Isolation of exosomes by differential centrifugation: Theoretical analysis of a commonly used protocol

TLDR

Exosomes (40–100 nm) are of great interest as disease markers and therapeutic delivery vehicles, yet differential centrifugation often yields inconsistent results because rotor type and K‑factor adjustments are not universally applicable. The study seeks to derive theoretical expressions for the proportion of pelleted vesicles and the cut‑off size as functions of centrifugation force, time, and rotor path length for both swinging‑bucket and fixed‑angle rotors. Using these expressions, the authors created a web‑based calculator that allows users to determine optimal centrifugation parameters for any rotor. Theoretical cut‑off size predictions accurately guided the selection of centrifugation conditions, and experimental NTA of HT29‑derived exosomes confirmed that the resulting concentration and size distributions matched the theory.

Abstract

Abstract Exosomes, small (40–100 nm) extracellular membranous vesicles, attract enormous research interest because they are carriers of disease markers and a prospective delivery system for therapeutic agents. Differential centrifugation, the prevalent method of exosome isolation, frequently produces dissimilar and improper results because of the faulty practice of using a common centrifugation protocol with different rotors. Moreover, as recommended by suppliers, adjusting the centrifugation duration according to rotor K-factors does not work for “fixed-angle” rotors. For both types of rotors – “swinging bucket” and “fixed-angle” – we express the theoretically expected proportion of pelleted vesicles of a given size and the “cut-off” size of completely sedimented vesicles as dependent on the centrifugation force and duration and the sedimentation path-lengths. The proper centrifugation conditions can be selected using relatively simple theoretical estimates of the “cut-off” sizes of vesicles. Experimental verification on exosomes isolated from HT29 cell culture supernatant confirmed the main theoretical statements. Measured by the nanoparticle tracking analysis (NTA) technique, the concentration and size distribution of the vesicles after centrifugation agree with those theoretically expected. To simplify this “cut-off”-size-based adjustment of centrifugation protocol for any rotor, we developed a web-calculator.