Cells release different types of membrane-bound vesicles in the extracellular environment. These membrane-bound vesicles are called extracellular vesicles (EVs), including exosomes and microvesicles. Extracellular vesicles are relatively small extracellular vesicles (30-150nm) that can transfer biomolecules such as nucleic acids, proteins, enzymes, and lipids between cells, and are an important mode of intercellular communication. In addition, they can be used as biomarkers for various diseases and as natural drug delivery systems for next-generation therapeutic agents.

In this article, we describe a method for rapidly and easily isolating extracellular vesicles from adipose derived stem cells by combining high-speed and ultracentrifugation. Cells in BioBLU ® Cultivate in a 0.3c disposable bioreactor and use DASbox ® Mini bioreactor system control. The DASbox mini bioreactor system can cultivate a large number of stem cells to obtain high-yield extracellular vesicles. Subsequently, we used a CR22N centrifuge to remove the conditioned medium. This is a high-speed centrifuge with an RCF of 32300 x g. In this article, we used R15A fixed angle rotation that can accommodate both 10 x 50 mL and 10 x 15 mL conical bottom tubes simultaneously. To further concentrate the extracellular vesicles, we used a CP100NX ultracentrifuge with a maximum RCF of 803000 x g and a P32ST horizontal rotor capable of accommodating 6 x 40 mL centrifuge tubes. In addition, compared to the control group without using sucrose buffer, pure and uniform extracellular vesicles can be quickly separated by using sucrose buffer in the ultracentrifugation step. Throughout the centrifugation process, we used a large capacity rotor to reduce repetitive centrifugation steps and shorten the extracellular vesicle separation time to less than 4 hours, faster than other separation methods such as precipitation or filtration.