2.1. Cell culture and EV isolation
Human BMSCs (ATCC, Manassas, VA) were purchased and expanded to passage 3 to 5 before being cultured in EV-free media prepared according to Wang X et al [33]. Human PSCs were obtained and isolated from healthy donors with informed consent approved according to the procedures of the Institutional Review Board (IRB) at Wake Forest University (Winston-Salem, NC), and cells from passage 11 were cultured in EV-free media [33]. Briefly, both BMSCs and PSCs were expanded in growth medium (GM) containing Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, Thermo Fisher Scientific, Waltham, MA), 10% (v/v) fetal bovine serum (FBS, ScienCell, Carlsbad, CA), 100 U/mL penicillin G, and 100 mg/mL streptomycin (Gibco, Thermo Fisher Scientific) at 37°C in an incubator with 5% CO2 atmosphere and 100% relative humidity. To induce osteogenic differentiation, BMSCs and PSCs were cultured in osteogenic medium (OM) including 10 nM dexamethasone, 100 μM ascorbic acid, and 2 mM β-glycerophosphate on T175 tissue culture flasks for 21 days. Fresh culture medium was replaced every third day. All chemical reagents were obtained from Millipore Sigma (St. Louis, MO) unless stated otherwise.
For EV isolation, the culture medium was replaced with a serum-free culture medium for 72 h. The media was then collected, and EVs were isolated by a series of differential centrifugation with filtration. Briefly, the supernatant was centrifuged at 1000g for 30 min at 4°C to eliminate cellular debris. This was then concentrated and purified with the KR2i TFF system (Spectrum Labs, High Point, NC) using the concentration–diafiltration–concentration mode. Typically, 500-200 mL supernatant from the hollow fiber bioreactor was concentrated to approximately 250 mL, diafiltrated with 500–1000 mL phosphate buffered saline (PBS), and finally concentrated to 10 mL. The hollow fiber filter modules were made from modified polyethersulfone, with a molecular weight cutoff of 500 kDa. The flow rate was 80 mL/min and the pressure limit was 8 psi for the filter module D02-E500-05-N (used to concentrate supernatant from large volumes to ~8 mL) [34]. After that supernatant was centrifuged at 500g for 20 min at 4°C, followed by passing through a 0.22 μm filter to eliminate debris. Then, the supernatant was ultracentrifuged at 120,000g for 70 min in a T-647.5 rotor (Sorvall WX Ultra series, Thermo Scientific, USA). The EV pellets were resuspended in PBS and stored at -80°C since it has been previously established that these are optimal long term storage conditions [35].