Industrial Biotechnology Centre and School of Biosciences, University of Kent, Canterbury, CT2 7NJ, UK.
Biotechnol Lett. 2023 Apr;45(4):425-437. doi: 10.1007/s10529-023-03353-3. Epub 2023 Jan 28.
Exosomes have previously been isolated from Chinese hamster ovary (CHO) cells and their anti-apoptotic properties reported. However, to further facilitate the study of CHO cell derived exosomes and allow their comparison across studies, it is necessary to characterise and define such exosomes using at least three criteria that can act as a reference for the generation of CHO cell produced exosomes. Here we report on the isolation of exosomes from CHO cells, an industrially relevant and widely used cell host for biopharmaceutical protein production, during the exponential and stationary phase of growth during batch culture using a Total Exosome Isolation (TEI) method. The resulting vesicles were characterized and visualized using a diverse range of techniques including Dynamic Light Scattering (DLS), Zeta potential, Electron Microscopy and immunoblotting, and their protein and RNA content determined. We also generated the lipid fingerprint of isolated exosomes using MALDI-ToF mass spectroscopy. We confirmed the presence of nano sized extracellular vesicles from CHO cells and their subsequent characterization revealed details of their size, homogeneity, surface charge, protein and RNA content. The lipid content of exosomes was also found to differ between exosomes isolated on different days of batch culture. This analysis provides a profile and characterisation of CHO cell exosomes to aid future studies on exosomes from CHO cells and improving the manufacturing of exosomes for biotherapeutic application.
先前已从中国仓鼠卵巢（CHO）细胞中分离出外泌体，并报道了其抗凋亡特性。然而，为了进一步促进对 CHO 细胞衍生的外泌体的研究，并允许在研究之间进行比较，有必要使用至少三个标准来对其进行特征描述和定义，这些标准可以作为 CHO 细胞产生的外泌体的生成参考。在这里，我们报告了使用总外泌体分离（TEI）方法，从 CHO 细胞中分离出外泌体的方法，CHO 细胞是生物制药蛋白生产中具有工业相关性和广泛应用的细胞宿主，在分批培养的指数期和静止期进行。使用多种技术对得到的囊泡进行了表征和可视化，包括动态光散射（DLS）、Zeta 电位、电子显微镜和免疫印迹，并测定了其蛋白质和 RNA 含量。我们还使用 MALDI-ToF 质谱法生成了分离出的外泌体的脂质指纹图谱。我们确认了 CHO 细胞存在纳米大小的细胞外囊泡，并且对其进行的后续表征揭示了它们的大小、均一性、表面电荷、蛋白质和 RNA 含量的详细信息。还发现，在不同批次培养天数分离出的外泌体的脂质含量存在差异。该分析提供了 CHO 细胞外泌体的特征描述，有助于未来对 CHO 细胞外泌体的研究，并提高外泌体用于生物治疗应用的制造水平。
