Mwco amicon ultra device

After preconditioning, cells were washed with PBS three times and cultured in serum-free DMEM without phenol red supplemented with 1% penicillin/streptomycin and 1% insulin–transferrin–selenium (ITS, Thermo Fisher Scientific) for 48 h. After this time, the conditioned medium was collected, centrifuged at 1000×g for 10 min at 4 °C, 5000×g for 20 min at 4 °C to remove dead cells and debris, and, subsequently filtered through 0.45- and 0.22-µm filters. After these steps, the secretome fraction was concentrated by centrifugation at 4000×g for 40 min at 4 °C, using a 3 kDa MWCO Amicon® Ultra device (Merck-Millipore, MA, USA). For EV isolation, the concentrated secretome was ultracentrifuged at 110,000×g for 2 h in a MAX-XP ultracentrifuge equipped with a TLA-45 fixed-angle rotor (Beckman Coulter, Krefeld, Germany), and the resulting pellet was washed with 0.1 µm filtered PBS (1 ml) and centrifuged again with the same settings. Finally, the supernatant was removed, and isolated EVs were suspended in 50–100 µl of filtered PBS and stored at − 80 °C for further analysis. When samples from different donors were pooled, concentrated secretome samples (AMICON samples) were equally mixed based on protein concentration (n = 5 donors) and EVs were isolated by ultracentrifugation as described.

EVs were obtained from cell lines cultured at a confluence of 80% at passages 12–15 as reported^{25 (link)}. Culture medium was replaced by exosome isolation medium (1% insulin-transferrin-selenium in DMEM with 10% Penicillin/Streptomycin). After 4 days, supernatants were collected and subjected to two steps of centrifugation at 4 °C, first at 1000 × g for 10 min, and then 5000 × g for 20 min. The obtained supernatants were then filtered through a 0.22 μm filter to eliminate dead cells and debris, and subsequently ultra-filtered through a 3 kDa MWCO Amicon^® Ultra device (Merck-Millipore, MA, USA) to concentrate the EVs. Concentrates were recovered and stored at − 20 °C until use.
For the intrapericardial administrations, protein quantifications were performed by Bradford assay, and the resulting protein concentration was diluted in Sodium Chloride 0.9% (w/v) to 1.832 mg/mL. Finally, 9.16 mg of exosomal proteins were intrapericardially administered in 5 mL of Sodium Chloride 0.9%.