Technical Reference #1683

1683.

Mechanosensitivity of Fibroblast Cell Shape and Movement to Anisotropic Substratum Topography Gradients Deok-Ho Kim; Karam Han; Kshitiz Gupta; Keon W. Kwon; Kahp-Yang Suh; Andre Levchenko;, Johns Hopkins University, Biomaterials, 30(1683), (2009)

Abstract:
In this report we describe using ultraviolet (UV)-assisted capillary force lithography (CFL) to create a model substratum of anisotropic micro- and nanotopographic pattern arrays with variable local density for the analysis of cell–substratum interactions. A single cell adhesion substratum with the constant ridge width (1 mm) and depth (400 nm) and variable groove widths (1 –9.1 mm) allowed us to characterize the dependence of cellular responses including cell shape orientation and migration on the anisotropy and local density of the variable micro- and nanotopographic pattern. We found that fibroblasts adhering to the denser pattern areas aligned and elongated more strongly along the direction of ridges vs. those on the sparser areas exhibiting a biphasic dependence of the migration speed on the pattern density. In addition cells responded to local variations in topography by altering morphology and migrating along the direction of grooves biased by the direction of pattern orientation (short term) and pattern density (long term) suggesting that single cells can sense the topography gradient. Molecular dynamic live cell imaging and immunocytochemical analysis of focal adhesions and actin cytoskeleton suggest that variable substratum topography can result in distinct types of cytoskeleton reorganization. We also demonstrate that fibroblasts cultured as monolayers on the same substratum retain most of the properties displayed by single cells. This result in addition to demonstrating a more sophisticated method to study aspects of wound healing processes strongly suggests that even in the presence of adhesive cell–cell interactions the cues provided by the underlying substratum topography continue to exercise substantial influence on cell behavior. The described experimental platform might not only further our understanding of biomechanical regulation of cell–matrix interactions but also contribute to bioengineering of devices with the optimally structured design of cell–material interface.

Keywords:
topography; focal adhesions; extracellular matrix; cell migration; wound healing

Materials & Methods:
2.6. Time-lapse microscopy of live cells For time-lapse analysis of individual cell movement NIH 3T3 fibroblasts were cultured on the glass coverslip covered with the topographical pattern substratum whichwas previously glued onto the bottom surface of the custom-made Mattek dish (P35G-20-C). For long-term observation the environmental chamber containing the custom-made Mattek dish integrated with topographically patterned substratumwas mounted onto the stage of a motorized inverted microscope (Zeiss Axiovert 200M) equipped with a Cascade 512B II CCD camera. Phase-contrast and epi-fluorescent images of the NIH 3T3 fibroblasts were automatically recorded using the Slidebook 4.1 (Intelligent Imaging Innovations Denver CO) for 12 h at 15 min intervals.

Microscopic Technique
Electron Microscopy

Cell Type(s)
primary corneal cell