Introduction.  Within 3D tissues, cells exert local, directional forces, which feed back to direct tissue shape and function. Current techniques are limited in their ability to read-out cell forces in physiologically relevant microenvironments: 3D traction force microscopy requires knowledge of matrix mechanical properties and is not compatible with dynamic microenvironments; and measuring the deformation of incompressible oil microdroplets cannot quantify absolute cell forces. We developed soft, compressible, and fluorescent hydrogel microdroplets that deform under cell-generated forces. These microgels were cultured within 3D engineered spheroids, and enable real-time, absolute read-outs of internal tissue force ‘maps’. 

Materials and methods.  Fluorescent polyacrylamide (PAA) hydrogel microgels of defined elastic moduli were fabricated by mechanically dispersing picolitre volumes of PAA prepolymer in an immiscible oil bath. Microgels were embedded into bulk PAA gels of known modulus undergoing uniaxial stretch to confirm sensor operation, and then in fibroblast-laden contractile collagen gels, and in fibroblast spheroids. The elastic modulus of the material was determined using bulk shear rheology, and microgel shape was monitored by fluorescent microscopy. Radial and circumferential tissue forces were extracted from the deformations using a finite element model. 

Results and discussion.  Microgels embedded within bulk PAA gels deformed under uniaxial planar tensile loads as expected. When microgels (E~0.6 kPa) were cultured within fibroblast-laden contractile collagen matrices, anisotropic forces were observed over 3 days of gel compaction; significant radial compressive forces were measured, compared to axial tensile forces. Sequential application of inhibitors and activators of cellular contractile forces demonstrate the real-time readout potential of the sensors. When microgels (E~0.1 kPa) were cultured in fibroblast spheroids, spatial differences in cell forces were observed. At the spheroid core, low radial and circumferential forces suggest necrosis in large spheroids. Compressive forces increased in both the radial and circumferential direction closer to the spheroid edge. At the edge, radial forces continued to be increasingly compressive, while circumferential forces dropped suggesting circumferential tension at the periphery. These results demonstrate that mechanical forces vary considerably within spheroid cultures, and spatial properties of spheroids should be considered in drug discovery for example, in which these model systems are increasingly being used. 

Conclusion.  These results experimentally quantify, for the first time to our knowledge, the mechanical heterogeneity present in 3D spheroid cultures. More generally, this technique may be applied to monitor real-time, biological forces in situ, to address a broad range of both fundamental and applied biological questions.