Isolation and quantification of human urinary exosomes by hydrophobic interaction chromatography on a polyester capillary-channeled polymer fiber stationary phase
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Exosomes are vesicles secreted by cells having a size range from 30 to 150 nm and carrying genetic materials that are important for intercellular functions, including cancer progression. Mounting evidence shows that tumor cells secrete more exosomes than normal cells. Thus, it is important to be able to efficiently isolate and quantify exosomes for potential use in clinical diagnostics, as well as to develop a deeper understanding of their role in intercellular processes. Current methods for exosome isolation and quantification are time-consuming and expensive. Few of these methods are able to combine exosome isolation and quantification into a singular operation scheme. However, a new efficient, rapid, and low-cost isolation and quantification method for exosomes in human urine samples using polyester (PET) capillary-channeled polymer (C-CP) fibers in a hydrophobic interaction chromatography (HIC) protocol has been developed. The process has been verified via scanning electron microscopy (SEM) before and after the capture of exosomes on the fiber surfaces. Sample load and elution rates were optimized to affect high resolution and throughput. Isolated exosomes were quantified based on a UV absorbance response curve created using a commercial human urine-derived exosome standard with an exosome concentration of 7.32 × 1011 mL−1. The loading capacity of a 30-cm C-CP PET column was ~ 7 × 1011 exosomes. An inter-injection washing method with PBS was developed to improve the reproducibility with a 2.9% RSD achieved for 7 complete isolation cycles.
KeywordsExosomes Urine Hydrophobic interaction chromatography Capillary-channeled polymers Fibers
The Gibson Foundation, the Prisma Health ITOR Biorepository, and the Greenville Hospital System are gratefully acknowledged. Special thanks are given to Dr. Larry Puls, Lori Allen, and Tina Pettry of Prisma Health for urine collection and storage. Matthew Haney and the Nanomedicines Characterization Core Facility at The University of North Carolina at Chapel Hill are gratefully acknowledged for completion of the NanoSight analysis. Special thanks are given to George Wetzel, Clemson University Electron Microscopy Facility, for assistance with EM; and to Tyler Slonecki for transportation and training.
This study received financial support for the chromatography development efforts from the National Science Foundation, Division of Chemistry, under grant CHE-1608663, and received financial support for the exosome isolation efforts from the Eppley Foundation for Scientific Research.
Compliance with ethical standards
The authors declare that they have no conflict of interest. All of the studies conducted in this manuscript were approved prior to their execution by the Clemson University Institutional Biosafety Committee, and have been performed in compliance with those approved protocols. All human samples were obtained via approved protocols and with informed patient consent as approved and required by the Prisma Hospital System Institutional Review Board (IRB). All patient samples were appropriately blinded for human subject protection.
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