Unveiling the Native Morphology of Extracellular Vesicles from Human Cerebrospinal Fluid by Atomic Force and Cryogenic Electron Microscopy

Abstract

Extracellular vesicles (EVs) are membranous structures in biofluids with enormous diagnostic/prognostic potential for application in liquid biopsies. Any such downstream application requires a detailed characterization of EV concentration, size and morphology. This study aimed to observe the native morphology of EVs in human cerebrospinal fluid after traumatic brain injury. Therefore, they were separated by gravity-driven size-exclusion chromatography (SEC) and investigated by atomic force microscopy (AFM) in liquid and cryogenic transmission electron microscopy (cryo-TEM). The enrichment of EVs in early SEC fractions was confirmed by immunoblot for transmembrane proteins CD9 and CD81. These fractions were then pooled, and the concentration and particle size distribution were determined by Tunable Resistive Pulse Sensing (around 10¹⁰ particles/mL, mode 100 nm) and Nanoparticle Tracking Analysis (around 10⁹ particles/mL, mode 150 nm). Liquid AFM and cryo-TEM investigations showed mode sizes of about 60 and 90 nm, respectively, and various morphology features. AFM revealed round, concave, multilobed EV structures; and cryo-TEM identified single, double and multi-membrane EVs. By combining AFM for the surface morphology investigation and cryo-TEM for internal structure differentiation, EV morphological subpopulations in cerebrospinal fluid could be identified. These subpopulations should be further investigated because they could have different biological functions.

Keywords: atomic force microscopy; cerebrospinal fluid; cryogenic transmission electron microscopy; extracellular vesicles; morphology; size-exclusion chromatography.

Figure 1

Tetraspanins CD9 and CD81 were present in the EV pool. Slot blot of the CSF pool and the eluates in the gravity-driven size-exclusion chromatography (SEC), with Ponceau S staining (A) and immunodetection based on CD9 (B) and CD81 (C); the fractions that composed the EV pool and the protein pool are marked with rectangles. Ponceau S staining of CSF pool, EV pool and protein pool separated by SDS-PAGE (D) and Western blot on albumin, CD9 and CD81 of CSF pool, EV pool and protein pool. Sizes of detected proteins are indicated in kilodaltons, kDa (E). CSF: cerebrospinal fluid; C: cerebrospinal fluid pool; EV: extracellular vesicle; SDS: sodium dodecyl sulfate; PAGE: polyacrylamide gel electrophoresis.

Figure 2

Concentrations in the EV pool obtained by Nanoparticle Tracking Analysis (NTA) and Tunable Resistive Pulse Sensing (TRPS) measured with nanopores NP150 and NP400 and combined, together with Atomic Force Microscopy (AFM).

Figure 3

Size distribution histograms of EV pool obtained by NTA (A); TRPS with NP150 (B), with NP400 (C) and combined (D); AFM in liquid (E); and cryo-TEM (F).

Figure 4

Near-native morphologies of EVs with preserved lumens. 3D AFM images of particles in the EV pool (row I) with different shapes and structures: multilobed (A), round (B), elongated bulging (C), single-lobed flat (D) and flat (E); dimensions in nm. Cryo-TEM images of different EV structures present in the EV pool (row II) with different structures marked with white arrows: onion-like internal structures (A), one or more vesicle inside one EV (B), which could have an electron-dense lumen (C), a deformed inner vesicle (D), two or more membranes (E). Bar 100 nm.

Figure 5

Near-native morphology of EVs with a collapsed lumen present in the EV pool. 3D AFM images of EVs (row I) with different cup-shaped structures (A,B), and partially open cup-shaped (C); dimensions in nm. Cryo-TEM images of different EVs (row II) with a single membrane bilayer (A); some appeared with an electron-dense lumen (B) or with damaged membrane (C). Bar 100 nm.