Abstract
Mesenchymal stem cell (MSC) exosomes are intracellular vesicles, which can regulate transcription and control gene expression through the molecules they carry, easily enter into the target cell, contain no regenerative effect, and do not produce an immune response. There are different methods in the literature to obtain these vesicles. However, studies on the isolation of MSC-derived exosomes and their comparative characterization using magnetically active cell sorting (MACS) and ultracentrifugation methods are lacking. The most appropriate isolation method for MSC-derived exosomes can be determined by comparing the isolation and characterization parameters of mesenchymal stem cells using magnetically active cell sorting and ultracentrifugation methods. The aim of this study was to define the advantages and disadvantages of the methods used for determining the purpose-oriented method. Human bone marrow-derived mesenchymal stem cells were cultured in standard MSC culture conditions (37ºC and 5% CO 2). Exosomal contamination was prevented by removal of exosomes from the serum that used in the standard growth medium. For exosome isolation of the cells reaching sufficient density, the media were replaced with new ones every two days, the old media were collected in liquid refrigerated with liquid nitrogen and stored at −80ºC. Part of the accumulated exosomes were isolated by using the MACS method, while the other was isolated by using the ultracentrifugation method, which included serial centrifugation steps. The amount of protein contained in the phosphate buffer solution in which the exosomes were reconstituted was determined by microplate reader using the BCA kit. Based on the protein concentration obtained, exosomes were read by means of a dye flow cytometer with fluorescent antibodies attached to surface markers specific to CD9, CD63, and CD81 specific for exosomes by latex beads. Finally, the exosomes were stained with uranyl acetate and phosphotungstic acid and then placed on 200 mesh and formvar-carbon film coated grids. Exosomes were isolated using both ultracentrifugation and MACS methods. While ultra-large amounts of exosomes can be isolated by ultracentrifugation method, MACS method provides a lower amount of isolation. Exosomes with magnetically active cell sorting are selected with specific surface markers, therefore, exosomal purity is thought to be higher. Exosomes which were isolated by both ultracentrifugation and MACS methods were monitored by using transmission electron microscopy and they were not found to be morphologically different. In conclusion, MACS and ultracentrifugation are effective methods for the isolation of human bone marrow-derived MSC exosomes. Both methods have advantages and disadvantages. Exosomes can be isolated together with magnetic beads using the MACS method. In the ultracentrifuge method, cleaner exosomes can be isolated. While the exosomes are isolated by MACS, they can also be characterized by beads.
