Monitoring matrix remodeling in the cellular microenvironment using microrheology for complex cellular systems

Multiple particle tracking (MPT) microrheology was employed for monitoring the development of extra-cellular matrix (ECM) mechanical properties in the direct microenvironment of living cells. A customized setup enabled us to overcome current limitations: (i) Continuous measurements were enabled using a cell culture chamber, with this, matrix remodeling by fibroblasts in the heterogeneous environment of macroporous scaffolds was monitored continuously. (ii) Employing tracer laden porous scaffolds for seeding human mesenchymal stem cells (hMSCs), we followed conventional differentiation protocols. Thus, we were, for the first time able to study the massive alterations in ECM elasticity during hMSC differentiation. (iii) MPT measurements in 2D cell cultures were enabled using a long distance objective. Exemplarily, local mechanical properties of the ECM in human umbilical vein endothelial cell (HUVEC) cultures, that naturally form 2D layers, were investigated scaffold-free. Using our advanced setup, we measured local, apparent elastic moduli G(0,app) in a range between 0.08 and 60 Pa. For fibroblasts grown in collagen-based scaffolds, a continuous decrease of local matrix elasticity resulted during the first 10 hours after seeding. The osteogenic differentiation of hMSC cells cultivated in similar scaffolds, led to an increase of G(0,app) by 100 %, whereas after adipogenic differentiation it was reduced by 80 %. The local elasticity of ECM that was newly secreted by HUVECs increased significantly upon addition of protease inhibitor and in high glucose conditions even a twofold increase in G(0,app) was observed. The combination of these advanced methods opens up new avenues for a broad range of investigations regarding cell-matrix interactions and the propagation of ECM mechanical properties in complex biological systems. Statement of Significance Cells sense the elasticity of their environment on a micrometer length scale. For studying the local elasticity of extracellular matrix (ECM) in the direct environment of living cells, we employed an advanced multiple particle tracking microrheology setup. MPT is based on monitoring the Brownian motion of tracer particles, which is restricted by the surrounding network. Network elasticity can thus be quantified. Overcoming current limitations, we realized continuous investigations of ECM elasticity during fibroblast growth. Furthermore, MPT measurements of stem cell ECM showed ECM stiffening during osteogenic differentiation and softening during adipogenic differentiation. Finally, we characterized small amounts of delicate ECM newly secreted in scaffold-free cultures of endothelial cells, that naturally form 2D layers. (C) 2020 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.