Tunable photoinitiated hydrogel microspheres for quantifying cell-generated forces in complex three-dimensional environments.

We present a high-throughput method using standard laboratory equipment and microfluidics to produce cellular force microscopy probes with controlled size and elastic modulus. Mechanical forces play crucial roles in cell biology but quantifying these forces in physiologically relevant systems remains challenging due to the complexity of the native cell environment. Polymerized hydrogel microspheres offer great promise for interrogating the mechanics of processes inaccessible to classic force microscopy methods. However, despite significant recent advances, their small size and large surface-to-volume ratio impede the high-yield production of probes with tunable, monodisperse distributions of size and mechanical properties. To overcome these limitations, we use a flow-focusing microfluidic device to generate large quantities of droplets with highly reproducible, adjustable radii. These droplets contain acrylamide gel precursor and the photoinitiator Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) as a source of free radicals. LAP provides fine control over microsphere polymerization due to its high molar absorptivity at UV wavelengths and moderate water solubility. The polymerized microspheres can be functionalized with different conjugated extracellular matrix proteins and embedded with fluorescent nanobeads to promote cell attachment and track microsphere deformation. As proof of concept, we measure the mechanical forces generated by a monolayer of vascular endothelial cells engulfing functionalized microspheres. Individual nanobead motions are tracked and analyzed to determine 3D traction forces via direct computation of stress from measured strain. These results reveal that the cell monolayer collectively exerts strong radial compression on the encapsulated probe, suggesting new biomechanical functions of endothelial cells that could modulate diapedesis or pathogen internalization. STATEMENT OF SIGNIFICANCE: Mechanical forces are crucial to many cell biology processes but quantifying them in complex native environments remains challenging. We address this by introducing linearly elastic probes with known mechanical properties, whose deformations can be accurately measured to infer local stresses. Specifically, we present a high-throughput method for producing polyacrylamide (PAAm) hydrogel microspheres embedded with fluorescent nanoparticles. To measure cell-generated forces in physiologically relevant systems, the probes are tracked using a 3D coherent point drift algorithm, yielding high-resolution deformation data with minimal computational cost. This method overcomes key barriers in PAAm microsphere fabrication by ensuring monodisperse size, tunable stiffness, and simple, reproducible processes suitable for most cell biology labs-making it a powerful tool for studying cellular mechanobiology.