Mesenchymal stromal cells (MSCs)-derived extracellular vesicles (EVs) are promising therapeutic nano-carriers for the treatment of osteoarthritis (OA). The assessment of their uptake in tissues is mandatory but, to date, available technology does not allow to track and quantify incorporation in real-time. To fill this knowledge gap, the present study was intended to develop an innovative technology to determine kinetics of fluorescent MSC-EV uptake by means of time-lapse quantitative microscopy techniques. Adipose-derived mesenchymal stromal cells (ASCs)-EVs were fluorescently labeled and tracked during their uptake into chondrocytes micromasses or cartilage explants, both derived from OA patients. Immunofluorescence and time-lapse coherent anti-Stokes Raman scattering, second harmonic generation and two-photon excited fluorescence were used to follow and quantify incorporation. EVs penetration appeared quickly after few minutes and reached 30–40 μm depth after 5 h in both explants and micromasses. In explants, uptake was slightly faster, with EVs signal overlapping both extracellular matrix and chondrocytes, whereas in micromasses a more homogenous diffusion was observed. The finding of this study demonstrates that this innovative technology is a powerful tool to monitor EVs migration in tissues characterized by a complex extracellular network, and to obtain data resembling in vivo conditions.

中文翻译：

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使用实时定量多峰非线性光学成像对软骨衍生骨关节炎样品中细胞外囊泡迁移的体外研究。

间充质基质细胞（MSCs）衍生的细胞外囊泡（EVs）是有望用于治疗骨关节炎（OA）的治疗性纳米载体。必须评估其在组织中的摄入量，但是迄今为止，可用的技术不允许实时跟踪和量化掺入量。为了填补这一知识空白，本研究旨在开发一种创新技术，通过定时定量显微镜技术确定荧光MSC-EV吸收的动力学。脂肪来源的间充质基质细胞（ASCs）-EVs被荧光标记，并在摄取到软骨细胞微团或软骨外植体中的过程中被追踪，两者均来自OA患者。免疫荧光和延时相干反斯托克斯拉曼散射，二次谐波的产生和双光子激发的荧光用于跟踪和量化掺入。几分钟后，EV的渗透迅速出现，并在5 h后在外植体和微团中达到30–40μm的深度。在外植体中，摄取略快，EV信号重叠细胞外基质和软骨细胞，而在微团中观察到更均匀的扩散。这项研究的发现表明，这项创新技术是监视电动汽车在以复杂细胞外网络为特征的组织中迁移并获得类似于体内条件的数据的强大工具。电动汽车的信号重叠细胞外基质和软骨细胞，而在微团中观察到更均匀的扩散。这项研究的发现表明，这项创新技术是监测电动汽车在以复杂细胞外网络为特征的组织中迁移并获得类似于体内条件的数据的强大工具。电动汽车的信号重叠细胞外基质和软骨细胞，而在微团中观察到更均匀的扩散。这项研究的发现表明，这项创新技术是监测电动汽车在以复杂细胞外网络为特征的组织中迁移并获得类似于体内条件的数据的强大工具。