Dynamic Exchange in 3D Cell Culture Hydrogels Based on Crosslinking of Cyclic Thiosulfinates.

Dynamic polymer materials are highly valued substrates for 3D cell culture due to their viscoelasticity, a time-dependent mechanical property that can be tuned to resemble the energy dissipation of native tissues. Herein, we report the coupling of a cyclic thiosulfinate, mono-S-oxo-4-methyl asparagusic acid, to a 4-arm PEG-OH to prepare a disulfide-based dynamic covalent hydrogel with the addition of 4-arm PEG-thiol. Ring opening of the cyclic thiosulfinate by nucleophilic substitution results in the rapid formation of a network showing a viscoelastic fluid-like behaviour and relaxation rates modulated by thiol content through thiol-disulfide exchange, whereas its viscoelastic behaviour upon application as a small molecule linear crosslinker is solid-like. Further introduction of 4-arm PEG-vinylsulfone in the network yields a hydrogel with weeks-long cell culture stability, permitting 3D culture of cell types that lack robust proliferation, such as human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). These cells display native behaviours such as cell elongation and spontaneous beating as a function of the hydrogel's mechanical properties. We demonstrate that the mode of dynamic cyclic thiosulfinate crosslinker presentation within the network can result in different stress relaxation profiles, opening the door to model tissues with disparate mechanics in 3D cell culture.

Shape-Morphing Photoresponsive Hydrogels Reveal Dynamic Topographical Conditioning of Fibroblasts.

The extracellular environment defines a physical boundary condition with which cells interact. However, to date, cell response to geometrical environmental cues is largely studied in static settings, which fails to capture the spatiotemporally varying cues cells receive in native tissues. Here, a photoresponsive spiropyran-based hydrogel is presented as a dynamic, cell-compatible, and reconfigurable substrate. Local stimulation with blue light (455 nm) alters hydrogel swelling, resulting in on-demand reversible micrometer-scale changes in surface topography within 15 min, allowing investigation into cell response to controlled geometry actuations. At short term (1 h after actuation), fibroblasts respond to multiple rounds of recurring topographical changes by reorganizing their nucleus and focal adhesions (FA). FAs form primarily at the dynamic regions of the hydrogel; however, this propensity is abolished when the topography is reconfigured from grooves to pits, demonstrating that topographical changes dynamically condition fibroblasts. Further, this dynamic conditioning is found to be associated with long-term (72 h) maintenance of focal adhesions and epigenetic modifications. Overall, this study offers a new approach to dissect the dynamic interplay between cells and their microenvironment and shines a new light on the cell's ability to adapt to topographical changes through FA-based mechanotransduction.

Generation of Multicue Cellular Microenvironments by UV-photopatterning of Three-dimensional Cell Culture Substrates.

The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand distribution and tissue geometry, have been increasingly shown to play critical roles in governing cell phenotype and behavior. However, these environmental cues and their effects on cells are often studied separately using in vitro platforms that isolate individual cues, a strategy that heavily oversimplifies the complex in vivo situation of multiple cues. Engineering approaches can be particularly useful to bridge this gap, by developing experimental setups that capture the complexity of the in vivo microenvironment, yet retain the degree of precision and manipulability of in vitro systems. This study highlights an approach combining ultraviolet (UV)-based protein patterning and lithography-based substrate microfabrication, which together enable high-throughput investigation into cell behaviors in multicue environments. By means of maskless UV-photopatterning, it is possible to create complex, adhesive protein distributions on three-dimensional (3D) cell culture substrates on chips that contain a variety of well-defined geometrical cues. The proposed technique can be employed for culture substrates made from different polymeric materials and combined with adhesive patterned areas of a broad range of proteins. With this approach, single cells, as well as monolayers, can be subjected to combinations of geometrical cues and contact guidance cues presented by the patterned substrates. Systematic research using combinations of chip materials, protein patterns, and cell types can thus provide fundamental insights into cellular responses to multicue environments.