Highly Tunable and Cell‐Remodelable Thiol‐ene Alginate‐Peptide Crosslinked Hydrogels to Recreate Cellular and Organoid Microenvironments for Biofabrication

In this study, a cell-mediated degradable alginate hydrogel system for organoid culture and amenable to biofabrication technologies is presented. Norbornene-functionalized alginate is crosslinked with a di-thiolated peptide sequence cleavable by matrix metalloproteinases and decorated with cysteine-terminated cell-adhesion peptide RGD, upon exposure to UV. Stiffness of the hydrogels can be controlled by tuning polymer and crosslinker concentrations. Pre-gel solutions are successfully bioprinted with a pneumatic extrusion-based system. The hydrogels are used to encapsulate a variety of sensitive cell types. Human endometrial organoids present high cell viability, grow in size over time, present spherical morphology, and express cell-cell contacts E-cadherin and proliferation marker Ki67. Encapsulated mouse embryonic stem cell-derived thyroid follicles produce thyroglobulin and T4. Mouse intestinal organoids adopt a proliferative phenotype. Vascularization inside the hydrogels is achieved using endothelial cells and supporting cells (single cell suspension and spheroids). Neurite outgrowth, both small and thick bundles, from encapsulated iPSC-derived neurospheres, demonstrates the reinnervation potential of the hydrogel. This polysaccharide hydrogel platform could be used as a defined, tunable, and ethical alternative to mouse sarcoma-extracted basement-membrane matrices.