Sarstedt). For a large scale of EV collection, microglia were plated in T175 flask (Sarstedt). For inflammatory activation, cells were challenged with 1 μg/ml LPS (Sigma-Aldrich, Clony 0127-B8) for 12 h and then grown for 12 h in serum-free media prior to collection of EVs. For TNF inhibition experiment, microglia were plated in growing medium either with 1 μg/ml LPS, 200 ng/ml etanercept, or both for 12 h. EVs were collected from serum-free media 12 h after treatment.