0.22 μm filter

After collection, explants were immediately placed in F12 media (Gibco, Waltham, MA) containing collagenase (1.25 mg/ml) (Sigma-Aldrich, St. Louis, MO) and incubated (37 °C and 5% CO₂) for 45 min. The collagenase step was repeated for another 45 min. Afterward, explants were incubated in F12 media containing trypsin (0.025%, Sigma-Aldrich, St. Louis, MO) mixture for 30 min followed by F12 media-containing FBS (Sigma-Aldrich, St. Louis, MO) for 15 min. Thereafter, explants were washed three times with F12 media and mechanically dissociated with a glass pipette until the solution turned cloudy indicating a successful dissociation. Dissociated cells were filtered through a 0.22-μm filter (BD Falcon, Franklin Lakes, NJ) and centrifuged (2400 rpm) for 2 min. After removing the supernatant, the cells were resuspended in neurobasal media (Sigma-Aldrich, St. Louis, MO) containing B-27 supplement (Life Technologies, Carlsbad, CA), PSN antibiotics (Gibco, Waltham, MA), 0.5 mM l-glutamine (Sigma-Aldrich, St. Louis, MO) and NGF (5 ng/ml, Alomone Labs, Jerusalem, Israel). The resuspended cells were plated on laminin-coated cover slides (Neuvitro, Vancouver, WA).

The collected fat particles were washed with phosphate‐buffered saline (PBS) and incubated for 5 min at room temperature. The lower layer of tumescent fluid and blood components were discarded. Then, the fat particles were mixed with an equal volume of prepared type IV collagenase solution (Gibco, USA). The solution was dissolved in low‐glucose Dulbecco's modified Eagle's medium (low‐glucose DMEM, HyClone, USA) at a final concentration of 0.2% and filtered twice with a 0.22‐μm filter (Falcon, USA). The cells were shaken on a 37°C shaker at 4 ×g for 1 h. After shaking, the solution was centrifuged at 239‐425 ×g for 5 min, and the supernatant and fat suspension were discarded. Ten millilitres of low‐glucose DMEM with 10% foetal bovine serum (FBS, Gibco, USA) and 1% antibiotic‐antimycotic (Gibco, USA) was added to the centrifuge tube (Falcon, USA), pipetted evenly and centrifuged at 106 ×g for 5 min. The supernatant was discarded, and cell pellets were seen at the bottom of the tube. Three millilitres of FBS‐containing medium was added and pipetted evenly; then, 8 ml of serum‐containing medium was added, mixed well and inoculated in a 10‐cm Petri dish. For the normoxic environment, cells were cultured in triplicate under normoxic conditions (21% O₂). For the hypoxic environment, cells were further cultured in triplicate under hypoxic conditions (1% O₂).