Culture of adipose derived stem cells:
Human adipose derived stem cells (hADSC) were purchase from ZenBio, Inc (Item # ASC-F). The cells screened negative for HIV-1, HIV-2, HTLC-1, HTLV-2, Hep-B, Hep-C, and mycoplasma. HADSC were isolated from subcutaneous adipose tissue of normal, nondiabetic donors between 25 and 45 years of age undergoing elective surgery and with BMI <23.3. Cells were cultured according to the manufacturer’s instructions.
Collection of conditioned media (CM):
Exosomes and microvesicles were isolated from conditioned media (CM). For CM collection, hADSC were grown to 90% confluence in T75 flasks, media was replaced with serum-free mesenchymal stem cell basal media, with StemFlex medium kit (Gibco, Cat# A33494-01) and CM was collected after 48 hours.
For GW4869-CM, the hADSC cells were treated with GW 4869 (1µM) (from Cayman Chemical Item No# 13127) in serum-free media for 48 hours. For C2-ceramide CM, hADSC were treated with C2-ceramide (Cayman Chemical Item No# 62510), a biologically active, cell permeable and less hydrophobic analog of natural ceramide, at 20 µM in serum-free media, 48 hrs. For C6-ceramide CM, hADSC were treated with C6-ceramide (Cayman Chemical Item No# 62525) at 20 µM with serum-free media, 48 hrs.
Isolation of exosomes and microvesicles:
Exosomes and microvesicles were isolated using Exo-Spin Exosome Purification Kit (Cell Guidance System, Cat# EX01-25). Briefly, 45 ml of CM was transferred to a conical tube and centrifuged at 300 x g for 10 min to remove any unattached cells. The supernatant was transferred to a new tube and spun at 16,000 x g for 30 min to remove any remaining cell debris. Next, the supernatant was transferred to a new tube, and ½ volume of Exo-Spin Buffer was mixed with it and incubated at 4°C overnight. This was then centrifuged at 16,000 x g for 1 hour, carefully aspirated and the supernatant was discarded. The exosome-containing pellet was resuspended in 100 µl of PBS. The Exo-Spin Column was prepared following manufacturer’s instructions. The exosome containing pellet (100 µl) was carefully applied to the top of column and the column was placed into the waste tube. This was then centrifuged at 50 x g for one min, and the eluate was discarded. The column was then placed into a 1.5 ml microcentrifuge collection tube, and 200 µl of PBS was applied to the column. This was centrifuged at 50 x g for one min. The 200 µl eluate contained the purified small extracellular vesicles containing exosomes and microvesicles.
Measurement of MALAT1 in cells and small extracellular vesicles (sEV):
Human ADSC were cultured in T-75 flasks. For CM collection, hADSC were grown to about 90% confluence, medium was replaced with serum-free mesenchymal stem cell basal medium, we used StemFlex medium kit (Gibco, Cat# A33494-01) and CM (15 ml) were collected after 48 hours. Total cellular RNA was isolated from sEV using RNeasy Mini Kit (Qiagen, Germantown, MD). One microgram of total RNA was used to synthesize complementary cDNA with iScript cDNA Synthesis Kit (Cat # 1708891, Bio-Rad, Hercules, CA) according to the manufacturer’s protocol.
The Real-Time PCR reaction was performed using 2 µl of cDNA with 1µl of 20X Taqman Human MALAT1 primer/probe set (Assay ID number Hs00273907_S1), (Applied Biosystems, Foster City, CA), 10 µL of TaqMan® Fast Advanced Master Mix (Applied Biosystems, Foster City, CA) and 7 µL of Nuclease-Free Water for a total volume of 20 uL. As an internal control, a second Real-Time PCR reaction was performed using 2 uL of cDNA with 1 µL 20X Taqman Human GAPDH primer/probe set(Assay ID number Hs03929097_g1), (Applied Biosystems, Foster City, CA). Thermocycling conditions were as follows: 50°C, 2 min; 95°C, 10 min; and 40 cycles of 95°C for 15 s and 60°C for 1 min using Applied BioSystems ViiA 7 System. The Human MALAT1 mRNA Relative expression (RQ) to GAPDH was calculated. Each sample was run in duplicate. SEV RNA was isolated as described above. Human MALAT1 mRNA relative expression (RQ) to GAPDH was calculated. Each sample was run in duplicate.
HDF scratch assays:
Human Dermal Fibroblasts-adult (HDF-a) cells were purchased from ScienCell (Catalog N0. 2320). HDF-a were from adult human skin and were negative for HIV-1, HBV, HCV, mycoplasma, bacteria, yeast and fungi. Cells were subcultured in Fibroblast Medium (FM, Catalog N0# 2301) until approximately 70% confluent and media changed every other day until the cells were about 85% confluent.
For performing a wound healing scratch assay, we used the Ibidi Culture-insert 2 well (Ibidi, Catalog N0. 80209). Use sterile tweezers for transfer, the sterile 2-well insert was placed into each well of a 12-well plate, The HDF-a cell suspension of about 3.6x105 /ml, was added (70 µl of cell suspension) to each well of the plate with inserts. Cells were cultured at 37°C with 5% CO2 as usual. After 24 hours cells were confluent, inserts were removed, and the plate was rinsed once with PBS to remove cell debris. Then cells were preincubated with mitomycin C (10µg/ml) for 2 hours to block further proliferation so that only migration was followed.
Isolated exosomes 20 µg/ml (protein) were then added to wells. Basic fibroblast growth factor (bFGF, Gibco, Cat # 13256-029) was used as the positive control, 20ng/ml. Wound healing assay photo images were acquired at 0, 8, 26, and 48 hours. Images were analyzed by Ibidi Metavi Wound Healing Analysis Automatic Cellular Analysis System and by counting cells that had migrated into the scratched field. Data from each time point and treatment were analyzed by two-tailed ANOVA with p<0.05.
Mitochondrial stress test:
Human Dermal Fibroblasts-adult (HDF-a) cells were purchased from ScienCell (Catalog N0. 2320) and cultured in a T-75 flask following the manufacture’s instructions. For the Mitochondrial Stress test the HDF-a, 5.5-6x103 cells, were plated into wells of a poly-L-lysine coated XFp cell culture miniplate with 80 µl of growth medium. In two of the 8 wells, no cells were plated for background correction. Sterile water (400 µl) was added to each chamber of the moat. Cells grew overnight in a cell culture incubator. Cells were then treated with exosomes prior to the stress test. Exosomes (20µg protein/ml) were added to the wells with serum free medium, and cultured in a CO2 incubator for 2-24 hours. The Seahorse Mito Stress Test used the Agilent Seahorse XFp Extracellular Flux Analyzer. Oxygen consumption rate (OCR) was measured using the mitochondrial stress test kit (Cat # 103010-100) following instructions. Agilent Seahorse XF DMEM media (Part # 103575-100) containing 10 mM glucose, 2 mM L-glutamine, and 1 mM sodium pyruvate were used prior to the assay and the compounds for loading the sensor cartridge ports for the Agilent Seahorse XFp analyzer were: 1.0 μM of Oligomycin (Port A), 1 μM of FCCP (Port B) and 0.5 μM of Rotenone/Antimycin A (Port C).
The Agilent Seahorse XF cell Mito Stress Test report generator automatically calculated the Agilent Seahorse XFp Cell Mito Stress Test parameters from WAVE data that was exported to Excel. Exosome preparations were tested in 3-4 separate assays. Significance was tested using a two-tailed Student’s t test (Microsoft Excel) with at least three independent biological replicates (n), with the Mann-Whitney U test or with one-way ANOVA as indicated (GraphPad Prism) (*p < 0.05, **p < 0.01, and ***p < 0.001).
SEV size and charge:
SEV size was determined using Malvern Nano Series Zetasizer Nano ZS90. A SEV aliquot of 50 µl was added to 450 µl nuclease-free water and mixed in a cuvette for determining the size of SEV in the instrument.