Comparison of the extraction methods of plasma exosomes from human umbilical cord blood

Abstract

We compared ultracentrifugation, sucrose gradient centrifugation, improved ultracentrifugation, and polyethylene glycol (PEG) precipitation in the extraction of plasma exosomes from human umbilical cord blood, aiming at screening out a stable and efficient method. The morphology, structure, and size of exosomes were observed based on transmission electron microscopy and dynamic light scattering. Total protein content of exosomes was determined by bicinchoninic acid (BCA) assay, and the expression of exosome markers CD63 and HSP70 and exosome negative marker GM130 (Golgi marker) by Western blotting. Results showed that sucrose gradient centrifugation was more stable and yielded exosomes of uniform particle size compared with ultracentrifugation which had been considered as the "gold standard" for exosome extraction. However, sucrose gradient centrifugation had the limitations of complex operation and time-intensiveness. The improved ultracentrifugation featured ease of implementation and the extracted exosomes were of high purity. PEG precipitation extracted the most exosomes in a shorter timeframe, but the purity of the exosomes was low. In conclusion, all the four methods can separate exosomes from human umbilical cord blood plasma, but they are different in operation time, product purity, and product content. Therefore, the method for extracting plasma exosomes from human umbilical cord blood should be selected based on the experimental purpose and specific requirements.