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.