Exploring Purification Methods of Exosomes from Different Biological Samples

Abstract

Objective. Exosomes were extracted from a variety of biological samples using several different purification processes, and our goal was to determine which method and sample were the most effective for exosome extraction. Methods. We used ExoQuick-TC combined with ultrafiltration to separate and purify exosomes from the supernatant of gastric cancer cells, while we used the ExoQuick kit and ultracentrifugation to purify exosomes from human serum samples. Furthermore, exosomes were isolated and purified from human urine samples by diafiltration and from postparturition human breast milk samples by the filtration-polyethylene glycol precipitation method. The isolated exosomes were morphologically analyzed using a transmission electron microscope, the particle size was measured by NanoSight, and the protein content was analyzed by western blotting. Results. The isolated exosomes showed an obvious cup holder shape, with a clear outline and typical exosome morphological characteristics. The sizes of exosomes derived from gastric cancer cell supernatant, serum, urine, and milk were $65.8\pm 26.9$  nm, $87.6\pm 50.9$  nm, $197.5\pm 55.2$  nm, and $184.1\pm 68.7$  nm, respectively. Western blot results showed that CD9 and TSG101 on the exosomes were expressed to varying degrees based on the exosome source. Exosome abundance was higher in the serum, urine, and breast milk than in the supernatant. It is suggested that its exosomes can be extracted to obtain an excellent potential biological source of exosomes. Conclusion. In this study, the extraction and separation methods of foreign bodies from different biological samples were obtained, and it was found that human breast milk was a potential excellent material for administration because of its high abundance.