Isolation and characterization of exosomes seperated from stem cells by ultra-centrifuge method

In recent years, Mesenchymal stem cell functions have been at the center of attention of researchers. Evidence shows that Mesenchymal stem cells act as paracrine manner and thus, biological agents such as exosomes and soluble factors are present in the supernatant that can act as same as Mesenchymal stem cells. Therefore, isolating exosomes from mesenchymal stem cells and determining their properties and their validation can be used to achieve therapeutic goals in autoimmune diseases and cancers. The use of exosomes isolated from soup and supernatant mesenchymal stem cell culture does not have any of the risks and problems of cell therapy, and also it is more effective than cell culture
In this study, adipose-derived Mesenchymal stem cells were used, after the identification of Mesenchymal stem cells, Mesenchymal stem cells were separated from supernatant using the ultra-centrifugation method. Concentration is determined by Bradford method and also their qualitative properties were determined using an electron microscope and Dynamic Light Scattering.
after culturing and passaging Mesenchymal stem cells, supernatant were collected after 48 hours and its exosomes were separated with a diameter of 40 to 100 nm using an ultra-centrifuge with an approximate acceleration of 60000g. Its Average concentration was 630 mg / ml, and the electron microscope images show that the membrane was not damaged during the separation process and spherical structure was maintained. Analyzed results from the Light differential device shows that almost 70% of the particles have an average diameter of 50-90 nm.
Comparing the two methods of isolating with acceleration of 100,000g and 60000g from the results obtained in other experiments, exosome particles are more pure using acceleration of 10000 g, but using the acceleration 60,000 g, larger particles of exosomes such as Microvesicle may be seen, but ultimately due to difficulty of accessing of a centrifuges with an approximate range of 100,000 gm, separation with the lower accelerations are also applicable. Also, the isolation of mesenchymal derived stem cell exosomes and its mass production may be considered as a bio-safe product in the treatment of diseases.