Ca2 mg2

The lipoaspirate [16 ] was diluted with sterile phosphate-buffered saline (PBS), Sigma-Aldrich, St. Louis, MO, USA) supplemented with antibiotics and centrifuged at 430 × g for 10 min (without brakes) to remove contaminating debris and red blood cells. The wash step was repeated 2–3 times depending on the quality of the specimen. The floating adipose tissue was digested with an equal volume of collagenase type I [10 mg/mL in PBS containing 5 mM Ca²⁺/Mg²⁺ (C0130, Sigma-Aldrich), final concentration 0.5%] at 37 °C for 30 min with shaking (250 rpm). The collagenase was inactivated by adding an equal volume of autologous serum, and the sample was centrifuged at 600 × g for 10 min. After centrifugation, the supernatant was discarded and the cell pellet was resuspended in NaCl 0.9% (Alpha Laboratories, Eastleigh, UK) and filtered through a 100 μm cell strainer (CS003 – PNC International Co. Ltd., Seoul, Korea) to remove debris. After centrifugation (300 RCF/5 min), 5 ml of the stromal vascular fraction were collected. All the processing must be realized within a maximal time of 90 min. The number of viable cells were determined manually (Trypan blue method) and validated on MACSQuant analyzer (Miltenyi-Biotech, Bergisch Gladbach, Germany) (7AAD staining method). All the quality control tests and injections were done with the obtained fresh samples.

Fetal dermal cells and adult dermal cells, previously cryopreserved in Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% dimethyl sulfoxide (DMSO) (CryoSure, WAK-Chemie Medical GmbH, Steinbach, Germany) and 30% fetal bovine serum (FBS), were grown in 75 cm² tissue flasks (SARSTEDT, Numbrecht, Germany) as previously described [11 (link)]. For CM collection, serum-free alpha-minimum essential medium (MEM) (Gibco, Thermo Fisher Scientific) was added to 80% confluent cells and collected 24 hours later. EVs were isolated by differential ultracentrifugation of CM according to a protocol [17 (link)] and as previously described [18 (link)]. In brief, CM was centrifuged at 1800 × g for 10 minutes to remove cell debris, centrifuged again at 17000 × g for 15 minutes, and then at 160000 × g for 1 hour in the ultracentrifuge (Optima MAX-XP, Beckman Coulter Inc., Irving, TX, USA). All centrifugation steps were done at 4°C. The pellet was resuspended in phosphate-buffered saline (PBS) without Ca^2+/Mg²⁺ (Sigma-Aldrich) or subjected to total protein extraction or total RNA extraction.

EVs were isolated by differential ultracentrifugation of CM, according to a protocol (53 (link)), and analyzed as previously described (37 (link)). In brief, cell culture was centrifuged at 300g for 10′ to remove cells, and the supernatant harvested was subsequently centrifuged at 1,800g for 10′ to remove debris, again at 20,000g for 30′, and then at 160,000g for 90′ in the ultracentrifuge (Optima MAX-XP, Beckman Coulter Inc., Irving, TX, USA). All centrifugation steps were performed at 4°C. The pellets were resuspended in phosphate-buffered saline (PBS) without Ca²⁺/Mg²⁺ (Sigma-Aldrich) or subjected to protein or RNA extraction.
Pellet particles resuspended in PBS were analyzed for size and concentration by nanoparticle tracking analysis (NTA) using the NanoSight (NS300, Malvern Instruments, Westborough, MA, USA). Samples were diluted in PBS, 300 μl of samples was loaded into the chamber, and five videos for each sample were recorded. Data analysis was done with the NTA software, and data were presented as mean ± standard deviation (SD) of the five videos.

Immortalized human HaCaT keratinocytes were kindly provided by Dr. Petra Boukamp [31 (link)] and were used to generate a HK5-EYFP expressing single cell clone B10 [32 (link)]. The cells were grown at 37 °C in a 5% CO₂ humidified atmosphere and DMEM containing l-alanyl-glutamine (Sigma-Aldrich) and 9% (v/v) fetal bovine serum SeraPlus (PAN Biotech). For passaging, cells were washed and incubated for 15 min in PBS without Ca²⁺/Mg²⁺ (Sigma-Aldrich) and thereafter trypsinized for ≈ 5 min in a solution of PBS without Ca²⁺/Mg²⁺ (Biochrom) containing 0.25% (w/v) trypsin (Biochrom) supplemented with 0.02% (w/v) EDTA (Sigma-Aldrich). Cells were passaged once per week 1 day after reaching confluence and were seeded at a concentration of 40,000–60,000 cells/cm² for DNA transfection. Cells were transfected on day 2 after seeding with 5 µg of plasmid DNA and 1.5 µl Xfect (Takara) in a total volume of 100 µl Xfect reaction buffer per 2 ml of cell culture medium in 35-mm diameter dishes.