Magnetic probe-based microrheology reveals local softening and stiffening of 3D collagen matrices by fibroblasts

Abstract

Changes in extracellular matrix stiffness impact a variety of biological processes including cancer progression. However, cells also actively remodel the matrices they interact with, dynamically altering the matrix mechanics they respond to. Further, cells not only react to matrix stiffness, but also have a distinct reaction to matrix viscoelasticity. The impact of cell-driven matrix remodeling on matrix stiffness and viscoelasticity at the microscale remains unclear, as existing methods to measure mechanics are largely at the bulk scale or probe only the surface of matrices, and focus on stiffness. Yet, establishing the impact of the matrix remodeling at the microscale is crucial to obtaining an understanding of mechanotransduction in biological matrices, and biological matrices are not just elastic, but are viscoelastic. Here, we advanced magnetic probe-based microrheology to overcome its previous limitations in measuring viscoelasticity at the cell-size-scale spatial resolution within 3D cell cultures that have tissue-relevant stiffness levels up to a Young’s modulus of 0.5 kPa. Our magnetic microrheometers exert controlled magnetic forces on magnetic microprobes within reconstituted extracellular matrices and detect microprobe displacement responses to measure matrix viscoelasticity and determine the frequency-dependent shear modulus (stiffness), the loss tangent, and spatial heterogeneity. We applied these tools to investigate how microscale viscoelasticity of collagen matrices is altered by fibroblast cells as they contract collagen gels, a process studied extensively at the macroscale. Interestingly, we found that fibroblasts first soften the matrix locally over the first 32 hours of culture, and then progressively stiffen the matrix thereafter. Fibroblast activity also progressively increased the matrix loss tangent. We confirmed that the softening is caused by matrix-metalloproteinase-mediated collagen degradation, whereas stiffening is associated with local alignment and densification of collagen fibers around the fibroblasts. This work paves the way for the use of measurement systems that quantify microscale viscoelasticity within 3D cell cultures for studies of cell–matrix interactions in cancer progression and other areas.

Cell mediated remodeling of stiffness matched collagen and fibrin scaffolds

“…Further, in contrast to our previous studies 10 and findings by Loeber et al for chondrocytes cultured in a hyaluronic acid gel 44 , stiffness was largely unaffected by distance from the cell, angular position θ , and axes of bead oscillation. Discrepancy in results could potentially stem from the difference in duration of cell culture, as supported by past research that found hydrogel stiffness to vary with cell incubation time 45 , 46 . Reported results are most likely by alternative factors not explored in this project,, including cell seeding density 46 , difference in collagen composition 47 , cell area 48 – 50 and expression levels of mesenchymal proteins including those for actin, actin polymerization, myosin motors as well as the state of regulatory proteins including ROCK.…”

Nanoscale Tracking Combined with Cell-Scale Microrheology Reveals Stepwise Increases in Force Generated by Cancer Cell Protrusions

“…The matrix displacements due to cellular forces are mediated by the local matrix viscoelastic properties; therefore, our technique uses a microscope-integrated system to measure those properties (Figure 2c). This system, a magnetic microrheometer, and its working principle is described in Pokki et al 46 (i.e., system type 2 for stiffer samples 46 ). Briefly, a pair of electromagnets is used to generate sinusoidal magnetic forces onto magnetic probes within the matrix with the cells (Equation S1).…”

“…Further, the methods probe one microscale location at the time. Pokki et al 46 recently introduced a magnetic-microrheology method using matrix-embedded, 10μm-diameter magnetic microspheres that simultaneously probe viscoelasticity of multiple microscale locations inside 3D cultures with stiffness levels as in a developing breast cancer.…”

DNA‐Engineered Hydrogels with Light‐Adaptive Plasmonic Responses

“…The tracked displacements were fitted to obtain the amplitude and the phase angle of magnetic-bead motion that allow for the calculation of complex shear modulus (G*) and loss tangent (tan δ) of the hydrogel networks using Equations (S1-S3), Supporting Information. [80] At thermal equilibrium, G* and tan δ are measures of elastic and viscous characteristics of the hydrogel networks that provide insights into the physical/chemical nature of crosslinks in the gel. [81][82][83] As indicated in Figure 2A, all three DNA-AuNR crosslinked networks displayed marked differences in their G* values, when compared to control gels without DNA-AuNR.…”