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.
Highlights
The physical properties of the local cell microenvironment regulate cell behavior in concert with autocrine and paracrine soluble or matrix bound signaling molecules [1,2,3,4,5]
We show that the dependency of actin cytoskeleton assembly, metabolic activity, and cell survival on the surface area of cell contact are altered in a 3-D environment
To determine how a 3-D arrangement of cell adhesion to ECM molecules influences cytoskeletal organization, we investigated the spatial arrangement of actin in fibroblast cells cultured in microwells in comparison with cells on 2-D patterned surfaces of the same projected area
Summary
The physical properties of the local cell microenvironment regulate cell behavior in concert with autocrine and paracrine soluble or matrix bound signaling molecules [1,2,3,4,5]. These properties are defined by a fibrillar ECM and adjacent cells and have implications for human health and disease [6] Our understanding of their role in regulating cell physiology resulted from technological advances which led to reductionist cell culture systems with tunable substrate stiffness, ligand density/topography, or cell adhesive area and shape in two dimensions (2-D). Most of these studies were performed on flat, 2-D culture surfaces where a number of seminal studies have shown that these properties regulate a seemingly endless variety of observable cell responses. 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)
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