Date of Award
8-2022
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Chemistry
Committee Chair/Advisor
Dr. R. Kenneth Marcus
Committee Member
Dr. Terri F. Bruce
Committee Member
Dr. Carlos Garcia
Committee Member
Dr. Byoungmoo Kim
Abstract
A number of recent works have emphasized the need to isolate nanometer-scale analytes, like extracellular vesicles (EVs), from various biologically-relevant fluids. Exosomes are a subset of small EVs that range from 30-200 nm in diameter that serve as biomolecular snapshots of their cell of origin containing mother cell-specific DNA, miRNA, mRNA, and proteins. As critical components of intercellular communication, exosomes and other EVs play significant roles in many physiological and pathological processes. Diverse populations of these vesicles can be collected from biofluids, including blood, saliva, and urine, from cell culture conditioned media and primary cells, and even from plant fluid stocks. With their characteristic vector-like activities and accessible collection from renewable sources, the large-scale processing of EVs from patient biofluids for clinical diagnostics and from plant fluids or high-yield bioreactors for use as therapeutic vectors has been previously proposed. However, these applications are limited by extremely impure, low-yield exosome recoveries, despite the large availability of exosome sources. Hence, an isolation method that provides high concentrations of pure, bioactive EVs from diverse sources on reasonable scales of time and cost is of much interest.
Employed in this work is a rapid EV isolation method using a hydrophobic interaction chromatography (HIC) workflow on a capillary-channeled polymer (C-CP) fiber spin-down tip. Here, EVs are isolated from several biofluid sources, including mock biofluid matrices, clinical patient biofluid samples, cellular milieu from mammalian and amoeba cell lines, and over 20 fruit and vegetable sample stocks. Representative populations of EVs are obtained using the C-CP tip method, where up to 12 samples are simultaneously processed in a standard tabletop centrifuge in less than 15 minutes. This batch solid-phase extraction technique allows up to 1 x 1012 EVs to be obtained from each μL-scale aliquot of the original biofluid. The tip-isolated EVs were characterized using transmission electron microscopy (TEM), multi-angle light scattering (MALS), nanoparticle tracking analysis (NTA), absorbance quantification, protein purity assay, and immunoassays to EV and source-specific proteins. The efficient HIC C-CP tip isolation method produces the required integrity and purity of recovered EVs to enable fundamental research to be performed and their therapeutic vector and clinical diagnostic potentials to be better explored.
Recommended Citation
Jackson, Kaylan, "Isolation and Characterization of Extracellular Vesicles from Various Biological Matrices using Capillary-Channeled Polymer (C-CP) Fiber Solid-Phase Extraction Spin-Down Tips" (2022). All Dissertations. 3066.
https://open.clemson.edu/all_dissertations/3066
Author ORCID Identifier
0000-0002-1159-3919