Comparison of extracellular vesicle isolation processes for therapeutic applications.

IF 6.7 1区 工程技术 Q1 CELL & TISSUE ENGINEERING Journal of Tissue Engineering Pub Date : 2023-05-23 eCollection Date: 2023-01-01 DOI:10.1177/20417314231174609
Soraya Williams, Maria Fernandez-Rhodes, Alice Law, Ben Peacock, Mark P Lewis, Owen G Davies
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Abstract

While extracellular vesicles (EVs) continue to gain interest for therapeutic applications, their clinical translation is limited by a lack of optimal isolation methods. We sought to determine how universally applied isolation methods impact EV purity and yield. EVs were isolated by ultracentrifugation (UC), polyethylene glycol precipitation, Total Exosome Isolation Reagent, an aqueous two-phase system with and without repeat washes or size exclusion chromatography (SEC). EV-like particles could be detected for all isolation methods but varied in their purity and relative expression of surface markers (Alix, Annexin A2, CD9, CD63 and CD81). Assessments of sample purity were dependent on the specificity of characterisation method applied, with total particle counts and particle to protein (PtP) ratios often not aligning with quantitative measures of tetraspanin surface markers obtained using high-resolution nano-flow cytometry. While SEC resulted in the isolation of fewer particles with a relatively low PtP ratio (1.12 × 107 ± 1.43 × 106 vs highest recorded; ATPS/R 2.01 × 108 ± 1.15 × 109, p ⩽ 0.05), EVs isolated using this method displayed a comparatively high level of tetraspanin positivity (e.g. ExoELISA CD63⁺ particles; 1.36 × 1011± 1.18 × 1010 vs ATPS/R 2.58 × 1010± 1.92 × 109, p ⩽ 0.001). Results originating from an accompanying survey designed to evaluate pragmatic considerations surrounding method implementation (e.g. scalability and cost) identified that SEC and UC were favoured for overall efficiency. However, reservations were highlighted in the scalability of these methods, which could potentially hinder downstream therapeutic applications. In conclusion, variations in sample purity and yield were evident between isolation methods, while standard non-specific assessments of sample purity did not align with advanced quantitative high-resolution analysis of EV surface markers. Reproducible and specific assessments of EV purity will be critical for informing therapeutic studies.

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比较用于治疗的细胞外囊泡分离过程。
尽管细胞外囊泡(EVs)在治疗方面的应用不断受到关注,但由于缺乏最佳的分离方法,其临床应用受到了限制。我们试图确定普遍应用的分离方法对EV纯度和产量的影响。我们采用超速离心法(UC)、聚乙二醇沉淀法、总外泌体分离试剂、带或不带重复洗涤的水性两相系统或尺寸排阻色谱法(SEC)分离了EV。所有分离方法都能检测到类 EV 颗粒,但其纯度和表面标志物(Alix、Annexin A2、CD9、CD63 和 CD81)的相对表达量各不相同。对样品纯度的评估取决于所用表征方法的特异性,总颗粒计数和颗粒与蛋白质(PtP)比率往往与使用高分辨率纳米流式细胞术获得的四泛素表面标记物的定量测量结果不一致。虽然 SEC 分离出的颗粒较少,PtP 比率相对较低(1.12 × 107 ± 1.43 × 106 vs 最高记录;ATPS/R 2.01 × 108 ± 1.15 × 109,p ⩽ 0.05),使用这种方法分离的 EVs 显示出相对较高的四泛素阳性水平(例如 ExoELISA CD63⁺ 颗粒;1.36 × 1011 ± 1.18 × 1010 vs ATPS/R 2.58 × 1010 ± 1.92 × 109,p ⩽0.001)。为评估与方法实施相关的实际考虑因素(如可扩展性和成本)而进行的配套调查结果显示,SEC 和 UC 在总体效率方面更受青睐。不过,这些方法在可扩展性方面还有所保留,可能会妨碍下游治疗应用。总之,不同分离方法的样本纯度和产量差异明显,而样本纯度的标准非特异性评估与先进的 EV 表面标记定量高分辨率分析不一致。EV纯度的可重复性和特异性评估对于为治疗研究提供信息至关重要。
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来源期刊
Journal of Tissue Engineering
Journal of Tissue Engineering Engineering-Biomedical Engineering
CiteScore
11.60
自引率
4.90%
发文量
52
审稿时长
12 weeks
期刊介绍: The Journal of Tissue Engineering (JTE) is a peer-reviewed, open-access journal dedicated to scientific research in the field of tissue engineering and its clinical applications. Our journal encompasses a wide range of interests, from the fundamental aspects of stem cells and progenitor cells, including their expansion to viable numbers, to an in-depth understanding of their differentiation processes. Join us in exploring the latest advancements in tissue engineering and its clinical translation.
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