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

Johanna Hafner; David Grijalva; Anita Ludwig-Husemann; Sarah Bertels; Lea Bensinger; Annamarija Raic; Julian Gebauer; Claude Oelschlaeger; Martin Bastmeyer; Karen Bieback; Cornelia Lee-Thedieck; Norbert Willenbacher

doi:10.1016/j.actbio.2020.04.053

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

Acta Biomater. 2020 Jul 15:111:254-266. doi: 10.1016/j.actbio.2020.04.053. Epub 2020 May 17.

Affiliations

¹ Institute of Mechanical Process Engineering and Mechanics, Applied Mechanics Group, Karlsruhe Institute of Technology, 76137 Karlsruhe, Germany. Electronic address: johanna.roether@kit.edu.
² Institute of Mechanical Process Engineering and Mechanics, Applied Mechanics Group, Karlsruhe Institute of Technology, 76137 Karlsruhe, Germany.
³ Institute of Functional Interfaces, Karlsruhe Institute of Technology, 76344 Eggenstein-Leopoldshafen, Germany; Institute of Cell Biology and Biophysics, Department of Cell Biology, Leibniz University Hannover, 30419 Hannover, Germany.
⁴ Department of Cell- and Neurobiology, Zoological Institute, Karlsruhe Institute of Technology, 76137 Karlsruhe, Germany.
⁵ Institute of Transfusion Medicine and Immunology, Medical Faculty Mannheim, Heidelberg University, German Red Cross Blood Service Baden-Württemberg - Hessen, 68167 Mannheim, Germany.
⁶ Institute of Functional Interfaces, Karlsruhe Institute of Technology, 76344 Eggenstein-Leopoldshafen, Germany; Department of Cell- and Neurobiology, Zoological Institute, Karlsruhe Institute of Technology, 76137 Karlsruhe, Germany.

PMID: 32434077
DOI: 10.1016/j.actbio.2020.04.053

Abstract

Multiple particle tracking (MPT) microrheology was employed for monitoring the development of extracellular 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 multipleparticle tracking microrheology setup. MPT is based on monitoring the Brownian motion oftracer particles, which is restricted by the surrounding network. Network elasticity can thusbe quantified. Overcoming current limitations, we realized continuous investigations of ECM elasticityduring fibroblast growth. Furthermore, MPT measurements of stem cell ECM showed ECMstiffening during osteogenic differentiation and softening during adipogenic differentiation.Finally, we characterized small amounts of delicate ECM newly secreted in scaffold-freecultures of endothelial cells, that naturally form 2D layers.

Keywords: ECM elasticity; HUVECs; MMP inhibitor; Mesenchymal stem cell differentiation; Micromechanics; Multiple particle tracking.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cell Differentiation
Cellular Microenvironment
Extracellular Matrix
Humans
Mesenchymal Stem Cells*
Osteogenesis*
Tissue Scaffolds