A novel "salting-out" procedure for the isolation of tumor-derived exosomes.

The last decade has seen an exponential growth in the number of exosome-related publications. Although many of these studies have used exosomes from biological fluids (blood, and ascites or pleural effusions) the vast majority employed vesicles isolated from large volumes of tissue culture supernatants. While several techniques are available for their isolation, all require a significant reduction in volume to obtain sufficient concentrations for study. One approach is to concentrate the medium before proceeding with their isolation, however, these procedures are very time consuming and require specialized laboratory equipment. Here we provide a new and effective method for the isolation of tumor-derived exosomes based on "charge neutralization" with acetate. We show that titration of tissue culture supernatants with 0.1M acetate to pH4.75 results in immediate precipitation of virtually all the exosomes. The precipitated exosomes can be washed to remove residual media and are readily "resolubilized" upon resuspension in acetate-free buffer at neutral pH. This simple cost effective method significantly increases the yield of exosomes from an unlimited quantity of culture supernatants. Exosomes isolated by this technique are indistinguishable from exosomes recovered by direct ultracentrifugation.