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

Abstract

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.

A unique binding mode of P1′ Leu-containing target sequences for Streptococcus pyogenes sortase A results in alternative cleavage

“…This mode of reactivity potentially has uses in the context of SML technology, where recent work has highlighted the utility of sortase-substrate pairs that offer alternatives to the canonical LPXTG motif. 8,45,46 This work also contributes to the fundamental understanding of substrate recognition by sortases, providing further evidence that sortases within the same class can exhibit significant differences in substrate selectivity. We anticipate that a thorough understanding of these differences, particularly at the structural level, may prove valuable for both the continued development of SML and the design of therapeutics that target sortases in vivo .…”

Degradable and Multifunctional PEG-Based Hydrogels Formed by iEDDA Click Chemistry with Stable Click-Induced Supramolecular Interactions

“…Nonetheless, this new iEDDA-CSI gelation may benefit light and radical-free hydrogel cross-linking and cell/protein encapsulation. In contrast, the highly tunable and predictable degradation of these hydrogels could be leveraged for autonomous cell/spheroid recovery without using external degradation triggers, such as temperature change or supplying proteases, two common approaches reported in the literature. , In addition, the new iEDDA-CSI assembled and degradable hydrogels may be leveraged for in vivo drug and cell delivery, , since the degradation time is ideal for the stability of protein-based therapeutics which range from days to months in an in vivo setting …”

Bioprinted Scaffold Remodels the Neuromodulatory Microenvironment for Enhancing Bone Regeneration

“…Degradation behavior plays an important role in controlling the rate of drug release and achieving tissue regeneration. [ 27 ] Therefore, the in vitro degradation kinetics of the composite hydrogels were studied in phosphate‐buffered saline (PBS) at 37 °C. The in vitro degradation curves showed that the pristine GelMA hydrogels degraded at a faster rate.…”