| Autor: |
Chou CY; Institute of Applied Mechanics, National Taiwan University, 1 Roosevelt Road, Sec. 4, Taipei 106319, Taiwan., Chiang PC; Reliance Biosciences, Inc., New Taipei City 231023, Taiwan., Li CC; Graduate Institute of Biomedical Electronics and Bioinformatics, National Taiwan University, Taipei 106319, Taiwan., Chang JW; Institute of Applied Mechanics, National Taiwan University, 1 Roosevelt Road, Sec. 4, Taipei 106319, Taiwan., Lu PH; Institute of Applied Mechanics, National Taiwan University, 1 Roosevelt Road, Sec. 4, Taipei 106319, Taiwan., Hsu WF; Institute of Applied Mechanics, National Taiwan University, 1 Roosevelt Road, Sec. 4, Taipei 106319, Taiwan.; Reliance Biosciences, Inc., New Taipei City 231023, Taiwan., Chang LC; Department of Internal Medicine, National Taiwan University Hospital, Taipei 100225, Taiwan.; Health Management Center, National Taiwan University Hospital, Taipei 100225, Taiwan., Hsu JL; Department of Neurology, New Taipei Municipal TuCheng Hospital, New Taipei City 236017, Taiwan.; Department of Neurology, Chang Gung Memorial Hospital Linkou Medical Center and College of Medicine, Neuroscience Research Center, Chang-Gung University, Linkou, Taoyuan 33302, Taiwan.; Graduate Institute of Mind, Brain, & Consciousness, Taipei Medical University, Taipei 110301, Taiwan., Wu MS; Department of Internal Medicine, National Taiwan University Hospital, Taipei 100225, Taiwan., Wo AM; Institute of Applied Mechanics, National Taiwan University, 1 Roosevelt Road, Sec. 4, Taipei 106319, Taiwan.; Reliance Biosciences, Inc., New Taipei City 231023, Taiwan. |
| Abstrakt: |
Extracellular vesicles (EVs) are present in blood at much lower concentrations (5-6 orders of magnitude) compared to lipoprotein particles (LP). Because LP and EV overlap in size and density, isolating high-purity EVs is a significant challenge. While the current two-step sequential EV isolation process using size-expression chromatography (SEC) followed by a density gradient (DG) achieves high purity, the time-consuming ultracentrifugation (UC) step in DG hinders workflow efficiency. This paper introduces an optimized magnetic bead reagent, LipoMin, functionalized with glycosaminoglycans (GAGs), as a rapid alternative for LP removal during the second-step process in about 10 minutes. We evaluated LipoMin's efficacy on two sample types: (a) EV fractions isolated by size exclusion chromatography (SEC + LipoMin) and (b) the pellet obtained from ultracentrifugation (UC + LipoMin). The workflow is remarkably simple, involving a 10 min incubation with LipoMin followed by magnetic separation of the LP-depleted EV-containing supernatant. Results from enzyme-linked immunosorbent assay (ELISA) revealed that LipoMin removes 98.2% ApoB from SEC EV fractions, comparable to the LP removal ability of DG in the SEC + DG two-step process. Importantly, the EV yield (CD81 ELISA) remained at 93.0% and Western blot analysis confirmed that key EV markers, flotillin and CD81, were not compromised. Recombinant EV (rEV), an EV reference standard, was spiked into SEC EV fractions and recovered 89% of CD81 protein. For UC + LipoMin, ApoA1 decreased by 76.5% while retaining 90.7% of CD81. Notably, both colorectal cancer (CRC) and Alzheimer's disease (AD) samples processed by SEC + LipoMin and UC + LipoMin displayed clear expression of relevant EV and clinical markers. With a 10 min workflow (resulting in a 96% time saving compared to the traditional method), the LipoMin reagent offers a rapid and efficient alternative to DG for LP depletion, paving the way for a streamlined SEC + LipoMin two-step EV isolation process. |
