Evaluation of exosome loading characteristics in their purification via a glycerol‐assisted hydrophobic interaction chromatography method on a polyester, capillary‐channeled polymer fiber phase

Abstract

Exosomes are membrane-secreted vesicles, with sizes ranging from 30 to 150 nm, which play key roles in intercellular communication. There is intense interest in developing methods to isolate and quantify exosomes toward clinical diagnostics, fundamental studies of intercellular processes, and use of exosomes as delivery vehicles for therapeutic agents. Current methods for exosomes isolation and quantification are time consuming and have operational high costs; few combine isolation and quantification into a singular operation unit. This report describes the use of hydrophobic interaction chromatography on a polyester capillary-channeled polymer fiber column, employing a step gradient for exosome elution, including use of glycerol as a solvent modifier. The entire procedure is completed in 8 min, while maintaining the structural integrity and biological activity of the isolated exosomes. Electron microscopy was used to verify the size and structural fidelity of single exosomes. Absorbance response curves for a commercial exosome sample were used for exosome quantification in the chromatographic separations. In order to determine the dynamic loading capacity for exosomes, different volumes of Dictyostelium discoideum cell culture milieu supernatant were loaded at different column lengths (5-30 cm) and loading flow rates (0.2-0.5 ml/min). A loading capacity of 5.4 × 10 12 exosomes derived from D. discoideum milieu was obtained on a 0.8 × 300 mm column; yielding recoveries of over 80%. It is believed that this isolation and purification strategy holds many advantages toward the use of exosomes across a wide breadth of medical and biotechnology applications. K E Y W O R D S capillary-channeled polymer fibers, exosomes, extracellular vesicles, hydrophobic interaction chromatography, purification 1 | INTRODUCTION Exosomes, a subset of extracellular vesicles (EVs), are membranesecreted vesicles, with sizes ranging from 30 to 150 nm, which exist in almost all cell types and body fluids and play key roles in intercellular communication and cancer progression. 1,2 Formed in the multivesicular body, exosomes encapsulate various species including proteins and genetic material, and carry along surface proteins representative of their source. Notably, tumor cells have been shown to produce and secrete exosomes in greater 3, 4 numbers than normal

Solid-phase extraction of exosomes from diverse matrices via a polyester capillary-channeled polymer (C-CP) fiber stationary phase in a spin-down tip format

“…Protein elution was induced by passage of 50 μL of 25% glycerol in PBS under the same centrifugation conditions, with the final elution of the captured EVs induced using 50 μL of 50% glycerol in PBS. The elution of proteins by 25% glycerol and exosomes by 50% glycerol has been confirmed by SEM imaging of the fiber surfaces after the various steps in this workflow as well as in the use of acetonitrile as the mobile phase modifier [42,43], as it is here. The eluted EVs were quantified by diluting a 3-μL aliquot to 1.5 mL with DI-H 2 O.…”

“…Though glycerol has been used as a biological preservative [66], it is not ideal for all downstream analyses (i.e., proteomic analysis) where necessary vesicle lysing may be hindered. In these cases, acetonitrile may be used as a substitute elution phase, as previously reported [42,43]. Essential to the quantification process of EVs in different matrices is the assumption that EVs may be quantitatively immobilized and recovered from the fiber surfaces.…”

“…The combination of high column permeability and low surface porosity provides high throughput and yield macromolecule separations [37][38][39]. Bruce, Marcus, and colleagues have also recently reported a method for exosome/ EV isolation using an HIC mode on a poly (ethylene terephthalate) (PET) C-CP fiber phase [40][41][42][43]. The use of the HIC elution strategy allows for exosome isolations based on the vesicles' inherent hydrophobicity (partially a function of their size), allowing non-destructive bulk recoveries of exosomes/EVs for further interrogation and applications.…”

Comparison of RNA content from hydrophobic interaction chromatography‐isolated seminal plasma exosomes from intrauterine insemination (IUI) pregnancies

“…Isolated fractions from the HPLC were collected using an R1 fraction collector (Teledyne Isco). PET C-CP columns were made according to previous study (Huang et al, 2020). The column was cut into a 5 cm segment for later use.…”

“…TEM imaging was used to verify the physical size and structural regularity of single exosomes and performed according to our previous studies (Bruce et al, 2019;Huang et al, 2020). The HIC methodeluted exosomes were negatively stained: 3.5 μl of sample was dropped on a carbon grid and rested for 5 min, and then filter paper was used to blot the fluids (Petersen et al, 2014)…”