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].