Manipulation and Optical Interrogation of the Cell Micromechanical Environment

Micromechanical environmental factors have significant impact on the determination of cell fate. Fluid coupled and extracellular matrix forces trigger intra- and intercellular signaling cascades critical in development, homeostasis, and disease. Microrheology is a set of tools designed to directly interrogate the micromechanical environment. Optical micromanipulation techniques offer exquisite sensitivity and high bandwidth. Oscillatory passive measures relate the Brownian motion of embedded probe particles to local complex shear modulus of viscoelastic materials. Active measures allow for the application of greater forces to actively engage the material in question. Rotational techniques provide measures of materials under flow. Herein we describe the development of an optomechanical platform for studying peri- and extracellular micromechanics. The endothelial glycocalyx layer is a complex 2 µm pericellular matrix expressed on the luminal surface of vascular endothelial cells that is thought to play critical roles in mechanotransduction and hemodynamics. Direct measures of glycocalyx material properties have been elusive. We describe passive, active, and rotational microrheology of glycocalyx mimetics to uncover micromechanical subtleties in hyaluronic acid, a primary glycocalyx constituent. Passive microrheology demonstrates the potential for transient network formation and the importance of hydrodynamic effects in hyaluronic acid solutions of physiologically relevant molecular weights and concentration. Active microrheology diverges from passive measures at frequencies above the inverse material relaxation time. Importantly, this divergence occurs at physiologic time scales. In contrast to oscillatory measures, rotational microrheology reports linear viscous behavior in glycocalyx mimetics under flow. This intrinsic micromechanical behavior of hyaluronic acid solutions may be critical in the determination of glycocalyx material properties. We develop a novel combinatorial approach to rotational microrheology allowing measurements in ‘thick’ biologically relevant samples. This technique is validated and used to map the viscosity as a function of radial distance from the endothelial cell membrane in excised porcine femoral artery. Viscosity is modulated at up to 6 µm from the endothelial cell membrane, well beyond the expected physical extent of the glycocalyx. This effect occurs at distances on the order of capillary diameters and may have profound hemodynamic consequences. Further implications with respect to glycocalyx material properties are discussed. Extracellular matrix stiffness is thought to be a profound regulator of cell fate. Bulk measures of matrix stiffness correlate well with cell phenotype and gene expression changes. State of the art techniques for tuning matrix stiffness rely on increasing protein concentration or the addition of exogenous crosslinkers, which successfully tune macroscale mechanics. However, these approaches confine cells in 3D culture, limit the availability of nutrients, and increase ligand density; confounding attempts to directly define the role of extracellular matrix mechanics in the determination of cell fate. The design of a novel mechanical device, which utilizes intrinsic extracellular matrix strain hardening to tune matrix mechanics is described in detail and demonstrated in fibrin hydrogels. Active microrheology reveals position dependent stiffening on cellular length scales within the device. Reflection confocal microscopy demonstrates that pore size is conserved. Smooth muscle and endothelial cells exhibit phenotypic changes in response device generated stiffness gradients. Active microrheology is used to probe the mechanics of extracellular matrix mimetic fibrin hydrogels at strains relevant to cellular physiology. Ensemble averages of microscale stiffnesses agree well with bulk measures. However, we find the micromechanical environment to be spatially heterogeneous and directionally dependent. We demonstrate softening in fibrin at physiologic strain amplitudes, in contrast to the generally accepted model of fibrin mechanics. This behavior is well fit by an empirical model. Further implications with respect to extracellular matrix mechanics is presented, along with an in depth discussion on the utility and limits of microrheology in the context of peri- and extracellular micromechanics