Scale extraction method for exosomes in fresh milk

A method for large-scale extraction of exosomes from fresh milk, wherein the method comprises:

• (1) filtering the fresh milk through a hydrophilic and lipophobic nano-interfacial net membrane to remove fat particles from the fresh milk, and collecting the filtrate;
• (2) ultrafiltering the filtrate collected in (1) using a tangential flow ultrafiltration device with a 100 to 300 kDa pore-size ultrafiltration membrane to concentrate the filtrate from which fat particles have been removed, thereby obtaining concentrated fresh milk;
• (3) thoroughly mixing the concentrated fresh milk collected in (2) with an aqueous two-phase solution, centrifuging the mixture for separation, discarding the upper phase, and collecting the lower phase;
• and repeating this procedure 1 to 2 times for the lower phase to further concentrate a solution of the exosome;
• wherein the aqueous two-phase solution is prepared by dissolving polyethylene glycol and dextran in distilled water or a PBS buffer, with polyethylene glycol having a concentration of 20 to 40 g/L and dextran having a concentration of 14 to 20 g/L;
• (4) diluting the concentrated solution of exosomes obtained from (3) with a diluting solution, and then further separating it through a gel column using highly cross-linked agarose as the a filler, wherein the diluting solution is a 20 mM Tris-HCl buffer with pH=7.2 containing 150 mM NaCl.