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Abstract As the most versatile and promising cell source, stem cells have been studied in regenerative medicine for two decades. Currently available culturing techniques utilize a 2D or 3D microenvironment for supporting the growth and proliferation of stem cells. However, these culture systems fail to fully reflect the supportive biological environment in which stem cells reside in vivo, which contain dynamic biophysical growth cues. Herein, a 4D programmable culture substrate with a self‐morphing capability is presented as a means to enhance dynamic cell growth and induce differentiation of stem cells. To function as a model system, a 4D neural culture substrate is fabricated using a combination of printing and imprinting techniques keyed to the different biological features of neural stem cells (NSCs) at different differentiation stages. Results show the 4D culture substrate demonstrates a time‐dependent self‐morphing process that plays an essential role in regulating NSC behaviors in a spatiotemporal manner and enhances neural differentiation of NSCs along with significant axonal alignment. This study of a customized, dynamic substrate revolutionizes current stem cell therapies, and can further have a far‐reaching impact on improving tissue regeneration and mimicking specific disease progression, as well as other impacts on materials and life science research.
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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.
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- PAR ID:
- 10403854
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Advanced Materials
- Volume:
- 35
- Issue:
- 19
- ISSN:
- 0935-9648
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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