Isolation and characterisation of exosomes from Chinese hamster ovary (CHO) cells

Abstract

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.

In-line coupling of capillary-channeled polymer fiber columns with optical absorbance and multi-angle light scattering detection for the isolation and characterization of exosomes

“…While CHO cell lines are commonly known for their production of antibodies, the cells also secrete exosomes as a natural by-product of the cell metabolism [ 10 , 46 , 60 , 61 ]. The workflow presented here aims to purify the exosomes from CHO supernatant and determine the concentration and sizing through in-line absorbance and MALS.…”

Quantitative Recoveries of Exosomes and Monoclonal Antibodies from Chinese Hamster Ovary Cell Cultures by Use of a Single, Integrated Two-Dimensional Liquid Chromatography Method

“…Exosomes have been used to treat various cancers ranging from ovarian to pancreatic cancers, − with tumor-derived exosomes proven to target the cell-types of origin . Recent studies have also looked at engineering exosomes for the targeted transport of their contents. , At present, a number of different cell cultivation approaches are under investigation for mass production, including those derived from human mesenchymal stem cells (MSCs) and embryonic kidney (HEK) cells as well as CHO cells . As exosomes are a natural byproduct of CHO cell culturing, in principle, the same bioreactor could be used for the large-scale production of two therapeutic products: exosomes and mAbs.…”

“…26,34 At present, a number of different cell cultivation approaches are under investigation for mass production, including those derived from human mesenchymal stem cells (MSCs) and embryonic kidney (HEK) cells 35 as well as CHO cells. 36 As exosomes are a natural byproduct of CHO cell culturing, in principle, the same bioreactor could be used for the large-scale production of two therapeutic products: exosomes and mAbs. Current exosome isolation methods include ultracentrifugation (UC), 37,38 size exclusion chromatography (SEC), 39,40 anion exchange chromatography (AEX), 34,41 and a variety of polymer-based precipitation methods.…”