Professor and Department Chair Tufts University Medford, Massachusetts, United States
Introduction: Hydrogels are water-swollen polymer networks that provide unique opportunities as separations modules, adhesives, and biomaterials. New hydrogel formulations are being developed every day, primarily for biomedical applications, where they are used as controllable bioactive environments to model cell-environment interactions and scale up tissue engineering toward hierarchical structures that cannot be efficiently produced with cells alone. These tissue-mimicking, 3D hydrogels are capable of modeling disease development and testing how cell-microenvironment interactions affect drug treatment efficacy. However, the 3D microenvironments introduce multifaceted design challenges, including simultaneously matching the stiffness, transport properties, and functional biochemical features of native tissue extracellular matrix. Prior work in the Peyton lab has established hydrogel formulations that match the stiffness and biochemical features of human tissues including brain, lung, and bone marrow, but solute transport effects in these hydrogels have not been investigated. Since synthetic polymer hydrogels typically have relatively homogeneous mesh sizes that are orders of magnitude smaller than those of gelatin, collagen, and Matrigel, they are likely much more restrictive to solute transport than those protein-based hydrogels, even when matching stiffness. Here, we use model-based, modular hydrogel design to investigate the role of microenvironment-restricted solute transport on hydrogel-encapsulated cell behaviors.
Materials and
Methods: Multi-arm, vinyl sulfone(VS)-terminated poly(ethylene glycol) (PEG) macromers were crosslinked with PEG-dithiol, matrix metalloproteinase (MMP)-degradable peptides, and pendant integrin-binding peptides to create cell-adhesive, biodegradable 3D hydrogel microenvironments. The number and length of arms per PEG-VS macromer were systematically varied to create hydrogels with equivalent shear modulus to a Matrigel hydrogel, measured by low-deformation force-indentation experiments. Gelation time was measured via rheology, and homogeneous gelation was confirmed via fluorescent peptide distribution and 3D microscopy of the distribution of gel-encapsulated HCC 1954 breast cancer cells. Solute transport through gels was measured using fluorescence recovery after photobleaching (FRAP) and transwell transport assays with a series of fluorescently tagged dextrans up to 150 kDa in size. Protein transwell transports assays using BSA and serum-containing media confirmed that solute restriction was not specific to dextrans.
Results, Conclusions, and Discussions: PEG-VS hydrogels spontaneously crosslink in 30 minutes under pH-neutral, physiological conditions without cytotoxic catalysts, making them ideal for encapsulating stress-sensitive cell types. Incorporation of integrin-binding peptides and MMP-degradable peptides do not significantly alter hydrogel stiffness or solute transport properties. However, solute transport is greatly restricted in PEG hydrogels compared to Matrigel, especially for large solute such as cytokines, serum proteins, and antibodies. Access to serum proteins is critical for cell growth and survival, indicating that solute transport remains a limiting factor in the applicability of synthetic hydrogels to 3D encapsulation culture of cells, even after issues of heterogeneity, gelation conditions cytotoxicity, and stiffness matching are addressed. Further optimization of solute transport in hydrogels while keeping other cell-relevant properties unchanged requires insightful hydrogel design and further basic research into how to manipulate and measure solute transport within hydrogels. Structurally decoupling stiffness and solute transport within synthetic hydrogels using the swollen polymer network model (see hydrogeldesign.org) and introducing high-transport void spaces via granular hydrogels provide two distinct, exciting avenues for further designing hydrogels for 3D encapsulation cell culture. Refining hydrogel design for predictable and adaptive solute transport alongside stiffness, biocompatibility, and other cell-relevant properties will support 3D scaling of in vitro cancer modeling and tissue engineering as well as drug delivery applications.
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