Cryogenic-Temperature Electron Microscopy Imaging of Extracellular Vesicles During and After Shedding

Naama Koifman, Chemical Engineering and the Russell Berrie Nanotechnology Institute, Technion, Israel Institute of Technology, Haifa, Israel
Idan Biran, Chemical Engineering And The Russell Berrie Nanotechnology Institute, Technion, Israel Institute Of Technology, Haifa, Israel
Anat Aharon, The Bruce Rappaport Faculty Of Medicine, Technion, Israel Institute Of Technology, Haifa, Israel
Benjamin Brenner, Department Of Hematology And Bone Marrow Transplantation, The Rambam Health Care Campus, Haifa, Israel
Yeshayahu Talmon, Chemical Engineering And The Russell Berrie Nanotechnology Institute, Technion, Israel Institute Of Technology, Haifa, Israel

Cryogenic scanning electron microscopy (cryo-SEM) is a unique imaging technique, by which cells can be imaged at a high resolution avoiding the addition of fixatives or contrast agents. Cryo-SEM is highly advantageous for imaging shedding cell membranes, which remain unaltered during specimen preparation, thus generating a more accurate and reliable morphological analysis. Cryogenic transmission electron microscopy (cryo-TEM) is tremendously useful in imaging extracellular vesicles (EVs), the products of cellular shedding, covering the whole range of EV diameters, while preserving their nanostructure.
The human leukemia monocytic cell line (THP1) is known to shed EVs under various stimulations. We study the effect of stimulation by exposure to the endotoxin lipopolysaccharide (LPS) or by starvation on THP1.
Unstimulated and stimulated cells were thermally fixed by high-pressure freezing, and imaged by cryo-SEM. EVs isolated from unstimulated and stimulated cells were imaged by cryogenic transmission electron microscopy (cryo-TEM). We also characterized the isolated EVs by nanoparticle tracking analysis (NTA).
Cryo-SEM images show blebbing of cells stimulated by LPS, which is in good agreement with previously suggested models. Micrographs show extensive membrane blebbing, as round, vesicular invaginations. Cells that underwent a 48-hour starvation stimulation exhibited a different morphology. EV morphologies were shown to be highly heterogenous in size and nanostructure. EVs isolated from cells undergoing starvation were fewer and larger than EVs isolated from LPS-stimulated cells.
Although both starvation and endotoxin-exposure are common stimulations, they most probably lead to a different cellular response, resulting in differences in size and concentration of the isolated EVs.