Dimensionality Controls Cytoskeleton Assembly and Metabolism of Fibroblast Cells in Response to Rigidity and Shape

BACKGROUND: Various physical parameters, including substrate rigidity, size of adhesive islands and micro-and nano-topographies, have been shown to differentially regulate cell fate in two-dimensional (2-D) cell cultures. Cells anchored in a three-dimensional (3-D) microenvironment show significantly altered phenotypes, from altered cell adhesions, to cell migration and differentiation. Yet, no systematic analysis has been performed that studied how the integrated cellular responses to the physical characteristics of the environment are regulated by dimensionality (2-D versus 3-D). METHODOLOGY/PRINCIPAL FINDINGS: Arrays of 5 or 10 µm deep microwells were fabricated in polydimethylsiloxane (PDMS). The actin cytoskeleton was compared for single primary fibroblasts adhering either to microfabricated adhesive islands (2-D) or trapped in microwells (3-D) of controlled size, shape, and wall rigidity. On rigid substrates (Young's Modulus = 1 MPa), cytoskeleton assembly within single fibroblast cells occurred in 3-D microwells of circular, rectangular, square, and triangular shapes with 2-D projected surface areas (microwell bottom surface area) and total surface areas of adhesion (microwell bottom plus wall surface area) that inhibited stress fiber assembly in 2-D. In contrast, cells did not assemble a detectable actin cytoskeleton in soft 3-D microwells (20 kPa), regardless of their shapes, but did so on flat, 2-D substrates. The dependency on environmental dimensionality was also reflected by cell viability and metabolism as probed by mitochondrial activities. Both were upregulated in 3-D cultured cells versus cells on 2-D patterns when surface area of adhesion and rigidity were held constant. CONCLUSION/SIGNIFICANCE: These data indicate that cell shape and rigidity are not orthogonal parameters directing cell fate. The sensory toolbox of cells integrates mechanical (rigidity) and topographical (shape and dimensionality) information differently when cell adhesions are confined to 2-D or occur in a 3-D space.