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Patterned deposition and 3D fabrication techniques have enabled the use of hydrogels for a number of applications including microfluidics, sensors, separations, and tissue engineering in which form fits function. Devices such as reconfigurable microvalves or implantable tissues have been created using lithography or casting techniques. Here, we present a novel open-microfluidic patterning method that utilizes surface tension forces to form hydrogel layers on top of each other, into a patterned 3D structure. We use a patterning device to form a temporary open microfluidic channel on an existing gel layer, allowing the controlled flow of unpolymerized gel in device-regions. After layer gelation and device removal, the process can be repeated iteratively to create multi-layered 3D structures. The use of open-microfluidic and surface tension-based methods to define the shape of each individual layer enables patterning to be performed with a simple pipette and with minimal dead-volume. Our method is compatible with unmodified (native) biological hydrogels, and other non-biological materials with precursor fluid properties compatible with capillary flow. With our open-microfluidic layer-by-layer fabrication method, we demonstrate the capability to build agarose, type I collagen, and polymer–peptide 3D structures featuring asymmetric designs, multiple components, overhanging features, and cell-laden regions. 

Stimuli‐responsive biomaterials show great promise for modeling disease dynamics ex vivo with spatiotemporal control over the cellular microenvironment. However, harvesting cells from such materials for downstream analysis without perturbing their state remains an outstanding challenge in 3/4‐dimensional (3D/4D) culture and tissue engineering. In this manuscript, a fully enzymatic strategy for hydrogel degradation that affords spatiotemporal control over cell release while maintaining cytocompatibility is introduced. Exploiting engineered variants of the sortase transpeptidase evolved to recognize and selectively cleave distinct peptide sequences largely absent from the mammalian proteome, many limitations implicit to state‐of‐the‐art methods to liberate cells from gels are sidestepped. It is demonstrated that evolved sortase exposure has minimal impact on the global transcriptome of primary mammalian cells and that proteolytic cleavage proceeds with high specificity; incorporation of substrate sequences within hydrogel crosslinkers permits rapid and selective cell recovery with high viability. In composite multimaterial hydrogels, it is shown that sequential degradation of hydrogel layers enables highly specific retrieval of single‐cell suspensions for phenotypic analysis. It is expected that the high bioorthogonality and substrate selectivity of the evolved sortases will lead to their broad adoption as an enzymatic material dissociation cue and that their multiplexed use will enable newfound studies in 4D cell culture.