Dynamics of Cellular Rigidity Sensing on the Micron and Sub-micron Scale
Creators: Ghassemi, Saba
Publisher: name: Columbia University
Year: 2011
Subjects: Engineering
Biology

description: This thesis describes a study of the effect of environmental cues including physical attribute of the cellular environment on cellular force and force transduction. Different mechanical parameters such as geometry and rigidity of the substrate are controlled independently and forces exerted by cells were measured. The experimental system for this study is based on fabrication of micron and submicron pillar substrates and their surface functionalization and finally measurement of forces that cells exert to these substrates. In chapter 2, the interplay between the rigidity of the substrate and the cell's force response was studied. Arrays of flexible PDMS pillars used to measure the pattern of traction force generation on matrices. Using three different pillar diameters (2, 1 and 0.5 micrometers), and three different pillar stiffnesses for each diameter, we showed that cells treat larger, fibronectin-coated pillars fundamentally differently than sub-micron pillars during initial contact formation. In the case of larger pillars, mouse embryo fibroblasts generated a constant force per unit area of about 1 nN/m2 on pillars of different stiffness by causing different displacements; whereas, the sub-micrometer pillars were displaced by about 60 nm irrespective of stiffness. In addition, micron-scale pillars are all pulled toward the center of the cell, whereas sub-micron pillars were also pulled toward each other locally. Further, the focal adhesion protein, paxillin, was concentrated at the edges of large pillars but it was focused on the tops of small pillars in a pattern analogous to the pattern on continuous substrates. Thus, we suggested that initial rigidity sensing involves measuring the force needed to produce displacements of about 60 nm in local regions (1m) of the substrate. In addition, these results suggested that, to examine the effects of substrate rigidity on cellular behavior, sub-micron pillars more closely approximate continuous substrates than do micron-scale pillars. In chapter 3, a technique was described for fabricating substrates whose rigidity can be controlled locally without altering the contact area for cell spreading. The substrates consist of elastomeric pillar arrays in which the top surface is uniform but the pillar height is changed across a sharp step. Results demonstrated the effects on cell migration and morphology at the step boundary. In chapter 4, a technique was described for the fabrication of arrays of elastomeric pillars whose top surfaces are treated with selective chemical functionalization to promote cellular adhesion in cellular force transduction experiments. The technique involves the creation of a rigid mold consisting of arrays of circular holes into which a thin layer of Au is deposited, while the top surface of the mold and the sidewalls of the holes are protected by a sacrificial layer of Cr. When an elastomer is formed in the mold, Au adheres to the tops of the molded pillars. This can then be selectively functionalized with a protein that induces cell adhesion, while the rest of the surface is treated with a repellent substance. An additional benefit is that the tops of the pillars can be fluorescently labeled for improved accuracy in force transduction measurements. The same fabrication process was used for fabrication of magnetically actuated pillars in order to be able to exert external force to cells and study the eect of localized mechanostimulation.