Automated Cryo‐EM and Supervised Machine Learning Enable Reproducible Characterization of Extracellular Vesicles and Co‐Isolating Particles

Journal of Extracellular Vesicles, vol. 15, pp. e70273

Abstract

Cryo-electron microscopy (cryo-EM) is the only technique capable of visualizing the lipid bilayer of extracellular vesicles (EVs), enabling their distinction from non-EV particles. However, the application of cryo-EM for EV sample characterization has been limited by a combination of low imaging throughput and complex image analysis of the structurally diverse EVs. To address these challenges, we developed a workflow combining automated cryo-EM image acquisition with supervised machine learning (sML)-assisted particle detection and classification. Automated image acquisition facilitates the routine acquisition of thousands of cryo-EM images with consistent quality, enabling the imaging of hundreds of EVs. sML-assisted particle detection enabled efficient and reproducible identification, size measurement, and structural classification of EVs. Furthermore, using sML we are able to differentiate EVs from non-EV particles, such as lipoproteins and protein aggregates, which might co-isolate due to overlapping physical properties or by physical association with EVs. In mixed EV-lipoprotein samples, we demonstrate that our pipeline can distinguish EVs and differentiate between high-density (HDL), low-density (LDL), and very low-density (VLDL) lipoproteins. Our automated cryo-EM and sML workflow overcomes key limitations of EV characterization using cryo-EM by increasing imaging throughput and enabling reproducible EV characterization. Furthermore, this method provides a tool for analysing EV heterogeneity, sample purity, and co-isolated contaminants, advancing the field of EV research.

Authors 8

1. Agustin Enciso‐Martinez corresponding Aachen Oncode Institute Department of Cell and Chemical Biology Leiden University Medical Center Leiden the Netherlands

   Leiden University Medical Center · Amsterdam UMC Location University of Amsterdam · Oncode Institute · Amsterdam University Medical Centers · Cancer Center Amsterdam · Amsterdam Cardiovascular Sciences · University of Amsterdam

   Affiliation as printed

   Biomedical Engineering & Physics; Amsterdam Cardiovascular Sciences, Cancer Center Amsterdam Amsterdam UMC, University of Amsterdam Amsterdam The Netherlands

   Laboratory of Experimental Clinical Chemistry Laboratory Specialized Diagnostics & Research Department of Laboratory Medicine Amsterdam UMC, University of Amsterdam Amsterdam The Netherlands

   Oncode Institute and Department of Cell and Chemical Biology Leiden University Medical Center Leiden The Netherlands

2. Frank G. A. Faas Aachen Electron Microscopy Facility Department of Cell and Chemical Biology Leiden University Medical Center Leiden the Netherlands

   Leiden University Medical Center

   Affiliation as printed

   Electron Microscopy Facility, Department of Cell and Chemical Biology Leiden University Medical Center Leiden The Netherlands

3. A van der Meulen - de de Jong Aachen Electron Microscopy Facility Department of Cell and Chemical Biology Leiden University Medical Center Leiden the Netherlands

   Leiden University Medical Center

   Affiliation as printed

   Electron Microscopy Facility, Department of Cell and Chemical Biology Leiden University Medical Center Leiden The Netherlands

4. Ton G. J. M. van Leeuwen

   Amsterdam UMC Location University of Amsterdam · Amsterdam University Medical Centers · University of Amsterdam

   Affiliation as printed

   Laboratory of Experimental Clinical Chemistry Laboratory Specialized Diagnostics & Research Department of Laboratory Medicine Amsterdam UMC, University of Amsterdam Amsterdam The Netherlands

5. Rienk Nieuwland

   Amsterdam UMC Location University of Amsterdam · Amsterdam University Medical Centers · University of Amsterdam

   Affiliation as printed

   Laboratory of Experimental Clinical Chemistry Laboratory Specialized Diagnostics & Research Department of Laboratory Medicine Amsterdam UMC, University of Amsterdam Amsterdam The Netherlands

6. Edwin van der Pol

   Amsterdam UMC Location University of Amsterdam · Amsterdam University Medical Centers · Cancer Center Amsterdam · Amsterdam Cardiovascular Sciences · University of Amsterdam

   Affiliation as printed

   Biomedical Engineering & Physics; Amsterdam Cardiovascular Sciences, Cancer Center Amsterdam Amsterdam UMC, University of Amsterdam Amsterdam The Netherlands

   Laboratory of Experimental Clinical Chemistry Laboratory Specialized Diagnostics & Research Department of Laboratory Medicine Amsterdam UMC, University of Amsterdam Amsterdam The Netherlands

7. Peter ten Dijke Aachen Oncode Institute Department of Cell and Chemical Biology Leiden University Medical Center Leiden the Netherlands

   Leiden University Medical Center · Oncode Institute

   Affiliation as printed

   Oncode Institute and Department of Cell and Chemical Biology Leiden University Medical Center Leiden The Netherlands

8. Roman I. Koning corresponding Aachen Electron Microscopy Facility Department of Cell and Chemical Biology Leiden University Medical Center Leiden the Netherlands

   Leiden University Medical Center

   Affiliation as printed

   Electron Microscopy Facility, Department of Cell and Chemical Biology Leiden University Medical Center Leiden The Netherlands