For miRNA analysis, DMEM-low glucose containing 10% FCS, 2 mM L-glutamine was filtrated using 0.22 µm filter cups to remove large EVs. 6x106 freshly isolated SVF cells were seeded in 22 mL filtrated medium in a T175 flask. The cell supernatant was collected after 24 h in a 50 ml-tube and centrifuged at 500 xg for 15 min (Eppendorf, 5804R; Vienna, Austria) to remove cellular debris. After transfer to a new tube (VWR, Vienna, Austria) and centrifugation at 14 000 xg for 15 min at 4°C (Heraeus Multifuge X3R; Thermo Fisher Scientific, Vienna, Austria) the supernatant was filtrated through a 0.8 µm strainer (VWR) to prepare clarified cell culture conditioned media (cCM), which was immediately frozen and stored at –80°C until further analysis. The frozen cCM samples were shipped on dry ice to TAmiRNA GmbH, Vienna Austria. cCM was concentrated to 1 mL using centrifugation-based ultrafiltration. Briefly, 20 mL of cCM were transferred to an Amicon 30 kDa ultrafiltration column and centrifuged at room temperature for 15 minutes. Residual volumes were measured and diluted to 1 mL using sterile PBS solution. Small extracellular vesicles (sEVs) were subsequently purified from cCM by size exclusion chromatography using qEV70s single columns (Izon): 150 µl of concentrated cCM were loaded based on recommendations from the manufacturer, and fraction 8 – 11 were collected and pooled to obtained the fraction of purified sEVs. Nanoparticle tracking analysis (NTA) was performed on all sEV samples to determine particle size and concentration.
Growth protocol
The collection of human adipose tissue was approved by the local ethical board with written patient’s consent. Subcutaneous adipose tissue was obtained during routine outpatient liposuction procedures from the hips and outer thighs (“saddlebags”) under local tumescence anaesthesia. Tumescence solution contained one vial Volon-A 10 mg (Dermapharm), three vials Suprarenin 1 mg/mL (Sanofi), 45 mL bicarbonate 8.4% (Fresenius Kabi) and 60 mL Xylocaine 2% including Lidocaine 0.04% (Astra Zeneca). The harvesting cannulas were triport and 4 mm in diameter (MicroAire System power-assisted liposuction). Table 1 provides patient characteristics. 100 mL liposuction material was transferred to a blood bag (Macopharma, Langen, Germany) and washed with an equal volume of phosphate buffered saline (PBS) to remove blood and tumescence solution. Next, for tissue digestion PBS was replaced with 0.2 U/mL collagenase NB4 (Nordmark, Uetersen, Germany) dissolved in 100 mL PBS containing Ca2+/Mg2+ and 25 mM N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES; Sigma, Vienna, Austria) resulting in a final collagenase concentration of 0.1 U/mL. The blood bag was incubated at 37 °C under moderate shaking (180 rpm) for 1 h. The digested tissue was transferred into 50 mL-tubes (Greiner, Kremsmünster, Austria). After centrifugation at 1200 xg for 7 min the cell pellet was incubated with 100 mL erythrocyte lysis buffer for 5 min at 37 °C to eliminate red blood cells. The supernatant was aspirated after centrifugation for 5 min at 500 xg. The pellet was washed with PBS and filtrated through a 100-µm cell strainer (Greiner). After another centrifugation step at 500 xg for 5 min the supernatant was removed. The isolated SVF was resuspended in filtrated (0.22 µm; Merck, Vienna, Austria) medium before: DMEM-low glucose (Lonza, Vienna, Austria) containing 10% fetal calf serum (FCS; Sigma, Vienna, Austria), 2 mM L-glutamine (Lonza). Cell number was determined using trypan blue exclusion and quantification in a cell counter (TC-20, Bio-Rad, Vienna, Austria).
Extracted molecule
total RNA
Extraction protocol
Total RNA extraction was performed using 200 µl cCM or 200 µl pooled sEV fractions together with the miRNeasy mini kit (Qiagen). Synthetic oligonucleotides obtained from the miRCURY Spike-In kit (Qiagen) were added to the Qiazol lysis buffer before homogenization of the cCM/sEV samples. Glycogen (5 mg/ml) was added to the chloroform extract at 1:100 dilution to enhance precipitation. All other steps were performed according to the recommendations of the manufacturer. Total RNA was eluted in 30 µl nuclease-free water and stored at -80°C in low-bind tubes (Eppendorf) until further analysis.
Label
SYBR Green
Label protocol
n/a
Hybridization protocol
n/a
Scan protocol
n/a
Description
control SAMPLE 2
Data processing
Cq-values were called using the second-derivate maximum method. Data quality was assessed using spike-in controls to determine RNA extraction efficiency, enzymatic inhibition, and overall variability. Data normalization was performed using the global mean (i.e. average Cq-value for all endogenous microRNAs with Cq<35) to obtain delta Cq-values. Values in the Matrix normalized are global-mean normalized delta Cq-value (dCqs). Fold Change 1 = sEVs: Lipedema vs Control Fold Change 2 = cCM: Lipidema/Control
