Kyykallio, H., 2021
Abstract
"The research of extracellular vesicles (EV) is heavily based on two-dimensional cell cultures although three-dimensional (3D) cell models are known to represent native tissues much more accurately. Recent research has shown that EVs from 3D cultured cells more closely represent in vivo EVs, but majority of current 3D models are not efficient in EV studies. In this work, a novel 3D culture model utilizing wood-derived nanofibrillar cellulose (NFC) was established and evaluated in study of EV secretion from MCF7 breast cancer cell spheroids. EV isolation from NFC cultures via enzymatic digestion was optimized and the impact of NFC derived material in EV analysis was assessed. Additionally, optically transparent anionic NFC (aNFC) was utilized in live cell imaging. Synthesis of abundant extracellular matrix component hyaluronan (HA) has been shown to induce shedding of EVs. NFC model was utilized to study the impact of secreted and external HA on EV secretion in 3D conditions by utilizing MCF7 cells with inducible GFP-Hyaluronan synthase 3 (HAS3) and NFC scaffolds with different HA concentrations. In addition, the impact of NFC matrix viscosity on EV secretion was analyzed. The analysis of EV secretion, size, and marker expression was performed combining electron and confocal microscopy and nanoparticle tracking analysis. HA synthesis induced EV secretion was found to be decreased in 3D conditions. Additionally, increasing viscosity and HA content in the matrix was found to decrease EV secretion. It is shown that NFC supports efficient EV secretion and allows EV isolation and imaging of spheroid cultures"
Kyykallio, H., Isolation and Analysis of Extracellular Vesicles from Nanofibrillar Cellulose 3D Cancer Cell Cultures, University of Eastern Finland, 2021