User-Controlled 4D Biomaterial Degradation with Substrate-Selective Sortase Transpeptidases for Single-Cell Biology.

Stimuli-responsive biomaterials show great promise for modelling 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 3D/4D culture and tissue engineering. In this manuscript, we introduce a fully enzymatic strategy for hydrogel degradation that affords spatiotemporal control over cell release while maintaining cytocompatibility. Exploiting engineered variants of the sortase transpeptidase evolved to recognize and selectively cleave distinct peptide sequences largely absent from the mammalian proteome, we sidestep many limitations implicit to state-of-the-art methods to liberate cells from gels. We demonstrate 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, we show that sequential degradation of hydrogel layers enables highly specific retrieval of single-cell suspensions for phenotypic analysis. We expect 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. This article is protected by copyright. All rights reserved.