Department of Medicine, Roswell Park Comprehensive Cancer Center, Buffalo, NY, USA.
Pathology and Anatomical Sciences, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY, USA.
Methods Mol Biol. 2021;2265:289-304. doi: 10.1007/978-1-0716-1205-7_22.
Cells release extracellular vesicles (EVs) that can be detected both in vivo and in cell culture medium. Among EVs, exosomes are 50-150 nm vesicles that are systematically packaged into multivesicular bodies for release into the external environment. In cancer, these intentionally packaged exosomes carry a payload of proteins such as RNAs and surface receptors that facilitate the reprogramming of proximal cells to assemble a protumor microenvironment. Exosomes have been implicated as an important intermediary extracellular communication pathway between cells, including in melanoma. Human melanoma-derived exosomes (HMEX) have been demonstrated to modulate the extracellular environment and inhibit immune cell activation. There are many methods to isolate and enrich for exosomes and the method applied can impact yield and purity of the isolates. In this chapter we describe the REIUS (rapid exosome isolation using ultrafiltration and size exclusion chromatography) method to isolate HMEX from melanoma cell cultures and then demonstrate their enrichment using molecular and microscopic approaches.
细胞释放细胞外囊泡（EVs），这些囊泡可在体内和细胞培养液中被检测到。在 EVs 中，外泌体是 50-150nm 的囊泡，它们被系统地包装到多泡体中，以便释放到外部环境中。在癌症中，这些被有意包装的外泌体携带蛋白质等有效载荷，如 RNAs 和表面受体，从而促进邻近细胞的重编程，以组装有利于肿瘤的微环境。外泌体被认为是细胞间重要的细胞外通讯途径之一，包括在黑色素瘤中。已经证明人黑色素瘤来源的外泌体（HMEX）可调节细胞外环境并抑制免疫细胞的激活。有许多方法可以分离和富集外泌体，所应用的方法会影响分离物的产量和纯度。在本章中，我们描述了使用超滤和尺寸排阻色谱法（REIUS）从黑色素瘤细胞培养物中分离 HMEX 的方法，然后使用分子和显微镜方法证明其富集。
