Production and purification of exosomes

Since exosomes mediate MHC class I- and class II-restricted T-cell stimulatory capacity (29-31) and efficiently substitute for whole DC cultures (5), good manufacturing process (GMP) laboratory procedures for exosome harvesting and purification have been set up for clinical implementations (32). Exosomes derived from DC and tumor cell culture supernatants can be readily purified within 4-5 h starting from 2-3 liters of culture supernatant based on their physical properties. Exosomes float on sucrose gradients at a density ranging from 1.13 (for B-cell-derived exosomes) to 1.210 g/cm3 (for dexosomes/texosomes/texas) (4,6,11,25,33,34). Ultrafiltration of the clarified culture supernatant through a 500-kDa hollow fiber membrane followed by ultracentrifugation onto a 30% sucrose/deuterium oxide cushion (density: 1.13-1.210 g/cm3) reduced the volume and protein concentration approx 200-fold and 1000-fold, respectively. The percentage recovery of exosomes is about 40-50%, based on the exosomal MHC class

II concentration of the starting clarified supernatant. This methodology was extended to a miniscale process with comparable results. Likewise, the classical sedimentation technique is a tedious and less productive process carrying over along with exosomes contaminants of the culture medium. Furthermore, the development of quality control assays allowed qualitative and quantitative standardization of exosome preparations. Immunocapture assays assessing exosomal contents in MHC class I and II molecules as well as FACS determination of exosomal protein patterns after coupling onto macroscopic beads are currently used to calibrate exosome dosages in the first phase I trial (24). Exosomes are secreted from monocyte-differentiating DCs from Day 5 to Day 6 using culture conditions devoid of maturing agents (granulocyte-macrophage colony-stimulating factor [GM-CSF]/interleukin-4 [IL-4]). The amounts of exosomal MHC class I molecules recovered in a 24-h DC culture supernatant using such a purification process were evaluated. From 2.1011 to 1012 exosomal MHC class I molecules were found in the pellets of 3.107-108 immature human DC culture/24 h. From 1014to 1015 exosomal MHC class II molecules were recovered from GMP human DC cultures (5 x 108- 109 cells) derived from a single leukopheresis in metastatic melanoma patients (35).

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