Tunable hydrogel networks by varying secondary structures of hydrophilic peptoids provide viable 3D cell culture platforms for hMSCs.

Hydrogels have excellent ability to mimic the extracellular matrix (ECM) during 3D cell culture, yet it remains difficult to tune their mechanical properties without also changing network connectivity. Previously, we developed 2D culture platforms based on tunable hydrogels crosslinked by peptoids with various secondary structures: helical, non-helical, and unstructured, which allowed control over hydrogel mechanics independent of network connectivity. Here, we extend our strategy to 3D matrices by modifying the peptoids with piperazine and homopiperazine residues to enhance water solubility without altering their secondary structure. Hydrogels crosslinked with helical peptoids exhibited significantly higher stiffness compared to hydrogels crosslinked with non-helical or unstructured peptoids. Human mesenchymal stem cells (hMSCs) encapsulated within these hydrogels were assessed for viability, proliferation, and immunomodulatory potential. The stiffest hydrogels promoted the highest rates of proliferation and increased yes-associated protein (YAP) nuclear localization. Softer hydrogels, however, showed enhanced production of indoleamine 2,3-dioxygenase (IDO), both with and without interferon gamma (IFN-γ) stimulation, highlighting their potential in immunomodulatory applications. The biomimetic platform developed here enables the study of how matrix mechanics influence stem cell behavior without confounding factors from network connectivity, leading to insights for hMSC-mediated immunomodulation.