Introduction: Changes in the mechanical properties of the extracellular matrix and cells are related to changes in tissue health, but monitoring real-time changes of stiffness in situ remain challenging. Current techniques are capable of both bulk and local measurements of stiffness, but require specialized equipment and are limited to end-point analyses. Furthermore, they are restricted in their ability to spatially resolve cellular-scale stiffness variations in 3D tissues, which would be critical to characterize the stiffness a cell would experience in situ. 

We have developed optically-measureable and dispersible sensors that actuate by “remote-control” to exert a small local force within 3D tissue. When embedded in soft materials, expansion is large, but is limited when embedded in stiff materials. By measuring the size change in these sensors, local tissue stiffness can be extrapolated. To demonstrate the utility of this technique, we measured internal stiffness in spheroids which is conventionally difficult to assay without specialized equipment. We further demonstrate the spatial and temporal capabilities of this technique by examining the evolving stiffness patterns in cell-laden collagen.

Methods: N-isopropylacrylamide (NIPAAM) hydrogels demonstrate size changes at a critical temperature just below body temperature. Fluorescent NIPAAM beads were made using a water-in-oil emulsion through agitation to produce spherical microgels roughly the size of a cell. The beads were embedded in a range of polyacrylamide hydrogels of known stiffness to create a calibration curve based on bead size change; and then in engineered tissues to measure stiffness evolution.

Results: Expansion of NIPAAM beads occurred as they transitioned from 37^oC to room temperature, with size changes beginning at 34^oC. Reversibility of bead size change allowed for repeated measurements over several days in cell-laden collagen gels. To date, we measured the stiffness of collagen gels to be 0.03±0.01 kPa, compared to cell-laden collagen remodelled after 2 days which stiffened to 3.2±2.5 kPa. Interestingly, spheroids had an internal stiffness of 6.7±4.4 kPa. Variations seen in compacted collagen and spheroids suggests spatial variation within each respective environment, which we are currently quantifying.

Conclusion: Smart material hydrogel beads offer a new way to measure 3D stiffness throughout the tissue without disrupting the underlying architecture, allowing for repeatable measurements within a dynamic environment. Stiffness changes can be measured as cell-laden collagen contracts and spatial variations in stiffness within the gel are apparent. This new technique offers a simple way to measure matrix stiffness at the cellular scale, with only a microscope and warming stage.