Determination of the Loading Capacity and Recovery of Extracellular Vesicles Derived from Human Embryonic Kidney Cells and Urine Matrices on Capillary-Channeled Polymer (C-CP) Fiber Columns
Determination of the Loading Capacity and Recovery of Extracellular Vesicles Derived from Human Embryonic Kidney Cells and Urine Matrices on Capillary-Channeled Polymer (C-CP) Fiber Columns
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DOI：
10.3390/separations9090251
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发表时间：
2022-09
期刊：
影响因子：
2.6
通讯作者：
Lacey S. Billotto;Kaylan K. Jackson;R. Marcus
中科院分区：
文献类型：
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作者：
Lacey S. Billotto;Kaylan K. Jackson;R. Marcus
细胞外囊泡 (EV) 是所有细胞分泌的 50-1000 nm 膜囊泡，在许多生物过程中发挥重要作用。外泌体是 EV 的一个较小的子集，由于其丰富的生物货物及其在细胞信号传导、维持稳态和调节细胞功能等过程中的作用，在基础生物化学和临床领域越来越受到关注。为了在基础生物化学和临床诊断研究领域中有效实施，以及它们在基因治疗中作为载体的用途，需要新的方法来及时从复杂基质中分离高浓度的高纯度外泌体。为了解决目前回收率和纯度方面的限制，本文描述了使用毛细管通道聚合物 (C-CP) 纤维固定相通过高效液相色谱 (HPLC) 对源自人胚胎肾 (HEK) 细胞培养物和人尿液样本的外泌体进行正面通量和回收率分析。使用 C-CP 纤维 HPLC 方法进行 EV 分离，克服了传统技术所带来的从小体积样品中回收纯化 EV 的挑战，同时在处理、经济性（每柱约 5 美元）、装载（约 1012 个颗粒）和从整个样本中回收（1011-1012 个颗粒）方面带来了显着的优势，无需进一步处理。
Extracellular vesicles (EVs) are 50–1000 nm membranous vesicles secreted from all cells that play important roles in many biological processes. Exosomes, a smaller-sized subset of EVs, have become of increasing interest in fundamental biochemistry and clinical fields due to their rich biological cargos and their roles in processes such as cell-signaling, maintaining homeostasis, and regulating cellular functions. To be implemented effectively in fundamental biochemistry and clinical diagnostics fields of study, and for their proposed use as vectors in gene therapies, there is a need for new methods for the isolation of large concentrations of high-purity exosomes from complex matrices in a timely manner. To address current limitations regarding recovery and purity, described here is a frontal throughput and recovery analysis of exosomes derived from human embryonic kidney (HEK) cell cultures and human urine specimens using capillary-channeled polymer (C-CP) fiber stationary phases via high performance liquid chromatography (HPLC). Using the C-CP fiber HPLC method for EV isolations, the challenge of recovering purified EVs from small sample volumes imparted by the traditional techniques was overcome while introducing significant benefits in processing, affordability (~5 $ per column), loading (~1012 particles), and recovery (1011–1012 particles) from whole specimens without further processing requirements.