Improved isolation of extracellular vesicles from cancer cells
A new combination of techniques for extracellular vesicle (EV) isolation is developed that is less complicated, time consuming, and harmful to the vesicles compared to existing methods.
Figure 1. Extracellular vesicles (arrows) from culture media from oral cancer cells captured by Transmission Electron Microscopy (TEM). Photo: Eduarda Guerreiro.
Extracellular vesicles (EVs) are miniscule particles surrounded by a membrane. These particles are continuously released by cells and can be identified in various biofluids, such as tears, saliva, blood, urine, etc., independent of the tissue where they originated. Extracellular vesicles (EVs) are important messengers when cells communicate with each other, both as part of normal functions and in disease. One such example is in cancer development and progression, where the cancer-associated EVs may help cancer cells to proliferate and migrate. To perform any kind of investigations into EVs, the first step is to isolate and purify them. This has been a challenge due to their small size. A new method to isolate EVs from cell culture media is presented in a recently published study by researchers from the Institute of Oral Biology. This method involves a new combination of techniques for EV isolation that is less complicated, time consuming, and harmful to the vesicles than existing methods.
Due to their properties, EVs are hard to retrieve from solutions (biofluids or cell culture). Previously, isolation methods have included highly specialized equipment – high-speed centrifuges – to which not all laboratories have access. In addition, the isolation conditions are so harsh that they are known to cause damage to the isolated exosomes. Newly developed methodologies such as vesicle precipitation and affinity-based capture methods may resolve these challenges. However, these methods seem to produce certain vesicle subpopulations. Due to these drawbacks, we selected a procedure of separation by size; called size-exclusion chromatography. This method allows us to preserve EV diversity and structure (Figure 1) while reducing the presence of contaminants.
To increase the final yield of isolated vesicles – which is important in EV studies - we modified the conventional cell culture conditions to increase the number of cells and, thus, the amount of EVs. In contrast to culture of cells in a two dimensional manner, with limited space and nutrients, our cells were grown in a specialized culture flask containing a mesh membrane that supported 3D cell-growth where the cells had access to a constant supply of nutrients and oxygen (Figure 2).
The new method developed allows for the isolation of high amounts of exosomes in a robust and reproducible manner, and at a much-reduced cost. This new cutting-edge method will facilitate research into EV detection in biofluids, and could bea potential cancer diagnostics tool.
Eduarda Guerreiro, Beate Vestad, Lilly A. Steffensen, Hans C.D. Aass, Muhammad Saeed, Reidun Øvstebø, Daniela E. Costea, Hilde K. Galtung,Tine M. Søland. Efficient extracellular vesicle isolation by combining cell media modifications, ultrafiltration, and size-exclusion chromatography i PLoS One. 2018 Sep 27;13(9)