干细胞和血清细胞外囊泡表征的局限性和挑战

IF 5.3 2区 化学 Q1 CHEMISTRY, ANALYTICAL
Sara Escudero-Cernuda, Noemi Eiro, María Fraile, Francisco J. Vizoso, Belén Fernández-Colomer, María Luisa Fernández-Sánchez
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引用次数: 0

摘要

外泌体是纳米级细胞外囊泡(ev)的一个亚群,由脂质双分子层形成,由细胞自然分泌。它们运输RNA、microrna、生物活性蛋白和脂质,并在细胞间通讯中发挥重要作用。外泌体在再生医学、免疫治疗和药物输送中是一种很有前途的无细胞治疗方法。实施新的外泌体治疗需要了解其浓度,纯度和完整的表征。然而,由于缺乏有效的分离和测定方法,以及缺乏表征良好的外泌体参考标准,比较不同的研究是极具挑战性的。在这项工作中,我们分离了人宫颈间充质干细胞(hUCESC) EVs,并对其进行了超离心鉴定,讨论了目前鉴定方法的局限性。首先,总蛋白测定受到游离蛋白和脂质污染的严重影响,这证实了采用几种方法测定水疱蛋白的必要性。这种纯度变化似乎受到囊泡起源的严重影响,因为更复杂的介质产生更多的基质干扰。大小排除高效液相色谱已被证明是一种新的方法,用于纯度评估hUCESC-EVs和商业EVs(脂肪干细胞和人血清)。结果发现商业外泌体的纯度较低,这表明商业ev必须包括蛋白质和脂质纯度。最后,将该方法与总蛋白测定相结合,证明了用囊泡蛋白浓度可以估计颗粒浓度。图形抽象
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Limitations and challenges in the characterization of extracellular vesicles from stem cells and serum

Exosomes are a subpopulation of nanosized extracellular vesicles (EVs), formed by a lipid bilayer and naturally secreted by cells. They transport RNA, microRNAs, bioactive proteins, and lipids and play an important role in intercellular communication. Exosomes are a promising alternative cell-free therapy in regenerative medicine, immunotherapy, and drug delivery. The implementation of new exosomes treatments requires knowledge of its concentration, purity, and full characterization. However, comparing different studies is highly challenging due to the lack of validated methodologies for isolation and determination, as well as a lack of well-characterized exosomes reference standards. In this work, human uterine cervical mesenchymal stem cell (hUCESC) EVs have been isolated by ultracentrifugation and characterized, discussing the current limitations of characterization methods. First, total protein assays are heavily influenced by free-protein and lipid contaminations which confirm the need of employing several methods for vesicular protein determination. This purity variation seems heavily influenced by the vesicles origin as more complex mediums originate more matrix interferences. Size exclusion high performance liquid chromatography has been demonstrated as a new methodology for purity assessment of hUCESC-EVs and commercial EVs (adipose stem cells and human serum). The results found low purity in the commercial exosomes highlighting that protein and lipid purity must be included in the commercial EVs. Finally, the combination of this chromatography method with total protein assays proved that particle concentration could be estimated using vesicular protein concentration.

Graphical abstract

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来源期刊
Microchimica Acta
Microchimica Acta 化学-分析化学
CiteScore
9.80
自引率
5.30%
发文量
410
审稿时长
2.7 months
期刊介绍: As a peer-reviewed journal for analytical sciences and technologies on the micro- and nanoscale, Microchimica Acta has established itself as a premier forum for truly novel approaches in chemical and biochemical analysis. Coverage includes methods and devices that provide expedient solutions to the most contemporary demands in this area. Examples are point-of-care technologies, wearable (bio)sensors, in-vivo-monitoring, micro/nanomotors and materials based on synthetic biology as well as biomedical imaging and targeting.
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