Hyaluronan Hydrogel "Safety Nets" for 3D Cell Culture Applications

Polymeric hydrogels can mimic features of native extracellular matrix (ECM), and their facile production and characterization enable customizable cell encapsulation and 3-dimensional (3D) culture. These have applications relevant to dentistry such as soft tissue engineering, cancer modeling, and drug screening. Hyaluronan (HA), a biologically-derived polymer found in native ECM, offers an optimal platform for this use, as it can be modified covalently with adhesive ligands, enzyme-degradable crosslinkers, and other biorelevant moieties, yielding tailored physical properties and biological response. Cell-directed degradation of these encapsulating matrices can be a prerequisite for phenotype preservation, but degradation kinetics may not match desired timelines for drug screening applications. This study focused on optimizing hydrogel composition, network structure, and gelation kinetics to preserve z-distribution of physiologically relevant cells within high-throughput microfluidic plates. Hydrogels were formed from aqueous solutions of thiolated HA (HA-SH), bifunctional acrylated poly(ethylene glycol)-peptide crosslinkers, and pendant acrylated peptides (RGD or YIGSR sequences) to support cell adhesion. By varying the absolute crosslinker concentration and relative proportions of high:low crosslinker degradation kinetics, hydrogels could be tuned to desired moduli (G: [~]10-120 Pa) and enzymatic degradation rate. Ratios of high:low degradable crosslinkers, at equivalent total crosslinker concentration, minimally impacted final modulus or gelation rate. Similarly, pendant adhesive ligands were swapped easily with negligible impact on hydrogel physical properties. Hydrogels with a 50:50 ratio of high:low degradable crosslinkers provided a "safety net" that preserved z-distribution of encapsulated bone marrow-derived fibroblasts, while maintaining expected phenotype. A comparable system supported the 3D co-culture of primary human salivary epithelial and mesenchymal cells within a perfusable microfluidic multiwell plate. This customizable bottom-up construction reduced confounding factors encountered in hybridoma-derived protein matrices, enabling modular customization of physiologically relevant, yet reproducible matrices to replicate native ECM. Our optimized model demonstrates workflows to improve future pharmaceutical screens, and tailor tissue engineering applications.