Extracellular vesicles (EVs) extruded by human placentae contain cargo that target them to specific maternal organs, where they may influence maternal adaptations during pregnancy. However, to explore EV bio-distribution in vivo, two fluorescent stains: sulfo-cyanine 7 NHS ester (Cy7) and CellTracker Red CMTPX (CMTPX), are required in order to visualise EVs in both the in vivo whole animal imager (AMI HTX) and fluorescent microscope, respectively. The removal of excess Cy7 involves a secondary isolation/purification of EVs. In this study we compared the recovery rate of EVs between different secondary isolation methods.
First trimester placental explants were cultured overnight with CMTPX. Placental EVs were harvested via differential-ultracentrifugation and protein content quantified by BCA. Harvested EVs were further labelled with Cy7 (20 μg). The following methods were used to remove excess Cy7 and purify labelled EVs: Vivaspin 20 mL [polyethersulfone membrane (PES) with 100 kDa molecular weight cut-off (MWCO)], vivaspin 6 mL (PES with 300 kDa MWCO), Amicon 4 mL [regenerated cellulose membrane (RC) with 100 kDa MWCO], and ultracentrifuge tubes (50 mL and 1.3 mL). Amount of protein recovered using the above methods were quantified.
Different doses of Cy7 were also used to stain placental-EVs: 0.625 – 20 μg Cy7 for large-EVs (100 nm- 1 μm), and 0.039 – 20 μg Cy7 for small-EVs (<100 nm). Free stain was removed using Amicon 4 mL. Fluorescence (AMI HTX) and protein (BCA) were quantified from the retenants and filtrates.
At 20 μg Cy7, the recovery rate of placental EVs using the methods in this study is between 10-30%. The recovery rate of Cy7-stained EVs from the secondary isolation does not differ to that of non-stained EVs. Recovery rate of small-EVs is highest at 0.3125 μg Cy7 (30%), and large-EVs at 2.5 μg Cy7 (14%) using Amicons with RC membrane.
To date, vivaspin/amicon concentrators with different membrane and ultracentrifugation with different volumes do not provide adequate yield of EV sample at a secondary isolation step.