0.22 μm pvdf filter

All freeze-
and oven-dried materials (grape stems, grape pomace, and wine lees)
were prepared following the procedure described by Milinčić
et al.^{12 (link)} with slight modifications. Briefly,
samples (100 mg) were first homogenized in 1 mL of ethanol, vortexed,
and sonicated at 40 kHz for 1 h (Branson 3510MT sonicator, Sigma-Aldrich,
St. Louis, MO) and maintained at 4 °C overnight. Afterward, the
samples were centrifuged at 8750 g, for 5 min, at
4 °C, and filtered through a 0.22 μm PVDF filter (Millipore,
MA, USA).

Ultracentrifugation methods were applied to isolate exosomes from the supernatants of SW620 and HCT8 cells as previous study^{46 (link)}. In brief, the cells were cultured in a complementary medium until about 80% confluence, and then the medium was replaced the defined medium without FBS. After 2 days of culture, we harvested the supernatants and centrifuged them at 300 × g for 15 min, 2000 × g for 15 min, and 10,000 × g for 30 min. The supernatants were filtrated through a 0.22 μm PVDF filter (Millipore, USA). Then the supernatants were collected to isolate exosomes by ultracentrifugation at 120,000 × g for 70 min (Beckman Coulter) twice. Particle Metrix (PMX), transmission electron microscopy (TEM), and western blotting were used to identify the exosomes.
The plasma exosomes and exosome RNA were isolated by SeraMir™ Exosome RNA Amplification Kit (SBI) according to the manufacturer’s instructions. In brief, 500 μl of plasma was used to combine with 120 μl ExoQuick incubated at 4 °C for 30 min and then we obtained the exosomes pellets by centrifuging at 13,000 r.p.m. for 2 min. The exosomes pellet was lysed with LYSIS Buffer, and next, we purified the exosome RNA. Plasma exosomes were also identified by PMX, TEM, and western blotting. The exosome RNA levels were normalized by exogenous λ polyA RNA (Takara, China) for qPCR^{47 (link)}.

Lentivirus preparation and infection was performed as previously described [15 (link)]. Briefly, lentiviral particles were generated by cotransfecting HEK 293FT cells with virus packaging vectors. HEK 293FT cells were maintained in Dulbecco’s modified eagle medium (DMEM) in 10% FBS, 100 units/ml streptomycin, and 100 mg/ml penicillin with 2 mM glutamax (Life Technologies). Transfection was performed using PEI (Polysciences, Warrington, PA). Five hours after transfection, the medium was changed. Virus supernatant was harvested 60 h posttransfection, filtered with a 0.22-μm PVDF filter (Millipore, Billerica, MA), ultracentrifuged at 25,000 rpm using a P28S rotor (Hitachi, Tokyo, Japan), and stocked in a final volume of 100 μl. The titer of the lentivirus used in all cell culture experiments was at least 5.0 × 10⁸ infectious units (IUs) per ml.

Exosomes were isolated as described previously39 (link). Briefly, conditioned medium was harvested 48 hours after cell seeding. After centrifugation at 3000 g to remove cellular debris, the supernatant was filtered through 0.22-μm PVDF filter (Millipore) to remove large vesicles. Exoquick Exosome Precipitation Solution (System Biosciences) was added to the filtered culture medium and mixed well. After refrigeration for 12 h, the mixture was centrifuged at 1500 g for 30 min. Exosome pellets were resuspended with PBS. The protein concentrations were measured by the Bradford method using a protein assay kit (Bio-Rad) as previously reported40 (link). To confirm the contents of the purified exosome samples, we performed immunoblotting to detect tetraspanin CD63 (Supplementary Fig. 6A). The size distribution of the exosomes in the culture supernatants was evaluated with the NanoSight LM10 system using Nanoparticle Tracking Analysis (NTA) software v2.3 (NanoSight Ltd, Amesbury, UK). The mean vesicle size in the samples was 89 nm (Supplementary Fig. 6B), in accordance with previous reports35 (link)41 (link). Purified exosomes were added to the culture medium at a concentration of 50 μg/ml. For plasma samples, exosome isolation was performed using ExoQuick Plasma prep and the Exosome precipitation kit (System Biosciences).

For exosome purification, CM was pre-cleared by filtration through a 0.22 μm PVDF filter (Millipore, USA). Exosomes were isolated from the CM by differential centrifugation steps as previously described [53 (link)]. The size and concentration of the exosomes were quantified using NanoSight NS300 instrument (Malvern Instruments Ltd., UK) equipped with NTA 3.0 analytical software (Malvern Instruments Ltd., UK). In addition, the plasma exosomes were isolated using ExoQuick Plasma prep and Exosome precipitation kit (SBI, USA). For exosomal RNA and protein extraction, exosomes were pretreated with RNase or Proteinase K, respectively. The exosome fraction protein content was assessed by Pierce® BCA Protein Assay kit (Thermo Scientific, USA).

The medium of HUVECs was collected and centrifuged at 3 000 × g for 15 min. The supernatant was filtered through 0.22-μm PVDF filter (Millipore, Bedford, MA, USA). The appropriate volume of Exoquick Exosome Precipitation Solution (System Biosciences, Mountain View, CA, USA) was added to the filtered culture medium and mixed well by inverting. After 10 h refrigeration, the mixture was centrifuged at 2 000 × g for 30 min. The supernatant was removed by aspiration. Exosome pellets were re-suspended using 500 μl of the serum-free AIM V medium (Life Technologies, Grand Island, NY, USA).

Concentrated excipient stock solutions were prepared, pH adjusted to 7.8 and sterile filtered using a 0.22 μm PVDF filter (Millipore). Calculated amounts of the excipient stocks were combined with media, which was then mixed with RV3-BB bulk drug substance in 50 mL sterile conical tubes. All formulation preparations were carried out in a class II biosafety cabinet (Labconco, USA). The two main formulations used in this study contained 60% (w/v) sucrose, 0.01% (w/v) PEG 3350, 25% (v/v) Dulbecco's Modified Eagle Medium (DMEM) in a sodium phosphate buffer at pH 7.8. Formulation A also contained 400 mM sodium succinate while formulation B does not contain sodium succinate (unless otherwise stated).