Grup de Sensors i Biosensors, Departament de Química, Universitat Autònoma de Barcelona, Bellaterra, Spain; Biosensing and Bioanalysis Group, Institute of Biotechnology and Biomedicine, Bellaterra, Spain.
Biosensing and Bioanalysis Group, Institute of Biotechnology and Biomedicine, Bellaterra, Spain.
Methods Mol Biol. 2023;2668:257-276. doi: 10.1007/978-1-0716-3203-1_18.
The analysis of the receptors on the surface of the cell-secreted vesicles provides valuable information of the cell signature and may also offer diagnosis and/or prognosis of a wide range of diseases, including cancer.Due to their low concentration, conventional procedures for extracellular vesicle (EV) detection usually require relatively large sample volumes, involving preliminary purification or preconcentration steps from complex specimens. Here, we describe the separation and preconcentration in magnetic particles of extracellular vesicles obtained from cell culture supernatants from MCF7, MDA-MB-231, and SKBR3 breast cancer cell lines, human fetal osteoblastic cell line (hFOB), and human neuroblastoma SH-SY5Y cell line, as well as exosomes from human serum. The first approach involves the covalent immobilization for the exosomes directly on micro (4.5 μm)-sized magnetic particles. The second approach is based on tailored magnetic particles modified with antibodies for further immunomagnetic separation of the exosomes. In these instances, micro (4.5 μm)-sized magnetic particles are modified with different commercial antibodies against selected receptors, including the general tetraspanins CD9, CD63, and CD81 and the specific receptors (CD24, CD44, CD54, CD326, CD340, and CD171). The magnetic separation can be easily coupled with downstream characterization and quantification methods, including molecular biology techniques such as immunoassays, confocal microscopy, or flow cytometry.
对细胞分泌囊泡表面受体的分析提供了有价值的细胞特征信息，也可能为包括癌症在内的多种疾病提供诊断和/或预后信息。由于其浓度较低，通常需要从复杂的标本中进行初步纯化或预浓缩步骤，才能进行传统的细胞外囊泡（EV）检测程序。在这里，我们描述了从 MCF7、MDA-MB-231 和 SKBR3 乳腺癌细胞系、人胎儿成骨细胞系（hFOB）和人神经母细胞瘤 SH-SY5Y 细胞系的细胞培养上清液以及人血清中分离和预浓缩磁性颗粒中的细胞外囊泡。第一种方法涉及将外泌体直接共价固定在微（4.5μm）尺寸的磁性颗粒上。第二种方法基于用抗体修饰的定制磁性颗粒，进一步对外泌体进行免疫磁性分离。在这些情况下，微（4.5μm）尺寸的磁性颗粒用不同的针对选定受体的商业抗体修饰，包括一般四跨膜蛋白 CD9、CD63 和 CD81 以及特异性受体（CD24、CD44、CD54、CD326、CD340 和 CD171）。磁分离可以很容易地与下游的表征和定量方法相结合，包括免疫测定、共聚焦显微镜或流式细胞术等分子生物学技术。
