Purity and yield of melanoma exosomes are dependent on isolation method

Abstract

Both exosomes and soluble factors have been implicated in the generation of an immunosuppressive tumour microenvironment. Determining the contribution of each requires stringent control of purity of the isolated analytes. The present study compares several conventional exosome isolation methods for the presence of co-enriched soluble factors while isolating exosomes from human melanoma-derived cell lines. The resultant preparations were analysed by multiplex bead array analysis for cytokine profiles, and by electron microscopy and nanotracking analysis for exosome size distribution and concentration. It is demonstrated that the amount and repertoire of soluble factors in exosome preparations is dependent upon the isolation method used. A combination of ultrafiltration and size exclusion chromatography yielded up to 58-fold more exosomes than ultracentrifugation, up to 836-fold lower concentrations of copurified soluble factors when adjusted for exosome yield, and a greater than twofold increase in PD-L1 expressing exosomes. Mechanistically, in context of the immunomodulatory effects of exosomes, the exosome isolation method should be carefully considered in order to limit any effects due instead to co-eluted soluble factors.

Optimized method for extraction of exosomes from human primary muscle cells

“…Several commercial kits have been developed to improve isolation efficacy and speed. The purity of vesicles isolated using these kits is often questioned in comparison to the ultracentrifugation methodology, especially when extracting from serum/plasma [49,50], but also in the in vitro context [51,52]. However, it is important to note that, whilst these studies do adhere strictly to the manufacturers' instructions for usage of the kits, they often fail to carry out identical sample preparations prior to the comparison -for example, carrying out centrifugations and/or filtration steps to remove microvesicles and other contaminants before ultracentrifugation but neglecting to do so before using the kits.…”

“…These additional steps removed the surplus of polymer [58], thereby rescuing the detection of exosomal markers (Fig. 3), and likely have the additional advantage of removing any cytokines [51] secreted by muscle cells.…”

Optimized method for extraction of exosomes from human primary muscle cells

“…Several commercial kits have been developed to improve isolation e cacy and speed. The purity of vesicles isolated using these kits is often questioned in comparison to the ultracentrifugation methodology, especially when extracting from serum/plasma [56,57], but also in the in vitro context [58,59]. However, it is important to note that, whilst these studies do adhere strictly to the manufacturers' instructions for usage of the kits, they often fail to carry out identical sample preparations prior to the comparison -for example, carrying out centrifugations and/or ltration steps to remove microvesicles and other contaminants before ultracentrifugation but neglecting to do so before using the kits.…”

“…These additional steps removed the surplus of polymer [65], thereby rescuing the detection of exosomal markers (Fig. 3), and likely have the additional advantage of removing any cytokines [58] secreted by muscle cells. These extra rinsing step may also improve the functionality of the exosome-like vesicles, for experiments involving the incorporation of vesicles into recipient cells (Fig.…”

Application of peptides with an affinity for phospholipid membranes during the automated purification of extracellular vesicles

“…To investigate the physicochemical and biochemical properties of EVs, a number of techniques, including ultracentrifugation-, size-, and immunoaffinity capture-based methods, have been developed for EV isolation. The results of EV isolation using multiple techniques suggest that the functional outcomes of EVs are technique-dependent 33 . The amount of EVs collected using the standard ultracentrifugation method is not consistent because the small, fragile EV pellets present after centrifugation can easily be lost during the decantation step 20 , 34 , and this further depends on the rotor type (fixed angle or swinging bucket) and even on the angle of the rotor 35 .…”