A quick pipeline for the isolation of 3D cell culture-derived extracellular vesicles.

Recent advances in cell biology research regarding extracellular vesicles have highlighted an increasing demand to obtain 3D cell culture-derived EVs, because they are considered to more accurately represent EVs obtained in vivo. However, there is still a grave need for efficient and tunable methodologies to isolate EVs from 3D cell cultures. Using nanofibrillar cellulose (NFC) scaffold as a 3D cell culture matrix, we developed a pipeline of two different approaches for EV isolation from cancer spheroids. A batch method was created for delivering high EV yield at the end of the culture period, and a harvesting method was created to enable time-dependent collection of EVs to combine EV profiling with spheroid development. Both these methods were easy to set up, quick to perform, and they provided a high EV yield. When compared to scaffold-free 3D spheroid cultures on ultra-low affinity plates, the NFC method resulted in similar EV production/cell, but the NFC method was scalable and easier to perform resulting in high EV yields. In summary, we introduce here an NFC-based, innovative pipeline for acquiring EVs from 3D cancer spheroids, which can be tailored to support the needs of variable EV research objectives.