多年来，研究人员一直对细胞外囊泡（EVs）进行研究，探讨其作为细胞间通讯的效应因子和调节因子的作用，并将其潜在应用于开发新型和性能越来越好的纳米技术系统，用于诊断和/或治疗多种疾病。鉴于EVs的诸多应用，如单独使用、功能化或通过工程手段制备的细胞来源药物，急需制定可靠且可重复的保存方法。本研究从健康的血细胞系B淋巴细胞中分离出EVs，并比较了不同储存方法和相对于冻干配方的有效性，以保留一些最重要的EVs关键特征，即浓度、平均粒径、蛋白质含量和表面抗原表达。为了开发最小程度影响EVs完整性和功能性的保存方法，我们结合不同的助剂进行了冻干处理。由于EVs不仅从体液中分离出来，还从细胞培养所产生的培养基中分离出来，因此我们决定测试传统药剂助剂和生物培养基的影响，以开发具有理想外观和性能特性的EVs固化产品。结果显示，一些经过测试的助剂，如糖与右旋糖酐和甘氨酸的组合，在冻干过程中成功维持了EVs的稳定性和完整性。此外，为了评估EVs的生物活性的保留情况，我们评估了重构的EVs在健康（B淋巴细胞）和肿瘤（Burkitt淋巴瘤）细胞中的细胞毒性和内吞能力。重构的EVs仅对癌细胞表现出毒性，为肿瘤领域开辟了新的治疗机会。此外，我们的研究还展示了一些在细胞培养领域常用的生物或细胞培养液，不仅可以作为抗冻剂，还可以作为活性药物成分，显著调节EVs的治疗效果，甚至增加其细胞内摄取能力。

Extracellular vesicles (EVs) have been studied for years for their role as effectors and mediators of cell-to-cell communication and their potential application to develop new and increasingly performing nanotechnological systems for the diagnosis and/or treatment of many diseases. Given all the EVs applications as just isolated, functionalized, or even engineered cellular-derived pharmaceuticals, the standardization of reliable and reproducible methods for their preservation is urgently needed. In this study, we isolated EVs from a healthy blood cell line, B lymphocytes, and compared the effectiveness of different storage methods and relative freeze-drying formulations to preserve some of the most important EVs' key features, i.e., concentration, mean size, protein content, and surface antigen's expression. To develop a preservation method that minimally affects the EVs' integrity and functionality, we applied the freeze-drying process in combination with different excipients. Since EVs are isolated not only from body fluids but also from culture media conditioned by the cells growing there, we decided to test both the effects of the traditional pharmaceutical excipient and of biological media to develop EVs solidified products with desirable appearance and performance properties. Results showed that some of the tested excipients, i.e., sugars in combination with dextran and glycine, successfully maintained the stability and integrity of EVs upon lyophilization. In addition, to evaluate the preservation of the EVs' biological activity, we assessed the cytotoxicity and internalization ability of the reconstituted EVs in healthy (B lymphocytes) and tumoral (Burkitt's lymphoma) cells. Reconstituted EVs demonstrated toxicity only toward the cancerous cells, opening new therapeutic opportunities for the oncological field. Furthermore, our study showed how some biological or cellular-conditioned fluids, commonly used in the field of cell cultures, can act not only as cryoprotectants but also as active pharmaceutical ingredients, significantly tuning the therapeutic effect of EVs, even increasing their cellular internalization.