Directing Multicellular Organization by Varying the Aspect Ratio of Soft Hydrogel Microwells

Abstract

Multicellular organization with precise spatial definition is essential to various biological processes, including morphogenesis, development, and healing in vascular and other tissues. Gradients and patterns of chemoattractants are well‐described guides of multicellular organization, but the influences of 3D geometry of soft hydrogels are less well defined. Here, the discovery of a new mode of endothelial cell self‐organization guided by combinatorial effects of stiffness and geometry, independent of protein or chemical patterning, is described. Endothelial cells in 2 kPa microwells are found to be ≈30 times more likely to migrate to the edge to organize in ring‐like patterns than in stiff 35 kPa microwells. This organization is independent of curvature and significantly more pronounced in 2 kPa microwells with aspect ratio (perimeter/depth) < 25. Physical factors of cells and substrates that drive this behavior are systematically investigated and a mathematical model that explains the organization by balancing the dynamic interaction between tangential cytoskeletal tension, cell–cell, and cell–substrate adhesion is presented. These findings demonstrate the importance of combinatorial effects of geometry and stiffness in complex cellular organization that can be leveraged to facilitate the engineering of bionics and integrated model organoid systems with customized nutrient vascular networks.

Hydrogels to Recapture Extracellular Matrix Cues That Regulate Vascularization

“…29,[40][41][42][43][44] To explore the impact of matrix stiffness observed with age and disease, researchers have varied 2-dimensional (2D) matrix stiffness to 2 to 3 orders of magnitude difference using various crosslinking densities. These studies have shown that matrix stiffness plays a significant role in regulating differentiation of endothelial progenitor/stem cells, [40][41][42] EC self-organization, 45 endothelial barrier integrity, 29,43 and vessel permeability. 29 For example, integrin α v β 1 activation on the 2D stiffer substrate (40 kPa) directed vascular progenitor cells into SMCs lineages.…”

Mechanoresponse of epithelial monolayers to in-plane and out-of-plane curvatures imposed by 3D microwells

“…Recent advances in hydrogel synthesis and microscale patterning have led to new advances in understanding the role of physical cues of the matrix in cell organization [18] . Among the role of stiffness and small-scale topographies, cumulative evidence has shown that matrix curvature affects spatiotemporal organization of cells and tissues [10] .…”

“…To address this issue, recent works have developed original physico-chemical strategies to pattern hydrogels of tunable rigidities with 3D micropatterns. Interestingly, it has been shown that endothelial cells can self-organize in response to combinatorial effects of stiffness and geometry, independent of protein or chemical patterning [18] . By forming well-defined corrugated hydrogels, it was shown that substrate curvature can affect the thickness of epithelial monolayers and that local curvature controls nuclear morphology and positioning.…”

Biomimetic Convex Implant for Corneal Regeneration Through 3D Printing

“…The resulting implants possessed curved structures that could guide cell organization and adhesion. First, as the printed implant was a dome-shaped cornea and cells tended to react to topographical cues, [22] we sought to document how the slope gradient of the convex structure mediated cell behavior by cytoskeleton/nuclei analysis, and a finite element method (FEM) simulation [23] was constructed to reveal dynamic interaction between cell tension and adhesion of cell-substrate, and a signaling pathway was also proposed to analyze adhesion behavior. Second, we showed that the superior convex structures could promote epithelialization, cell adhesion, and neuron regeneration to heal corneal defects after transplantation of printed convex implants into a rabbit partial keratectomy corneal injury model within 180 days of observation, which opens new avenues for material design.…”