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

Abstract

Exosomes, a subset of the extracellular vesicle (EV) group of organelles, hold great potential for biomarker detection, therapeutics, disease diagnosis, and personalized medicine applications. The promise and potential of these applications are hindered by the lack of an efficient means of isolation, characterization, and quantitation. Current methods for exosome and EV isolation (including ultracentrifugation, microfiltration, and affinity-based techniques) result in impure recoveries with regard to remnant matrix species (e.g., proteins, genetic material) and are performed on clinically irrelevant time and volume scales. To address these issues, a polyethylene terephthalate (PET) capillary-channeled polymer (C-CP) fiber stationary phase is employed for the solid-phase extraction (SPE) of EVs from various matrices using a micropipette tip-based format. The hydrophobic interaction chromatography (HIC) processing and a spin-down workflow are carried out using a table-top centrifuge. Capture and subsequent elution of intact, biologically active exosomes are verified via electron microscopy and bioassays. The performance of this method was evaluated by capture and elution of exosome standards from buffer solution and three biologically relevant matrices: mock urine, reconstituted non-fat milk, and exosome-depleted fetal bovine serum (FBS). Recoveries were evaluated using UV-Vis absorbance spectrophotometry and ELISA assay. The dynamic binding capacity (50%) for the 1-cm-long (~ 5 μL bed volume) tips was determined using a commercial exosome product, yielding a value of ~ 7 × 10 11 particles. The novel C-CP fiber spin-down tip approach holds promise for the isolation of exosomes and other EVs from various matrices with high throughput, low cost, and high efficiency. Graphical abstract Electronic supplementary material The online version of this article (10.1007/s00216-020-02728-z) contains supplementary material, which is available to authorized users.

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

“…Marcus and coworkers have demonstrated an exosome isolation method using poly(ethylene terephthalate) (PET) capillary‐channeled polymer (C‐CP) fibers in a hydrophobic interaction chromatography (HIC) method 25‐28 . Previous works have employed C‐CP fibers extensively in intact protein separations via various modalities 29‐33 .…”

“…HIC is a gentler method than its sister reversed phase chromatography where organic solvent elution is employed, maintaining the structural integrity and biological activity of the EVs. Based on our initial method, 25 subsequent efforts have affected isolation and quantification of EVs from complex biological fluids, including human urine, 25,27,28 human plasma, 26 reconstituted milk, 28 and cell culture milieu 25 . The efficacy of the C‐CP fiber HIC approach relative to the physical morphology and the retention of the biological activity of the vesicular surfaces has been validated by electron microscopy, NTA, dot blots, western blots, and immunoaffinity fluorescence measurements 28 .…”

“…Marcus and coworkers have demonstrated an exosome isolation method using poly(ethylene terephthalate) (PET) capillary-channeled polymer (C-CP) fibers in a hydrophobic interaction chromatography (HIC) method. [25][26][27][28] Previous works have employed C-CP fibers extensively in intact protein separations via various modalities. [29][30][31][32][33] HIC is a mode of separation in which molecules (usually proteins) in a high salt concentration environment interact hydrophobically with the lesspolar surfaces of the stationary phase 34 ; a variant of the common "salting out" mode of protein purification.…”

“…25 The efficacy of the C-CP fiber HIC approach relative to the physical morphology and the retention of the biological activity of the vesicular surfaces has been validated by electron microscopy, NTA, dot blots, western blots, and immunoaffinity fluorescence measurements. 28 Online and postisolation quantification is readily performed based on UV absorbance response curves created using commercial exosome standards. These efforts provide a relatively easy, low cost, and efficient means for EV isolation and quantification using a simple high performance liquid chromatography (HPLC) platform.…”

“…This concept has been successfully demonstrated in this laboratory in the isolation of EVs from human plasma, mock urine, and reconstituted milk. 26,28 In this report, we describe an efficient, low-cost, and gentle isolation and quantification method for EVs derived from Dictyostelium discoideum cell culture milieu using PET C-CP fibers in a glycerol assisted HIC protocol. D. discoideum is a nonpathogenic eukaryotic microorganism, which offers a simple model system of individual eukaryotic cells during growth.…”