MAGIC matrices: freeform bioprinting materials to support complex and reproducible organoid morphogenesis.

Organoids are powerful models of tissue physiology, yet their applications remain limited due to a lack of complex tissue morphology and high organoid-to-organoid structural variability. To address these limitations we developed a soft, composite yield-stress extracellular matrix that supports freeform 3D bioprinting of cell slurries at tissue-like densities. Combined with a custom piezoelectric printhead, this platform allows more reproducible and complex morphogenesis from uniform and spatially organized organoid "seeds." At 4 °C the material exhibits reversible yield-stress behavior to support long printing times without compromising cell viability. When transferred to cell culture at 37 °C, the material cross-links and exhibits similar viscoelasticity and plasticity to basement membrane extracts such as Matrigel. We use this setup for high-throughput generation of intestinal and salivary gland organoid arrays that are morphologically indistinguishable from those grown in pure Matrigel, but exhibit dramatically improved homogeneity in organoid size, shape, maturation time, and budding efficiency. The reproducibility of organoid structure afforded by this approach increases the sensitivity of assays by orders of magnitude, requiring less input material and reducing analysis times. The flexibility of this approach additionally enabled the fabrication of perfusable intestinal organoid tubes. Combined, these advances lay the foundation for the efficient design of complex tissue morphologies in both space and time.

A Balance between Inter- and Intra-Microgel Mechanics Governs Stem Cell Viability in Injectable Dynamic Granular Hydrogels.

Injectable hydrogels are increasingly explored for the delivery of cells to tissue. These materials exhibit both liquid-like properties, protecting cells from mechanical stress during injection, and solid-like properties, providing a stable 3D engraftment niche. Many strategies for modulating injectable hydrogels tune liquid- and solid-like material properties simultaneously, such that formulation changes designed to improve injectability can reduce stability at the delivery site. The ability to independently tune liquid- and solid-like properties would greatly facilitate formulation development. Here, such a strategy is presented in which cells are ensconced in the pores between microscopic granular hyaluronic acid (HA) hydrogels (microgels), where elasticity is tuned with static covalent intra-microgel crosslinks and flowability with mechanosensitive adamantane-cyclodextrin (AC) inter-microgel crosslinks. Using the same AC-free microgels as a 3D printing support bath, the location of each cell is preserved as it exits the needle, allowing identification of the mechanism driving mechanical trauma-induced cell death. The microgel AC concentration is varied to find the threshold from microgel yielding- to AC interaction-dominated injectability, and this threshold is exploited to fabricate a microgel with better injection-protecting performance. This delivery strategy, and the balance between intra- and inter-microgel properties it reveals, may facilitate the development of new cell injection formulations.

Correction: 3D aggregation of cells in packed microgel media.

Correction for '3D aggregation of cells in packed microgel media' by Cameron D. Morley et al., Soft Matter, 2020, DOI: 10.1039/d0sm00517g.

3D aggregation of cells in packed microgel media.

In both natural and applied contexts, investigating cell self-assembly and aggregation within controlled 3D environments leads to improved understanding of how structured cell assemblies emerge, what determines their shapes and sizes, and whether their structural features are stable. However, the inherent limits of using solid scaffolding or liquid spheroid culture for this purpose restrict experimental freedom in studies of cell self-assembly. Here we investigate multi-cellular self-assembly using a 3D culture medium made from packed microgels as a bridge between the extremes of solid scaffolds and liquid culture. We find that cells dispersed at different volume fractions in this microgel-based 3D culture media aggregate into clusters of different sizes and shapes, forming large system-spanning networks at the highest cell densities. We find that the transitions between different states of assembly can be controlled by the level of cell-cell cohesion and by the yield stress of the packed microgel environment. Measurements of aggregate fractal dimension show that those with increased cell-cell cohesion are less sphere-like and more irregularly shaped, indicating that cell stickiness inhibits rearrangements in aggregates, in analogy to the assembly of colloids with strong cohesive bonds. Thus, the effective surface tension often expected to emerge from increased cell cohesion is suppressed in this type of cell self-assembly.

Anthracene-based mechanophores for compression-activated fluorescence in polymeric networks.

The recent attention given to functionalities that respond to mechanical force has led to a deeper understanding of force transduction and mechanical wear in polymeric materials. Furthermore, polymers have been carefully designed such that activation of "mechanophores" leads to productive outputs, such as material reinforcement or changes in optical properties. In this work, a crosslinker containing an anthracene-maleimide linkage was designed and used to prepare networks that display a fluorescence response when damaged. The pressure-dependent damage of poly(N,N-dimethylacrylamide) networks was monitored using solid-state fluorescence spectroscopy, with increasing compressive forces leading to higher degrees of mechanophore activation. When a stamp was used to compress the networks, only the areas in contact with the raised portion of the stamp underwent mechanophore activation, resulting in the generation of patterns that were only visible under UV light. Finally, an isomeric "flex" mechanophore was designed and used to prepare networks that were compressed and compared to the previously described networks.

Quantitative characterization of 3D bioprinted structural elements under cell generated forces.

With improving biofabrication technology, 3D bioprinted constructs increasingly resemble real tissues. However, the fundamental principles describing how cell-generated forces within these constructs drive deformations, mechanical instabilities, and structural failures have not been established, even for basic biofabricated building blocks. Here we investigate mechanical behaviours of 3D printed microbeams made from living cells and extracellular matrix, bioprinting these simple structural elements into a 3D culture medium made from packed microgels, creating a mechanically controlled environment that allows the beams to evolve under cell-generated forces. By varying the properties of the beams and the surrounding microgel medium, we explore the mechanical behaviours exhibited by these structures. We observe buckling, axial contraction, failure, and total static stability, and we develop mechanical models of cell-ECM microbeam mechanics. We envision these models and their generalizations to other fundamental 3D shapes to facilitate the predictable design of biofabricated structures using simple building blocks in the future.