Biomechanical and Biological Characterization of XGel, a Human-Derived Hydrogel for Stem Cell Expansion and Tissue Engineering.

Hydrogels are 3D scaffolds used as alternatives to in vivo models for disease modeling and delivery of cells and drugs. Existing hydrogel classifications include synthetic, recombinant, chemically defined, plant- or animal-based, and tissue-derived matrices. There is a need for materials that can support both human tissue modeling and clinically relevant applications requiring stiffness tunability. Human-derived hydrogels are not only clinically relevant, but they also minimize the use of animal models for pre-clinical studies. This study aims to characterize XGel, a new human-derived hydrogel as an alternative to current murine-derived and synthetic recombinant hydrogels that features unique physiochemical, biochemical, and biological properties that support adipocyte and bone differentiation. Rheology studies determine the viscosity, stiffness, and gelation features of XGel. Quantitative studies for quality control support consistency in the protein content between lots. Proteomics studies reveal that XGel is predominantly composed of extracellular matrix proteins, including fibrillin, collagens I-VI, and fibronectin. Electron microscopy of the hydrogel provides phenotypic characteristics in terms of porosity and fiber size. The hydrogel demonstrates biocompatibility as a coating material and as a 3D scaffold for the growth of multiple cell types. The results provide insight into the biological compatibility of this human-derived hydrogel for tissue engineering.

Generation of 3D Spheroids Using a Thiol-Acrylate Hydrogel Scaffold to Study Endocrine Response in ER+ Breast Cancer.

Culturing cancer cells in a three-dimensional (3D) environment better recapitulates in vivo conditions by mimicking cell-to-cell interactions and mass transfer limitations of metabolites, oxygen, and drugs. Recent drug studies have suggested that a high rate of preclinical and clinical failures results from mass transfer limitations associated with drug entry into solid tumors that 2D model systems cannot predict. Droplet microfluidic devices offer a promising alternative to grow 3D spheroids from a small number of cells to reduce intratumor heterogeneity, which is lacking in other approaches. Spheroids were generated by encapsulating cells in novel thiol-acrylate (TA) hydrogel scaffold droplets followed by on-chip isolation of single droplets in a 990- or 450-member trapping array. The TA hydrogel rapidly (∼35 min) polymerized on-chip to provide an initial scaffold to support spheroid development followed by a time-dependent degradation. Two trapping arrays were fabricated with 150 or 300 µm diameter traps to investigate the effect of droplet size and cell seeding density on spheroid formation and growth. Both trapping arrays were capable of ∼99% droplet trapping efficiency with ∼90% and 55% cellular encapsulation in trapping arrays containing 300 and 150 µm traps, respectively. The oil phase was replaced with media ∼1 h after droplet trapping to initiate long-term spheroid culturing. The growth and viability of MCF-7 3D spheroids were confirmed for 7 days under continuous media flow using a customized gravity-driven system to eliminate the need for syringe pumps. It was found that a minimum of 10 or more encapsulated cells are needed to generate a growing spheroid while fewer than 10 parent cells produced stagnant 3D spheroids. As a proof of concept, a drug susceptibility study was performed treating the spheroids with fulvestrant followed by interrogating the spheroids for proliferation in the presence of estrogen. Following fulvestrant exposure, the spheroids showed significantly less proliferation in the presence of estrogen, confirming drug efficacy.