4.2.1 Preparation of ADSCs-derived nanovesicles (ANVs)
The Sprague−Dawley (SD) rats weighting about 100 g were used to take adipose tissue from the groin. The adipose tissue was cut into 1mm3 small masses and digested with 1% type I collagenase and stop by PBS after 30 min. After centrifuge at 300 xg for 10 min, the supernatant and undigested tissue were removed, precipitated cells were resuspended with DMEM containing 10% FBS. The residual red blood cells were lysed with 1×RBC lysis buffer, and the collected cells were resuspended and filtered with 200 mesh cell filter and centrifuged at 300 xg for 10 min to obtain the ADSCs. The ADSCs were purified by collecting adherent cells after amplification culture in low-sugar DMEM containing 10% FBS and and 1% (v/v) penicillin/streptomycin and used at passages 4~5 in all experiments. The stem cell properties were identified by morphological observation and induced differentiation experiment.
The ADSCs were lysed with hypotonic cell lysis buffer (1 mmol/L NaHCO3, 0.2 mmol/L EDTA, 1 mmol/L PMSF, 200 μL/107 cells) at 4 ℃ after cultured to 70~80%, and then further ground with a tissue grinder. Centrifuge the solution with 3200 xg at 4 ℃ for 5 min to collect the supernatant. By repeatedly extruding the supernatant through 400 and 200 nm polycarbonate trace etching (PCTE) membrane of liposome extruder in sequence, the collected cell membrane can be reconstructed into nanovesicles (ANVs). SDS-PAGE of ADSCs and ANVs protein samples extracted by RIPA lysis buffer were further mixed with 6× loading buffer and placed at 95 ◦C for 10 min. Gels were running at 150 V for 30 min with SDS-PAGE rapid electrophoresis buffer.