Biomedicines (May 2022)

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

  • Mario Kurtjak,
  • Sami Kereïche,
  • Damir Klepac,
  • Hrvoje Križan,
  • Marko Perčić,
  • Vedrana Krušić Alić,
  • Teja Lavrin,
  • Metka Lenassi,
  • Karmen Wechtersbach,
  • Nika Kojc,
  • Marija Vukomanović,
  • Siniša Zrna,
  • Maša Biberić,
  • Robert Domitrović,
  • Kristina Grabušić,
  • Mladenka Malenica

DOI
https://doi.org/10.3390/biomedicines10061251
Journal volume & issue
Vol. 10, no. 6
p. 1251

Abstract

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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.

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