Optiprep

Pericyte isolation from mouse livers were carried out as described in (Graham, 2002 ). Briefly, livers from 4 mice were harvested in cold Hanks’ Balanced Salt Solution post perfusion. Organs were minced, enzyme digested and filtered in a similar way to the lungs. The suspension was centrifuged at 600g for 7 minutes at room temperature. A gradient was prepared using 60% Optiprep (Alere) with HBSS in the lower phase and 39.86% Optiprep in the middle phase homogenised with the cell pellet. The top was layered with 0.5 ml HBSS followed by centrifugation at 1400g for 20 minutes at 4°C without break. The interphase between the top and middle layers were collected and washed by centrifugation at 600g for 7 minutes. Finally, the pellet was treated with RBC lysis buffer (Sigma) and washed. Cells were isolated and plated in tissue culture treated 12 well plates (Corning) in pericyte medium i.e., DMEM with 20% FBS, 10mM L-Glutamine, 100units Penicillin Streptomycin (Gibco) and were allowed to settle overnight at 37°C, in humidified atmosphere at 5% CO₂. The cells were then washed thoroughly with PBS and then treated as indicated.

To further analyze the groups separated with the PreSEC method, the EVs corresponding to the SG1 and SG2 were separated by floatation into a iodixanol density gradient (Kowal et al., 2016). Briefly, EV‐enriched fractions were concentrated by UC and the pellet was resuspended in 2.5 ml of a solution of 30% Optiprep (Alere Technologies), 250 mM sucrose, 20 mM HEPES, and 1 mM EDTA (pH 7.4). This vesicle suspension was covered with 1.3 ml of a 20% and 1.2 ml of a 10% iodixanol solutions. The tubes were centrifuged for 1 h at 4°C at 200,000 RCF (46,000 RPM) in a SW55Ti rotor (deacceleration without brake). Then, 490 μl fractions were collected from the top to the bottom. The fractions were diluted with PBS and centrifuged at 118,000 RCF for 39 min in a TLA 100.3 rotor. Pellets were resuspended in lysis solution and protein content was determined by BCA assay.

AAVs were generated in HEK293t cells with triple-transfection methods with some modifications (Xiao et al., 1998 (link)). Briefly, AAV2/2, AAV2/9, and AAV2retro were generated by transfection of HEK293t cells with pHelper (Cell BioLabs, San Diego, CA, USA), the rep/cap vector (pXR2 for AAV2/2, pAAV2.9 for AAV2/9, or rAAV2-retro helper for AAV2retro) and pAAV genomic vector. Three days post-transfection, virus-producing cells were collected and lysed by freeze and thaw for purification. After centrifugation, the supernatant was loaded on gradients (15%, 25%, 40%, and 58%) of iodexanol OptiPrep (Alere Technologies AS, Stirling, Scotland). After centrifugation at 16000 g at 15°C for 3 h, 100–150 μl upper from 58% iodexanol layer was collected. Virus aliquots were stored at −80°C until use. The titers of AAV used in the present study were 5.0 × 10² infectious units/ml (HEK293t cells) or 1.0 × 10¹¹–1.8 × 10¹⁴ viral genome/ml quantitative PCR (qPCR; Aurnhammer et al., 2012 (link)).