For the suspension culture, HEK293F cells (ATCC, ACS-4500™) were cultivated in 50-mL Tubespin containing 10 mL ProCHO5 medium (Lanza Co.) at a density of 0.5×106cells/mL. Cultures were maintained in a shaking incubator at 37°C with stirring speed at 180 rpm. For adherent cell culture, HEK293T cells (ATCC, CRL-11268™) were incubated in 5mL DMEM medium containing 10% fetal calf serum (Gibco) in an incubator at 37°C and cells were passaged twice a week. The cell density and viability were determined by the Trypan Blue exclusion method.
All plasmids were constructed with plasmid pCDNA3.4 (Invitrogen co.), including pCDNA 3.4/MFG-E8, pCDNA3.4/MFG-E8-EGFP, pCDNA3.4/EGFP, pCDNA3.4/MFG-E8-Gaussia luciferase(GL), and pCDNA3.4/CD9-GL. After digestion with KpnI and XhoI, the open reading frame (ORF) of MFG-E8, EGFP, MFG-E8-GL or CD9-GL was inserted into the multiple cloning site (MCS) of pCDNA3.4. For fusion expression, a flexible linker (GGGGSGGGGSGGGGS) was used to link DNA sequences of two genes.
Transient expression of protein in HEK293F cells
One day prior to transfection, HEK 293F Cells were seeded in fresh ProCHO5 medium at a density of 2×106 cells/mL. On the day of transfection, cells were centrifuged at 800 rpm for 5 min and resuspended in 2mL RPMI1640 media at the indicated cell density in TubeSpins. The plasmid DNA and 25 kDa linear polyethyleneimine (PEI, Polysciences, Warrington, PA) were mixed and stood for 10min, and then added to the culture. The transfected culture was first incubated for 3h at 37°C with 5% CO2, 85% humidity, and agitation at 180 rpm, followed by adding EX-Cell HEK293 medium (Sigma) to 10mL.
Cell culture medium was collected and centrifuged at 3,000g for 15 min to remove cellular debris, and the supernatants were transferred to an appropriate vessel for the MF-600 ultracentrifuge (Hanil Science Industrial, Incheon, Korea) according to the method described by Théry C. Successive centrifugations at increasing speeds were performed to throw the pellet away (300g for 10 min- 2000g for10 min–10,000g for 30 min). In the last step, the supernatant was collected and centrifuged one more time at 100,000g for 70 min and only the pellets were kept. The pellet was washed in a large volume of PBS for three times to eliminate contaminating proteins, and centrifuged one last time at the same high speed. The final sEvs pellets were resuspended in 100 ml PBS and filtered through a syringe filter (0.2 mm, Sartorius). The morphology of sEvs was observed by Transmission Electronic Microscopy (FEI co., CZ). The number and size of sEvs particles were measured by nanoparticle-tracking analysis with a Nanosight NS300 (Malvern Instruments).
Cells were cultured for up to 72 or 96h post-transfection before the supernatant was collected. Cell lysates and isolated sEvs were subjected to 12% SDS-PAGE and western blotting according to standard protocols. Western blots were incubated at 4℃ for 16 h with the indicated primary antibodies against MFG-E8, EGFP, CD9, CD63, luciferase or αvβ3 (Invitrogen, Cat.No. PA5-82036; Proteintech, Cat.No. 66002-1-Ig; 60232-1-Ig; 67605-1-Ig; 67293-1-Ig; Abcam, Cat.No. ab190147) and then washed for three times in Tris-buffered saline T (TBS-T), followed by 1 h incubation with Goat Anti-Mouse IgG(H + L) (Proteintech, Cat.No. SA00001-1) at room temperature.
Cells were successively incubated with 4% paraformaldehyde, 0.25% TritonX-100, and 1% BSA. At the end of each step, cells were centrifuged and washed with PBS buffer. In the end, cells were incubated with anti-M8 antibodies solution for 12h at 4℃, followed by incubation with Goat Anti-Mouse IgG H&L (FITC) (Abcam, Cat.No. ab6785) and DAPI solution respectively, then observed by Zeiss laser confocal microscopy.
Transmission Electronic Microscopy (TEM) analysis with gold nanoparticles labeled sEvs
The preparation of gold nanoparticles (AuNPs) and gold nanoparticles labeled mAb (AuNPs-mAb) was done according to reference . Briefly, 1 mL of 1% HAuCl4 was quickly added to the 50mL boiled ultrapure water and 1.2 mL trisodium citrate dihydrate (10 mg/mL) was added after a few seconds. The mixture was heated for 10 min, and then diluted with ultrapure water to 50 mL.
For preparation of AuNPs-antibody, 1mL of AuNPs (0.02mg/mL) was adjusted to pH 8.5 with 0.25M K2CO3, and 10µL anti-MFG-E8 antibody was diluted with 1×TBST to 100µL. Then the antibody was quickly added to the AuNPs solution. The mixture was rotated for 15min and kept still for 15 min at room temperature. Subsequently, 100 µL of 10% BSA were added to cover the unconjugated site, and rotated for another 15 min and then kept still for 15 min. Finally, the mixture was centrifuged at 12000 rpm for 30 min and the precipitate was resuspended in 50–100 µL PBS, followed by incubation with isolated sEvs overnight at 4℃. 10µL of labeled sEvs was dropped on GRID,and applied to TEM after air-drying.
Gaussia Luciferase (GL) activity analysis
The GL-loaded sEvs were rinsed with 100µL/well of 1X DPBS buffer, followed by cell lysis by adding 50–100µL/well of 1X Cell Lysis Buffer and shaking for 15-30min.
Gaussia Luciferase (GL) activity was analyzed according to the protocol Pierce™ Gaussia Luciferase Glow Assay Kit (Thermo Scientific). In brief, the Working Solution was prepared by adding 50µL of 100X Coelenterazine to 5mL of Gaussia Glow Assay Buffer firstly. 10–20µL/well of cell lysate was added to an opaque 96-well plate, then 50µL of Working Solution was added to each well. After 10 minutes for signal stabilization, we detect the light output in a Luminometer (Berthold, Bad Wildbad, Germany).
To confirm whether recombinant MFG-E8 adhered to the cell membrane, cells were adjusted in advance and cultured in an incubator at 37°C with 5% CO2. Then 1*106 cells were placed in a 1.5 ml tube. The cells were centrifuged at 1000*g for 5 mins and washed thrice with 1x PBS. Next, the cells were resuspended in 100µl PBS., 5µl of the anti-MFG-E8 antibody was added to the cell suspension. The sample was mix thoroughly at 37 ℃ in the dark for 30 mins. Then the sample was centrifuged at 1000*g for 5 mins and washed thrice with 1x PBS. Finally, 5µl anti-Mouse IgG H&L(FITC) was added to the cell suspension, and the sample was mix again at 37 ℃ in the dark for 30 mins. The sample was centrifuged at 1000*g for 5 mins and washed thrice with 1x PBS. The cells were resuspended in 500 µL of PBS and examined using flow cytometry. To demonstrate sEvs with MFG-E8 had αvβ 3 targeting, cells were placed in a 1.5 ml tube after incubating with MFG-E8-EGFP-sEvs of EGFP-sEvs. The cells were centrifuged at 1000*g for 5 mins and washed thrice with 1x PBS. Then, the cells were resuspended in 500 µL of PBS and examined using flow cytometry.