Tomographic volumetric bioprinting (VBP) enables fast photofabrication of cell‐laden hydrogel constructs in one step, addressing the limitations of conventional layer‐by‐layer additive manufacturing. However, existing biomaterials that fulfill the physicochemical requirements of VBP are limited to gelatin‐based photoresins of high polymer concentrations. The printed microenvironments are predominantly static and stiff, lacking sufficient capacity to support 3D cell growth. Here a dynamic resin based on thiol–ene photo‐clickable polyvinyl alcohol (PVA) and thermo‐sensitive sacrificial gelatin for fast VBP of functional ultrasoft cell‐laden hydrogel constructs within 7–15 s is reported. Using gelatin allows VBP of permissive hydrogels with low PVA contents of 1.5%, providing a stress‐relaxing environment for fast cell spreading, 3D osteogenic differentiation of embedded human mesenchymal stem cells and matrix mineralization. Additionally, site‐specific immobilization of molecules‐of‐interest inside a PVA hydrogel is achieved by 3D tomographic thiol–ene photopatterning. This technique may enable spatiotemporal control of cell‐material interactions and guides in vitro tissue formation using programmed cell‐friendly light. Altogether, this study introduces a synthetic dynamic photoresin enabling fast VBP of functional ultrasoft hydrogel constructs with well‐defined physicochemical properties and high efficiency.

T cells sense and respond to their local environment at the nanoscale by forming small actin-rich protrusions, called microvilli, which play critical roles in signaling and antigen recognition, particularly at the interface with the antigen presenting cells. However, the mechanism by which microvilli contribute to cell signaling and activation is largely unknown. Here, we present a tunable engineered system that promotes microvilli formation and T cell signaling via physical stimuli. We discovered that nanoporous surfaces favored microvilli formation and markedly altered gene expression in T cells and promoted their activation. Mechanistically, confinement of microvilli inside of nanopores leads to size-dependent sorting of membrane-anchored proteins, specifically segregating CD45 phosphatases and T cell receptors (TCR) from the tip of the protrusions when microvilli are confined in 200-nm pores but not in 400-nm pores. Consequently, formation of TCR nanoclustered hotspots within 200-nm pores allows sustained and augmented signaling that prompts T cell activation even in the absence of TCR agonists. The synergistic combination of mechanical and biochemical signals on porous surfaces presents a straightforward strategy to investigate the role of microvilli in T cell signaling as well as to boost T cell activation and expansion for application in the growing field of adoptive immunotherapy.

T cells sense and respond to their local environment at the nanoscale by forming small actin-rich protrusions, called microvilli, which play critical roles in signaling and antigen recognition, particularly at the interface with the antigen presenting cells. However, the mechanisms by which microvilli contribute to cell signaling and activation is largely unknown. Here, we present a tunable engineered system that promotes microvilli formation and T cell signaling via physical stimuli. We discovered that nanoporous surfaces favored microvilli formation, and markedly altered gene expression in T cells and promoted their activation. Mechanistically, confinement of microvilli inside of nanopores leads to size-dependent sorting of membrane-anchored proteins, specifically segregating CD45 phosphatases and T cell receptors (TCR) from the tip of the protrusions when microvilli are confined in 200 nm pores, but not in 400 nm pores. Consequently, formation of TCR nanoclustered hotspots within 200 nm pores, allows sustained and augmented signaling that prompts T cell activation even in the absence of TCR agonists. The synergistic combination of mechanical and biochemical signals on porous surfaces presents a straightforward strategy to investigate the role of microvilli in T cell signaling as well as to boost T cell activation and expansion for application in the growing field of adoptive immunotherapy.

Tomographic volumetric bioprinting (VBP) enables fast photofabrication of 3D cell-laden hydrogel constructs in one step, addressing the limitations of conventional layer-by-layer additive manufacturing. However, existing biomaterials that fulfill the physicochemical requirements of VBP are limited to photocurable protein derivatives. Reported here is the first synthetic resin composed of thiol-ene photo-clickable polyvinyl alcohol (PVA) for ultrafast VBP of defined 3D tissue models within 7-15 seconds. The incorporation of gelatin as a temporary thermo-reversible network allows VBP of perfusable hydrogels with PVA contents as low as 1.5%, providing a stress-relaxing environment for fast cell spreading and 3D growth of embedded human mesenchymal stem cells. Additionally, tomographic 4D photopatterning of a thiolated molecule of interest is demonstrated for site-specific immobilization of chemical cues within a pre-printed PVA hydrogel within seconds. Altogether, this study introduces a synthetic biomaterial enabling ultrafast VBP of functional hydrogel constructs with well-defined physicochemical properties with unprecedented efficiency.