The therapeutic potential of the conditioned medium (CM) derived from MSCs (mesenchymal stem/stromal cells) in disparate medical fields, from immunology to orthopedics, has been widely suggested by in vitro and in vivo evidences. Prior to MSC-CM use in clinical applications, appropriate quality controls are needed in order to assess its reproducibility. Here, we evaluated different CM characteristics, including general features and precise protein and lipid concentrations, in 3 representative samples from adipose-derived MSCs (ASCs). In details, we first investigated the size and distribution of the contained extracellular vesicles (EVs), lipid bilayer-delimited particles whose pivotal role in intercellular communication has been extensively demonstrated. Then, we acquired Raman signatures, providing an overlook of ASC-CM composition in terms of proteins, lipids, and nucleic acids. At last, we analyzed a panel of 200 molecules including chemokines, cytokines, receptors, and inflammatory and growth factors and searched for 32 lipids involved in cell signalling and inflammation. All ASC-CM contained a homogeneous and relevant number of EVs ( particles per million donor ASCs) with a mean size of , suggesting the appropriateness of the method for EV retaining and concentration. Furthermore, also Raman spectra confirmed a high homogeneity among samples, allowing the visualization of specific peaks for nucleic acids, proteins, and lipids. An in depth investigation that focused on 200 proteins involved in relevant biological pathways revealed the presence in all specimens of 104 factors. Of these, 26 analytes presented a high degree of uniformity, suggesting that the samples were particularly homogenous for a quarter of the quantified molecules. At last, lipidomic analysis allowed the quantification of 7 lipids and indicated prostaglandin-E2 and N-stearoylethanolamide as the most homogenous factors. In this study, we assessed that ASC-CM samples obtained with a standardized protocol present stable features spanning from Raman fingerprint to specific marker concentrations. In conclusion, we identified key ingredients that may be involved in ASC-CM therapeutic action and whose consistent levels may represent a promising quality control in the pipeline of its preparation for clinical applications.

中文翻译：

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迈向分泌组标准化：确定 MSC 衍生治疗鸡尾酒的关键成分

来自 MSC（间充质干/基质细胞）的条件培养基 (CM) 在不同医学领域（从免疫学到骨科）中的治疗潜力已被体外和体内研究广泛表明证据。在 MSC-CM 用于临床应用之前，需要适当的质量控制以评估其可重复性。在这里，我们评估了来自脂肪来源的 MSC (ASC) 的 3 个代表性样品中不同的 CM 特征，包括一般特征和精确的蛋白质和脂质浓度。具体而言，我们首先研究了所含细胞外囊泡 (EV) 的大小和分布，这种脂质双层分隔的颗粒在细胞间通讯中的关键作用已得到广泛证实。然后，我们获得了拉曼特征，在蛋白质、脂质和核酸方面提供了对 ASC-CM 组成的忽略。最后，我们分析了一组 200 个分子，包括趋化因子、细胞因子、受体、以及炎症和生长因子，并搜索了 32 种参与细胞信号传导和炎症的脂质。所有 ASC-CM 都包含同质且相关数量的 EV（ 每百万供体 ASC 中的粒子数），平均大小为 ,表明 EV 保留和浓缩方法的适当性。此外，拉曼光谱也证实了样品之间的高度同质性，可以显示核酸、蛋白质和脂质的特定峰。一项针对参与相关生物途径的 200 种蛋白质的深入调查揭示了所有样本中都存在 104 种因子。其中，26 种分析物呈现出高度的均一性，表明样品对于四分之一的定量分子而言特别均匀。最后，脂质组学分析允许对 7 种脂质进行量化，并表明前列腺素-E2 和 N-硬脂酰乙醇酰胺是最同质的因素。在这项研究中，我们评估了使用标准化协议获得的 ASC-CM 样本呈现出从拉曼指纹到特定标记浓度的稳定特征。总之，我们确定了可能参与 ASC-CM 治疗作用的关键成分，其一致的水平可能代表其临床应用准备过程中的有希望的质量控制。