Extracellular vesicle and particle isolation from human and murine cell lines, tissues, and bodily fluids

Abstract

We developed a modified protocol, based on differential ultracentrifugation (dUC), to isolate extracellular vesicles and particles (specifically exomeres) (EVPs) from various human and murine sources, including cell lines, surgically resected tumors and adjacent tissues, and bodily fluids, such as blood, lymphatic fluid, and bile. The diversity of these samples requires robust and highly reproducible protocols and refined isolation technology, such as asymmetric-flow field-flow fractionation (AF4). Our isolation protocol allows for preparation of EVPs for various downstream applications, including proteomic profiling. For complete details on the use and execution of this protocol, please refer to Hoshino et al. (2020).

Keywords: Cell Membrane; Cell culture; Cell isolation; Exomeres; Exosomes; Extracellular vesicles and particles; Isolation.

Graphical abstract

Figure 1

Workflow for EVP isolation from cell culture, tissue explants, and bodily fluids

Figure 2

Careful handling of the EVP pellet (A) After ultracentrifugation, carefully decant supernatant and (B and C) re-suspend the pellet in a small volume of PBS (0.5–1 mL depending on tube size) by pipetting and flushing the marked area of the lower side of the ultracentrifugation tube. Avoid generating bubbles.

Figure 3

Comparison of cell viability between whole tissue versus tissue dissected into smaller pieces (A) Nanosight (NTA) profiles and protein concentration by bicinchoninic acid (BCA) assay for human colon that was cultured as whole tissue or dissected into smaller pieces. (B) Immunoblotting of lysates (20 μg of proteins) from baseline tissue (directly after collection, timepoint 0 h) immediately placed into lysing buffer without culturing, whole tissue, and dissected tissue (after 12 h culture) derived from the human colon. PARP antibody (#9542, Cell Signaling Technology [CST]), cleaved caspase-3 antibody (#9661S, CST), GAPDH antibody (#2118S, CST) were used for immunoblotting. (C) Comparison of human colon whole tissue and dissected tissue cell viability as determined by dual Acridine Orange / Propidium Iodide fluorescence and brightfield optics, using the LUNA automated cell counter. Live cells are green and dead cells are red. (D) Comparison of human colon whole tissue and dissected tissue cell viability by apoptotic flow staining shows similar viability between baseline tissue directly after collection and whole and dissected tissues after 12 h tissue explant culture. (E) Comparison of cell viability between baseline and 24 h cultures of mouse lung tissue explant. (F) Cell viability, as determined by flow cytometry with LIVE/DEAD™ Fixable Blue Dead Cell Stain, shows increased viability in dissected tissue cultures compared to whole tissue culture from mice.

Figure 4

Ultracentrifugation of tissue sample EVPs over a sucrose/deuterium oxide density layer (A) EVPs re-suspended in PBS could be further purified by overlaying onto a sucrose/deuterium oxide cushion and centrifuging at 100,000 × g, resulting in a lower EVP-enriched layer that could be aspirated from the upper layer containing any potential contaminants. (B) Aspiration of the lower fraction using an 18G needle and 3 mL syringe.

Figure 5

Dissecting tissues into 2 mm³ pieces improves EVP yields without affecting EVP morphology (A) Nanosight (NTA) profiles for whole organs and tissue dissected into smaller pieces for murine normal lung and metastasis-bearing lung. (B) Transmission electron microscopy images of the same samples as in b, normal lung (upper row) and metastasis-bearing lung (lower row). (C and D) (C) Particle size for EVPs from normal lung and metastasis-bearing lung and (D) EVP protein yields from whole tissue versus tissue dissected into smaller pieces. n = 3, mean and SD is shown; ∗p < 0.05.

Figure 6

Representative AF4 separation of EVPs prepared from B16F10 cells using differential ultracentrifugation (A) Representative AF4 fractionation profile showing hydrodynamic radius (Rh) and fractograms of UV absorbance and QELS plotted against time. (B) Representative negative staining TEM images of combined fractions resolved by AF4 for corresponding exomeres, Exo-S, and Exo-L subpopulations. Colored arrows: representative EVPs in each subpopulation (adapted from Zhang et al., 2018).

Figure 7

Example of “contaminated” samples purified over a sucrose/deuterium oxide density layer Aggregates and insoluble material could contaminate the EVP pellet, especially in complex samples, such as tissue explants or bodily fluids. Purification methods, such as density cushion, could be performed to improve sample purity.