Compositions comprising small extracellular vesicles derived from umbilical cord blood mononuclear cells with anti-inflammatory and immunomodulatory properties and process for obtaining them

A method for isolating Small Extracellular Vesicles (SEVs) secreted by umbilical cord blood mononuclear cells (UCBMNCs) comprising:

• i) a first step of culturing the UCBMNCs under ischemia conditions;
• ii) a second step of obtaining culture media from the UCMBNCs;
• iii) a third step of sequentially centrifuging and concentrating the culture media, to clear the culture media from large particles and cell debris, the third step comprising:
• a) sequentially centrifuging the culture media, including a first centrifuging at 200 g-400 g for 10-20 min, and a second centrifuging at 1500 g-2500 g, for 20-30 min, at a temperature of 4-10° C., thereby generating a cleared supernatant;
• b) sequentially twice microfiltering and ultrafiltering the cleared supernatant, including:
• a first microfiltering with a first filter having a pore size of 800 nm-450 nm, thereby generating a first filtrate;
• and
• a second microfiltering with a second filter having a pore size of 250 nm-200 nm, thereby generating a second filtrate;
• and
• ultrafiltering the second filtrate, using a membrane with a molecule size cut-off of 100 KDa, at an adequate constant pressure, under stable stirring conditions of 100-200 rpm, to obtain a mixture of particles larger than 100 KDa and smaller than 200 nm;
• and
• c) concentrating the mixture to obtain a final volume of up to 10× less than initial volume of the mixture, thereby generating a concentrated mixture;
• and
• iv) a fourth step of further purifying the concentrated mixture by size exclusion chromatography (SEC), to remove small soluble proteins from the SEVs, the fourth step comprising:
• a) separating SEVs from soluble proteins m the filtrate by Size Exclusion Chromatography (SEC), using a resin comprising a cross-linked porous agarose-based material with a biomolecular separation resolution between 5000 Da and 5000000 Da, and using a 280 nm absorbance signal for detection of SEVs, and
• b) collecting an SEVs fraction from the SEC at a stable temperature of 4 to 10° C., starting at an increase leading to a first peak of the 280 nm absorbance signal, and ending when the 280 nm absorbance signal of the first peak goes down to zero, thereby producing SEVs with anti-inflammatory properties.