间充质基质细胞衍生的细胞外囊泡（MSC-EV）是一种有前途的神经炎症治疗方法。 MSC-EV 可以与大脑常驻免疫细胞小胶质细胞相互作用，发挥其免疫调节作用。为了响应细胞因子等炎症信号，小胶质细胞会发生表明其功能的表型变化。形态和分泌。然而，这些针对 MSC-EV 的变化尚不清楚。此外，不存在评估 MSC-EV 生物活性的疾病相关筛查工具，这进一步阻碍了临床转化。在这里，我们开发了一种定量、高通量形态学分析方法来评估小胶质细胞对神经炎症相关信号的反应，以及这种形态学反应是否可用于指示 MSC-EV 的生物活性。使用永生化的人小胶质细胞系，我们观察到用干扰素-γ (IFN-γ) 和肿瘤坏死因子-α (TNF-α) 刺激后尺寸（周长、长轴长度）和复杂性（形状因子）增加。用 MSC-EV 治疗后，总体形态评分（使用主成分分析确定）转向未刺激的形态，表明 MSC-EV 具有生物活性并调节小胶质细胞。 MSC-EV 在 TNF-α/IFN-γ 刺激的细胞中的形态学效应伴随着 14 种趋化因子/细胞因子（例如 CXCL6、CXCL9）的分泌减少和 12 种趋化因子/细胞因子（例如 CXCL8、CXCL10）的分泌增加。细胞裂解物的蛋白质组学分析显示，经 MSC-EV 处理后，192 种蛋白质（例如 HIBADH、MEAK7、LAMC1）显着增加，257 种蛋白质（例如 PTEN、TOM1、MFF）显着减少。值得注意的是，许多这些蛋白质参与细胞形态和迁移的调节。基因集变异分析揭示了与免疫反应相关途径的上调，例如细胞因子产生的调节、免疫细胞浸润（例如 T 细胞、NK 细胞）和形态变化（例如 Semaphorin、RHO/Rac 信号传导）。此外，测量了小胶质细胞线粒体形态的变化，表明 MSC-EV 调节线粒体代谢。本研究全面论证了 MSC-EV 对人类小胶质细胞形态、细胞因子分泌、细胞蛋白质组和线粒体含量的影响。我们的高通量、快速、低成本的形态测量方法能够筛选 MSC-EV 批次和制造条件，以增强 EV 功能并以疾病相关的方式减轻 EV 功能异质性。这种方法具有高度通用性，可以根据疾病相关信号、靶细胞和治疗产品的选择进一步调整和完善。© 2024。这是美国政府的作品，在美国不受版权保护；可能适用外国版权保护。

Mesenchymal stromal cell derived extracellular vesicles (MSC-EVs) are a promising therapeutic for neuroinflammation. MSC-EVs can interact with microglia, the resident immune cells of the brain, to exert their immunomodulatory effects. In response to inflammatory cues, such as cytokines, microglia undergo phenotypic changes indicative of their function e.g. morphology and secretion. However, these changes in response to MSC-EVs are not well understood. Additionally, no disease-relevant screening tools to assess MSC-EV bioactivity exist, which has further impeded clinical translation. Here, we developed a quantitative, high throughput morphological profiling approach to assess the response of microglia to neuroinflammation- relevant signals and whether this morphological response can be used to indicate the bioactivity of MSC-EVs.Using an immortalized human microglia cell-line, we observed increased size (perimeter, major axis length) and complexity (form factor) upon stimulation with interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α). Upon treatment with MSC-EVs, the overall morphological score (determined using principal component analysis) shifted towards the unstimulated morphology, indicating that MSC-EVs are bioactive and modulate microglia. The morphological effects of MSC-EVs in TNF-α /IFN-γ stimulated cells were concomitant with reduced secretion of 14 chemokines/cytokines (e.g. CXCL6, CXCL9) and increased secretion of 12 chemokines/cytokines (e.g. CXCL8, CXCL10). Proteomic analysis of cell lysates revealed significant increases in 192 proteins (e.g. HIBADH, MEAK7, LAMC1) and decreases in 257 proteins (e.g. PTEN, TOM1, MFF) with MSC-EV treatment. Of note, many of these proteins are involved in regulation of cell morphology and migration. Gene Set Variation Analysis revealed upregulation of pathways associated with immune response, such as regulation of cytokine production, immune cell infiltration (e.g. T cells, NK cells) and morphological changes (e.g. Semaphorin, RHO/Rac signaling). Additionally, changes in microglia mitochondrial morphology were measured suggesting that MSC-EV modulate mitochondrial metabolism.This study comprehensively demonstrates the effects of MSC-EVs on human microglial morphology, cytokine secretion, cellular proteome, and mitochondrial content. Our high-throughput, rapid, low-cost morphometric approach enables screening of MSC-EV batches and manufacturing conditions to enhance EV function and mitigate EV functional heterogeneity in a disease relevant manner. This approach is highly generalizable and can be further adapted and refined based on selection of the disease-relevant signal, target cell, and therapeutic product.© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.