Scrc 4000

Bone marrow was obtained from discarded and de-identified waste tissue from adult bone marrow transplant donors in accordance with the Institutional Review Board of Duke University Medical Center. Adherent cells were expanded and maintained in expansion medium: DMEM-low glucose (Gibco), 1% penicillin/streptomycin (Gibco), 10% fetal bovine serum (FBS) (ThermoFisher), and 1 ng/mL basic fibroblast growth factor (Roche) (Hagmann et al., 2013 (link)).
Adipose-derived stem cells (ASCs) were purchased from ATCC (SCRC-4000) and cultured in complete growth medium: mesenchymal stem cells basal medium (ATCC PCS-500–030), mesenchymal stem cell growth kit (ATCC PCS-500–040) (2% FBS, 5 ng/mL basic recombinant human FGF, 5 ng/mL acidic recombinant human FGF, 5 ng/mL recombinant human EGF, 2.4 nM L-alanyl-L-glutamine), and 0.2 mg/mL G418.

The human immortalized ASC cell line ASC52telo (SCRC-4000; ATCC, Manassas, VA, USA) was used for ASC-CM extraction. SCRC-4000 cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin until they reached confluence. The waste culture medium was removed, and the cells were washed twice in PBS. Subsequently, fresh serum-free DMEM was added to the cells. The cells were then incubated at 37 °C in a humidified atmosphere containing 5% CO₂ for 48 h. After 48 h of incubation, the medium was collected and ultra-filtrated with a 0.45 µm filter. The supernatant obtained after filtering the cell components was used as the ASC-CM without further dilution. FBS was then added to achieve a concentration of 10% when used for experiments.

ASCs and AMCs kept under experimental conditions (72 h and 21 days TNFα 300 U/mL) were scraped and incubated in ice for 30 min with RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P40) and a protease inhibitor cocktail (Complete EDTA-free, Roche Molecular Biochemicals, Merck KGaA, Darmstadt, Germany). The total cellular lysate was centrifuged at 14,000 rpm for 1 h to clear cell debris. The protein concentration was determined using the Bradford assay. Proteins were denatured in Laemmli sample buffer (2% SDS, 10% glycerol, 5% 2-mercaptoethanol, 62.5 mM Tris-HCl pH 6.8, 0.004% bromophenol blue), separated on 12% polyacrylamide gels, transferred to nitrocellulose membranes (TransBlot Transfer Medium Bio-Rad Laboratories S.r.l., Segrate, MI, Italy), and blotted with the primary antibodies listed in Table 2. Antigen–antibody complexes were visualized using the SuperSignal West Femto Maximum Sensitivity Substrate (Pierce) on a CCD camera (Chemidoc, Bio-Rad Laboratories S.r.l., Segrate, MI, Italy).
The ASC52telo (SCRC-4000, ATCC, Manassas, VA, USA) hTERT immortalized adipose derived mesenchymal stem cell line was used as a positive control. Western blot bands were quantified by densitometry using ImageJ software V1.52 and the results were presented as histograms using GraphPad Prism 5 software (California).

Extracellular vesicles (EVs) isolated from cell cultures were used to evaluate material properties on membrane particle absorption and mimic virus particles. EVs were isolated and characterized from ASC52-telo (SCRC-4000, ATCC, Manassas, VA, USA) cell culture similarly to previously described protocols [38 (link),39 (link)]. Single-channel devices were washed with 70% ethanol overnight, dried, and washed with sterile 1× PBS overnight. Then, 20 µL of 1.02 × 10⁸ EVs/mL in 0.02 µm filtered 1× PBS were injected into channels and incubated for 1 h at +37 °C. After incubation, EV solutions from channels were collected and measured by nanoparticle tracking analysis (NTA) with an NS 300 instrument (Malvern, Philadelphia, PA, USA) equipped with green (532 nm) laser and scientific Complementary metal–oxide–semiconductor (sCMOS) camera and compared with input sample. Here, 0.02 µm filtered 1× PBS was used as a negative control. Measurements were performed on five 30 s videos that were recorded using camera level 12. The data were analyzed using NTA software v3.0 with the detection threshold 8 and screen gain at 5. Experiments were performed in biological duplicates and measured in technical duplicates. p-value was calculated by the Mann–Whitney test.