DataSheet2_Orchestrating Extracellular Vesicle With Dual Reporters for Imaging and Capturing in Mammalian Cell Culture.PDF

Background: Recent technological advancements have enabled live-cell imaging of intracellular organelles to monitor their biogenesis in mammalian cells. However, applying this method to gain insight into extracellular organelles, such as extracellular vesicles (EVs), presents unique challenges that require special considerations in design and engineering.
Results: We have developed a dual-reporter system that combines genetic fusion, fluorescence microcopy and magnetic beads capture of EVs to study the biogenesis of EVs in mammalian cell cultures. First, we genetically produced a series of reporters by fusing a green fluorescent protein (GFP) and an affinity peptide (6xHis), with either the endogenous transmembrane protein, CD63, or EVs targeting vesicular stomatitis viral glycoprotein (VSVG). Transfection of these reporters into human 293T cells resulted in expression and integration of these reporters into pre-exosome compartments, which were subsequently released into the culture medium. Confocal imaging and nano-particle tracking analysis demonstrated that EVs were appropriately labeled and exhibited a single dominant peak in the 80–110 nm size range, indicating that isolated EVs were comprised of micro-vesicles and/or exosome subpopulations. Incubation of isolated EVs with nickel-coated magnetic beads resulted in successful capture of GFP-positive EVs. Finally, addition of EVs into culture medium was able to reveal the cellular uptake of GFP-labeled EVs by recipient cells. Taken together, our dual-reporter system provides a powerful method for both monitoring and capturing of EVs in mammalian cell culture systems.
Conclusion: A dual-reporter system provides a robust tool to study the life cycle of EVs in mammalian cells from biogenesis and excretion to cellular uptake.

研究背景：近年来，技术进步使得活细胞成像技术可用于监测哺乳动物细胞内细胞器的生物发生过程。然而，将该方法应用于胞外细胞器（如细胞外囊泡（extracellular vesicles, EVs））的研究时，面临独特的挑战，需要在实验设计与工程优化方面进行特殊考量。
研究结果：本研究开发了一种双报告系统，该系统结合了基因融合技术、荧光显微术与磁珠捕获技术，以研究哺乳动物细胞培养体系中细胞外囊泡的生物发生过程。首先，我们通过基因工程构建了一系列报告蛋白：将绿色荧光蛋白（green fluorescent protein, GFP）与亲和肽（6xHis）融合，分别连接至内源性跨膜蛋白CD63或靶向细胞外囊泡的水泡性口炎病毒糖蛋白（vesicular stomatitis viral glycoprotein, VSVG）。将这些报告质粒转染至人293T细胞后，报告蛋白得以表达并整合至前外泌体区室，随后被释放至细胞培养上清中。共聚焦成像与纳米颗粒追踪分析结果显示，细胞外囊泡被成功标记，且在80–110 nm粒径范围内呈现单一主导峰，表明分离得到的细胞外囊泡包含微囊泡和/或外泌体亚群。将分离得到的细胞外囊泡与镍包被磁珠共孵育，可成功捕获GFP阳性的细胞外囊泡。最后，将分离的细胞外囊泡添加至细胞培养基中，可实现受体细胞对GFP标记细胞外囊泡的细胞摄取过程的可视化。综上，本研究开发的双报告系统为哺乳动物细胞培养体系中细胞外囊泡的监测与捕获提供了一种强有力的研究方法。
研究结论：该双报告系统为研究哺乳动物细胞内细胞外囊泡的完整生命周期（从生物发生、分泌至细胞摄取）提供了一款稳健的研究工具。