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

Abstract

BackgroundVarious 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 FindingsArrays 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/SignificanceThese 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.

Three-Dimensional Cell Culture Environment Promotes Partial Recovery of the Native Corneal Keratocyte Phenotype from a Subcultured Population

“…The 3D culture has similarly been shown to be a strong signal in a number of other cell lines. [22][23][24][25] Wound-healing response (a-SMA) is downregulated in both low-and high-density cultures Our density study results show that the wound-healing response is downregulated in the 3D culture both at low seeding density and at high seeding density when compared with intermediate seeding densities and normal TC culture (Fig. 4C).…”

“…It has been well established that the observed cell phenotype changes when the 2D and 3D cell cultures are compared for a given cell population, [22][23][24] including human foreskin fibroblasts. 25 We hypothesized that by placing a subcultured corneal cell population in a 3D environment, we could downregulate the myofibroblast phenotype.…”

Micropatterning as a tool to decipher cell morphogenesis and functions

“…A recent study has shown that the level of confinement below which cells have difficulties to form stress fibres is lower in 3D microwells than in 2D micropatterns, supporting the idea that stimulation along the z-axis matters (Ochsner et al, 2010). However, in these experiments, the difference in the ability of a cell to form stress fibres is only visible when the microwells are molded in soft polymers and not when rigid polymers are used, indicating a crucial role for the stiffness of the microenvironment in organizing the cellular actin architecture.…”

Molecular composition of GAG-collagen I multilayers affects remodeling of terminal layers and osteogenic differentiation of adipose-derived stem cells

“…After washing with PBS and distilled water, samples were mounted and examined and photographed with CLSM using a 63× oil immersion objective. To assess the degree of rearrangement of Col I by hADSCs a method was adapted from published papers [29,30], with quantification being performed in ImageJ. Briefly, the images were first normalised to ensure the same total range of pixel values.…”