Amicon ultra 15 100 kda

Cells were cultured in conditioned medium containing 10% exosome-depleted FBS for 72 h at 90–100% density. The conditioned medium was harvested and centrifuged at 2,000 × g for 10 min followed by 10,000 × g for 30 min at 4°C. Then, the supernatant was filtered using a 0.22-µm filter to eliminate cellular debris thoroughly and concentrated using centrifugal ultrafiltration (Amicon^® Ultra-15 100 KDa; Merck KGaA) to minimize the potential contamination, respectively.
Exosomes were isolated from processed conditioned medium with Ribo™ Exosome Isolation Reagent (Guangzhou RiboBio Co., Ltd., Guangzhou, China), according to the manufacturer's instructions. Briefly, processed conditioned medium was mixed with Ribo™ Exosome Isolation Reagent at a ratio of 3:1 and incubated at 4°C overnight. After centrifugation at 1,500 × g for 30 min, exosomes pellets were resuspended in appropriate phosphate-buffered saline (PBS; HyClone; GE Healthcare Life Sciences) for TEM, NTA and further research. The exosome pellets were used immediately or stored at −80°C until use.

Purified CAFs and NFs were cultured in BEGM medium (containing 10% exosome‐depleted FBS) for 72 h. When each cell monolayer was near confluence, the culture supernatant was collected and centrifuged (1500 g, 10 min), followed by centrifugation (10,000 g, 30 min) at 4°C. The resulting exosome supernatant was then filtered (0.22 μm filter) to eliminate cellular debris, followed by centrifugal ultrafiltration (Amicon^® Ultra‑15 100 KDa; Merck KGaA) to prevent potential contamination. Exosomes were isolated from the ultrafiltered supernatant with Ribo™ Exosome Isolation Reagent (RiboBio Co., Ltd.), as previously described.¹⁵ Briefly, the collected conditioned medium was mixed with Ribo™ Exosome Isolation Reagent (ratio 3:1), followed by an incubation at 4°C overnight. The medium was then centrifuged (1500 g, 30 min), and the resulting exosome pellets were suspended in phosphate‑buffered saline (PBS; HyClone; GE Healthcare Life Sciences) and analysed immediately or stored at −80°C for subsequent research.

To produce the EVs for in vivo injection, the corresponding stable cells were seeded in 15‐cm dishes with 10 million cells/ dish with full DMEM medium. Two days late, the medium was changed to Opti‐MEM medium (Gibco, USA) with 1% Anti‐anti. And then the conditional medium (CM) was harvested 48 h late, serially centrifuged at 700 × g for 5 min and 2000× g at 4°C for 10 min. The CM was filtered with bottle top filters system (Corning, low protein binding) with cellulose acetate membrane. This system contains a 0.22 μm pore size within the membrane and can remove the remaining larger particles. The EVs in the filtered CM were isolated with Tangential flow filtration (TFF, MicroKross, 20 cm², Spectrum labs). The cut‐off of TFF is 300 kDa and the particles bigger than 300 kDa are remained in the system and concentrated. And then the concentrated particles were further concentrated by Amicon Ultra‐15 100 kDa (Millipore) spin filter, centrifuged at 4000× g for 30 min to several hours at 4°C depending on the amount EVs in the samples. At last, the concentrated EVs were collected in maxirecovery 1.5 ml Eppendorf tubes (Axygene, USA) and the concentrations were detected by Nanoparticle Tracking Analysis (NTA).

GNPs were purchased from BBI Solutions (Crumlin, UK). The glucose-modified GNPs were prepared as previously described with some modifications (41 (link)-43 ). Briefly, 4 mL GNP solution was filtered through Amicon Ultra-15 100 kDa centrifugal filter devices (Millipore, Billerica, MA, USA), and ultrafiltration was carried out at 5,000 ×g for 10 min. The GNPs were then resuspended in 4 mL deionized water. This process was repeated twice to remove the excess coating of polymers in the GNP solution. Next, 200 µL 1 mM glucose-monofunctional polyethylene glycol-thiol (glucose-mPEG-SH) was added to 1 mL 0.3 mM GNP solution. The mixture was stirred for 24 h at room temperature (25 °C) and ultrafiltration was conducted as described above to remove excess glucose-mPEG-SH.