Spherical microwell arrays for studying single cells and microtissues in 3D confinement

Abstract

Microwell arrays have emerged as three-dimensional substrates for cell culture due to their simplicity of fabrication and promise for high-throughput applications such as 3D cell-based assays for drug screening. To date, most microwells have had cylindrical geometries. Motivated by our previous findings that cells display 3D physiological characteristics when grown in the spherical micropores of monodisperse foam scaffolds (Lee et al 2013 Integr. Biol. 5 1447–55 and Lin et al 2011 Soft Matter 7 10010–6), here we engineered novel microwells shaped as spherical caps with obtuse polar angles, yielding narrow apertures. When used as bare substrates, these microwells were suitable for culturing cell spheroids; the narrow apertures sterically hindered unattached cultured cells from rolling out of microwells under agitation. When only the walls of the microwell were conjugated with extracellular matrix proteins, cells remained confined in the microwells. Epithelial cells proliferated and burst out of the aperture, and cell polarity was oriented based on the distribution of extracellular matrix proteins in the microwells. Surprisingly, single fibroblast cells in spherical wells of various diameters (40–100 μm) underwent cell-cycle arrest, while cells in circular cylindrical microwells continued to proliferate. Spatial confinement was not sufficient to cause cell-cycle arrest; however, confinement in a constant negative-curvature microenvironment led to cell-cycle arrest. Overall, these investigations demonstrate that this spherical microwell substrate constitutes a novel basic research tool for elucidating how cells respond to dimensionality and microenvironment with radii of curvature at the cellular length scale.

3D printed silk-gelatin hydrogel scaffold with different porous structure and cell seeding strategy for cartilage regeneration

“…Cells seeded on scaffolds or cultured in monolayers, microarrays, or pellets assume varied morphologies and secrete different signaling molecules [ 34 , 37 ]. Recently, 3D cellular aggregates, instead of 2D cell sheets, have been employed as building blocks in order to understand the principles of cell–cell and cell–matrix adhesion [ 58 , 59 ]. Cell aggregates can better simulate real cell morphology, fate, and metabolic activities in vivo .…”

A matrigel-free method to generate matured human cerebral organoids using 3D-Printed microwell arrays

“…Specifically, the provided concave spherical curvature and high spatial confinement were found to promote 3D cell aggregation in spheroids [ 25 , 30 , 38 ], very similar to the vital influence of the skull in the brain development [ [75] , [76] , [77] , [78] ]. In EB formation, these two features were further shown to increase the cell packing density [ 37 ], form tighter junctions, and modulate ECM networks [ 36 , 37 ]. Previous studies also established that microwell device's 3D topography and surface coating could affect cell behaviors (e.g., proliferation, differentiation) [ 17 , 41 , 79 , 80 ] and tissue morphogenesis (e.g., surface wrinkling) [ 10 , 81 ].…”

“…Lately, microwell-derived EBs were further cultured to develop into organoids [ 17 , 34 ], however, still relying on Matrigel-embedding for modulating the organoid micro-environment [ 13 , 17 , 34 ]. In addition, most of the microwell generated spheroids (structureless aggregation) [ 36 , 37 ] or spheroid-like organoids (e.g. Islet organoids [ 21 , 38 ]) have much lower structural and developmental complexity compared to brain organoids.…”

Surface curvature and basal hydraulic stress induce spatial bias in cell extrusion

“…Being water and solute permeable materials, hydrogels can buffer against drastic increase in solute concentrations, thereby lowering osmosis induced hydraulic stress. To eliminate the possibility that any extrusion differences were due to hydrogels being a much softer material (∼30 kPA [16]), MDCK monolayers cultured on silicone substrates of similar stiffness (∼25 kPA [17]) were also tracked (Fig. 3e).…”