Invited Paper BI1+NS-ThM3
Engineering Cell Behavior in Microfabricated Substrates: Adding Dimensionality to the Sensory Toolbox

Thursday, October 21, 2010, 8:40 am, Room Taos

The physical properties of the local cell microenvironment regulate cell behavior in concert with soluble or matrix bound signaling molecules. In vivo, these properties are defined by a fibrillar ECM and adjacent cells and have implications for human health and disease. 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, or cell adhesive area and shape in two dimensions (2-D). Most of these studies were performed on flat, 2-D culture surfaces where studies have shown that these properties regulate a seemingly endless variety of observable cell responses. Regulating these processes with engineered cell culture platforms might prove useful in tissue engineering or regenerative medicine applications where a specific cell phenotype needs to be stimulated or maintained.

The extent to which observations made in 2-D Petri dishes can be transferred to predict cell behavior in a 3-D environment is a focus of current research. However, only a limited number of studies investigated the different microenvironmental parameters as a function of dimensionality, often with no or limited control of cell shape and substrate stiffness, and thus cannot be directly compared to observations made on (patterned) 2-D culture systems.

The focus of this talk is to demonstrate how the surface area of adhesive contact and substrate rigidity differentially regulate actin cytoskeleton assembly in 2-D versus 3-D environments, and how this impacts cell phenotype and function. PDMS polymeric substrates (compatible with inverted stage microscopy) for the organization of single cells in engineered quasi-3-D microenvironments were fabricated presenting arrays of microwells of different shape/aspect ratios, and stiffness (typically 1 MPa to 10 kPa Young’s Modulus). The walls and bottom of wells were backfilled with extracellular matrix proteins such as fibronectin or mobile lipid bilayers.

On rigid substrates cytoskeleton assembly within single fibroblast cells was found to occur in 3-D microwells at shapes 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), but did so on flat, 2-D substrates that were otherwise equivalent. These data indicate that neither cell shape nor rigidity are orthogonal parameters directing cell fate. The sensory toolbox of cells seems to integrate mechanical (rigidity) and topographical (shape and dimensionality) information differently when cell adhesions are confined to 2-D or occur in a 3-D space.