Detection Results Evaluation of Adipose Mesenchymal Stem Cells Derived Extracellular Vesicles by Nanoparticle Tracking Analysis and Nano-flow Cytometry

OBJECTIVE To explore the detection capability of nanoparticle tracking analysis(NTA) and nano-flow cytometry(nanoFCM) in detecting standard microspheres and adipose mesenchymal stem cells(AdMSCs) derived extracellular vesicles(EVs), provide experimental basis for the identification and quality control of EVs. METHODS Firstly, the detection capabilities of NTA and nanoFCM were verified by detecting standard microspheres. For the AdMSCs-EVs produced via PEG6000-based ultracentrifugation method, the general characterizations were performed with Western blotting and transmission electron microscope. The particle size distributions as well as the post-PKH67 labeling positive EV ratios were analyzed by NTA and nanoFCM respectively. RESULTS NTA and nanoFCM had similar capacity regarding to particle concentration detection, while nanoFCM had higher accuracy and better particle size differentiation ability. The EVs size distribution results showed that, samples analyzed by NTA presented a wider distribution while nanoFCM presented a narrower distribution. For detection of PKH67-positive EVs, the positive ratios detected by NTA were significantly lower than by nanoFCM. CONCLUSION NanoFCM has higher accuracy along with some application limitations. NTA is relatively less capable at particle size distinction, but has a wider particle size detection range. Therefore, for detection of EVs, NTA can satisfy the needs of size distribution characterization, while nanoFCM with high sensitivity for fluorescence detection is more suitable for analyzing the proportion of surface markers in EVs.