Isolation of biologically-active exosomes from human plasma

Abstract

Effects of exosomes present in human plasma on immune cells have not been examined in detail. Immunological studies with plasma-derived exosomes require their isolation by procedures involving ultracentrifugation. These procedures were largely developed using supernatants of cultured cells. To test biologic activities of plasma-derived exosomes, methods are necessary that ensure adequate recovery of exosome fractions free of contaminating larger vesicles, cell fragments and protein/nucleic acid aggregates. Here, an optimized method for exosome isolation from human plasma/serum specimens of normal controls (NC) or cancer patients and its advantages and pitfalls are described. To remove undesirable plasma-contaminating components, ultrafiltration of differentially-centrifuged plasma/serum followed by size-exclusion chromatography prior to ultracentrifugation facilitated the removal of contaminants. Plasma or serum was equally acceptable as a source of exosomes based on the recovered protein levels (in μg protein/mL plasma) and TEM image quality. Centrifugation on sucrose density gradients led to large exosome losses. Fresh plasma was the best source of morphologically-intact exosomes, while the use of frozen/thawed plasma decreased exosome purity but not their biologic activity. Treatments of frozen plasma with DNAse, RNAse or hyaluronidase did not improve exosome purity and are not recommended. Cancer patients' plasma consistently yielded more isolated exosomes than did NCs' plasma. Cancer patients' exosomes also mediated higher immune suppression as evidenced by decreased CD69 expression on responder CD4+ T effector cells. Thus, the described procedure yields biologically-active, morphologically-intact exosomes that have reasonably good purity without large protein losses and can be used for immunological, biomarker and other studies.

Keywords: Exosome characteristics; Exosome isolation; Human plasma; Immunological studies; Size-exclusion chromatography.

Figure 1

Schema of the isolation procedure for human plasma-derived exosomes.

Figure 2

Characterization of isolated exosomes. A) Western blot of isolated cancer plasma-derived exosomes floated on a continuous sucrose gradient (0.25–2.5M). Exosomes expressing CD81 are located in fractions 6–7. B) A representative TEM image of isolated exosomes. Exosomes range in size from 20–80nm. C) Flow cytometry of exosomes captured on beads. I. Ungated forward and side scatter of single and aggregated exosome-carrying beads. II. A representative histogram of exosomes (16μg). Note a clear right-shift of the CD9-FITC signal compared to the isotype control (light gray). The gate is set on single beads. III. A sigmoidal curve defines the relationship of exosome input (μg protein) and MIF values for the CD9-FITC signal. D) Exosomes isolated from plasma of a cancer patient were evaluated by NanoSight. The plot shows a broad size distribution (mean 102nm, SD ±46) and the concentration of cancer exosomes (5.6x10¹⁰/ml of plasma). The data are means of 6 measurements ± SE.

Figure 3

Effects of ultrafiltration and size-exclusion chromatography on isolated exosomes. A) TEM images of plasma-derived exosomes from a representative NC of 5 examined. Note the presence of thrombocytes in non-filtered plasma (upper image: low mag. and lower image: high mag.) B) TEM of epon-embedded and sectioned exosomes from plasma of a representative cancer patient isolated by size-exclusion chromatography (upper image) or omitting chromatography (lower image). Note increased purity of the exosomal preparation obtained after chromatography. C) Protein levels in exosomes isolated from plasma of patients with cancer (n=3) ± size-exclusion chromatography. The data are means ± SE.

Figure 4

Comparison of serum-derived with plasma-derived exosomes. A) TEM of serum-derived exosomes (upper image) or plasma-derived exosomes (lower image) obtained from the same cancer patient. Note the similar morphology of the isolated exosomes. B) The presence of platelet IIb/IIIa complex in exosomes (10ug) derived from serum (S1, S2) versus plasma (P1, P2) of two cancer patients as seen in a Western blot. An exosomal marker TSG101 and a housekeeping protein GAPDH were used as loading controls. C) A box plot shows no significant (N.S.) difference in total proteins of exosomal fractions isolated from paired serum or plasma specimens of the same 6 patients with cancer. Note a considerably larger SE in values for serum-derived exosomes than those isolated from plasma. The bar inside the box indicates the median, the box shows interquartile range (25–75%) and whiskers extend to 1.5 x the interquartile range.

Figure 5

Exosome quality and recovery before and after freezing of plasma. A) TEM of isolated exosomes before (upper image) and after a single freeze/thaw cycle (lower image). B) The increase in total protein levels of exosome fractions are higher when fresh plasma is frozen after low-speed centrifugation than in plasma frozen after differential centrifugation. The values are from 3 independent experiments with plasma of different NC. The data are presented as box plots (see the legend to Figure 4). C) Differences in morphology of exosomes in TEM (negative staining) from fresh (upper image) as compared to exosomes from frozen plasma samples examined after differential centrifugation (lower image). Plasma was obtained from a representative cancer patient. The arrows indicate exosomes surrounded by exogenous material.

Figure 6

Enzymatic treatments of frozen/thawed plasma. A) TEM (negative staining) of a representative frozen sample examined before (upper image) and after (lower image) treatment with DNAse and RNAse. Note decrease in the background and also a considerable loss of exosomes. B) Change in total exosomal protein levels ± enzyme treatment. A representative experiment of 3 independent experiments performed with samples of different NC. C) TEM (negative staining) of exosomes isolated from cancer patients’ plasma stored frozen for 7 years: without enzymatic treatment (upper image) and after treatment with h DNAse/RNAse or Heparinase (lower image).

Figure 7

Functional activity of isolated exosomes co-incubated with activated T effector cells. Flow cytometry showing the differential suppressive effects of TEX, cancer patient’s plasma-derived or NC exosomes on T effector cells (CD4+) represented by CD69-expression. Note the higher suppressive effect of cancer plasma-derived exosomes vs. NC exosomes. The curve in light gray represents the isotype control.

Figure 8

Levels of total protein in exosomes isolated as shown in Figure 1 from fresh plasma specimens obtained from NC or HNSCC patients with active disease (AD) or no evident disease after therapy (NED). n=3 for each group, * p<0.05 and ** p<0.01. These data are presented as box plots (see the legend to Figure 4).