Advanced hydrogel systems that allow precise control of cells and their 3D microenvironments are needed in tissue engineering, disease modeling, and drug screening. Multiphoton lithography (MPL) allows true 3D microfabrication of complex objects, but its biological application requires a cell-compatible hydrogel resist that is sufficiently photosensitive, cell-degradable, and permissive to support 3D cell growth. Here, an extremely photosensitive cell-responsive hydrogel composed of peptide-crosslinked polyvinyl alcohol (PVA) is designed to expand the biological applications of MPL. PVA hydrogels are formed rapidly by ultraviolet light within 1 min in the presence of cells, providing fully synthetic matrices that are instructive for cell-matrix remodeling, multicellular morphogenesis, and protease-mediated cell invasion. By focusing a multiphoton laser into a cell-laden PVA hydrogel, cell-instructive extracellular cues are site-specifically attached to the PVA matrix. Cell invasion is thus precisely guided in 3D with micrometer-scale spatial resolution. This robust hydrogel enables, for the first time, ultrafast MPL of cell-responsive synthetic matrices at writing speeds up to 50 mm s . This approach should enable facile photochemical construction and manipulation of 3D cellular microenvironments with unprecedented flexibility and precision.
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