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

Abstract

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.

Beyond Basic Characterization and Omics: Immunomodulatory Roles of Platelet-Derived Extracellular Vesicles Unveiled by Functional Testing

“…In vitro functional assay 2.12.1 Cells THP-1 cells (ATCC TIB-202), a human monocytic cell line, were grown in complete RPMI media supplemented with 10% EV-depleted fetal bovine serum and 1% GlutaMAX™ Supplement (Gibco). EV depletion from fetal bovine serum was performed using polyethylene glycol 55 . THP-1 cells were plated to 24-well plate at a concentration of 2 x 10 5 viable cells per well and incubated for 48 h in 1 ml/well of media supplemented with 50 nM phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) for differentiation into macrophages.…”

Extracellular vesicles and their cells of origin: Open issues in autoimmune diseases

“…It should be noted that the various isolation methods of EVs end with different characteristics and potencies in terms of application ( 96 , 97 ). It was shown that EVs from 3D cultures are functionally different and are more similar to the patient-derived EVs compared to those obtained from traditional 2D-culture conditions ( 98 – 100 ). These imply that spending time for optimization of isolation method/s ( 101 , 102 ) to reach pure EVs with higher efficiency ( 103 ) in a reproducible manner ( 104 ) is of great importance.…”

Tetraspanin profiles of serum extracellular vesicles reflect functional limitations and pain perception in knee osteoarthritis

“…Moreover, classifying EVs according to biogenesis pathways remains difficult, and, for this reason, they have been recommended to be categorized based on physical characteristics, such as size, to “small EVs” (sEVs) and “large EVs” (lEVs) by the MISEV2018 guidelines [ 21 ]. Here, we used two complementary EV methods capable of single EV detection: ( i ) high-sensitive flow cytometry with light scatter calibration based on the Mie theory allowing to accurately size lEVs (200–1000 nm in diameter) [ 22 ] and ( ii ) single particle interferometric reflectance imaging sensor (SP-IRIS) allowing the single detection of sEVs (50–200 nm) [ 23 ].…”