Granular Hydrogels for Harnessing the Immune Response.

This review aims to understand the current progress in immune-instructive granular hydrogels and identify the key features used as immunomodulatory strategies. Published work is systematically reviewed and relevant information about granular hydrogels used throughout these studies is collected. The base polymer, microgel generation technique, polymer crosslinking chemistry, particle size and shape, annealing strategy, granular hydrogel stiffness, pore size and void space, degradability, biomolecule presentation, and drug release are cataloged for each work. Several granular hydrogel parameters used for immune modulation: porosity, architecture, bioactivity, drug release, cell delivery, and modularity, are identified. The authors found in this review that porosity is the most significant factor influencing the innate immune response to granular hydrogels, while incorporated bioactivity is more significant in influencing adaptive immune responses. Here, the authors' findings and summarized results from each section are presented and suggestions are made for future studies to better understand the benefits of using immune-instructive granular hydrogels.

Functional Interrogation of Ca2+ Signals in Human Cancer Cells In Vitro and Ex Vivo by Fluorescent Microscopy and Molecular Tools.

Genetically encoded calcium indicators (GECIs) and high-resolution confocal microscopy enable dynamic visualization of calcium signals in cells and tissues. Two-dimensional and 3D biocompatible materials mimic the mechanical microenvironments of tumor and healthy tissues in a programmable manner. Cancer xenograft models and ex vivo functional imaging of tumor slices reveal physiologically relevant functions of calcium dynamics in tumors at different progression stages. Integration of these powerful techniques allows us to quantify, diagnose, model, and understand cancer pathobiology. Here, we describe detailed materials and methods used to establish this integrated interrogation platform, from generating transduced cancer cell lines that stably express CaViar (GCaMP5G + QuasAr2) to in vitro and ex vivo calcium imaging of the cells in 2D/3D hydrogels and tumor tissues. These tools open the possibility for detailed explorations of mechano-electro-chemical network dynamics in living systems.

Single versus dual microgel species for forming guest-host microporous annealed particle PEG-MAL hydrogel.

Inter-particle secondary crosslinks allow microporous annealed particle (MAP) hydrogels to be formed. Methods to introduce secondary crosslinking networks in MAP hydrogels include particle jamming, annealing with covalent bonds, and reversible noncovalent interactions. Here, we investigate the effect of two different approaches to secondary crosslinking of polyethylene glycol (PEG) microgels via reversible guest-host interactions. We generated a dual-particle MAP-PEG hydrogel using two species of PEG microgels, one functionalized with the guest molecule, adamantane, and the other with the host molecule, ß-cyclodextrin (Inter-MAP-PEG). In a different approach, a mono-particle MAP-PEG hydrogel was generated using one species of microgel functionalized with both guest and host molecules (Intra-MAP-PEG). The Intra-MAP-PEG formed a homogenous distribution due to the single type of microgels used. We then compared the mechanical properties of these two types of MAP-PEG hydrogels and found that Intra-MAP-PEG resulted in significantly softer gels with lower yield stress. We investigated the effect of intra-particle guest-host interactions through titrated weight percentage and the concentration of functional groups added to the hydrogel. We found that there was an ideal concentration of guest-host molecules that enables intra- and inter-particle guest-host interactions with sufficient covalent crosslinking. Based on these studies, Intra-MAP-PEG provides a homogeneous guest-host hydrogel that is shear-thinning with reversible secondary crosslinking.

Injectable Microporous Annealed Particle Hydrogel Based on Guest-Host-Interlinked Polyethylene Glycol Maleimide Microgels.

Microporous annealed particle (MAP) hydrogels have emerged as a versatile biomaterial platform for regenerative medicine. MAP hydrogels have been used for the delivery of cells and organoids but often require annealing post injection by an external source. We engineered an injectable, self-annealing MAP hydrogel with reversible interparticle linkages based on guest-host functionalized polyethylene glycol maleimide (PEG-MAL) microgels. We evaluated the effect of guest-host linkages on different types of microgels fabricated by either batch emulsion or mechanical fragmentation methods. Batch emulsion generated small spherical microgels with controllable 10-100 µm diameters and mechanical fragmentation generated irregular microgels with larger diameters (100-200 µm). Spherical microgels (15 µm) showed self-healing behavior and completely recovered from high strain while fragmented microgels (133 µm) did not recover. Guest-host interactions significantly contributed to the mechanical properties of spherical microgels but had no effect on fragmented microgels. Spherical microgels were superior to the fragmented microgels for co-injection of immune cells and pancreatic islets due to their lower force of injection, demonstrating more homogeneously distributed cells and greater cell viability after injection. Based on these studies, the spherical guest-host MAP hydrogels provide a controllable, injectable scaffold for engineered microenvironments and cell delivery applications.

Guest-host interlinked PEG-MAL granular hydrogels as an engineered cellular microenvironment.

We report the development of a polyethylene glycol (PEG) hydrogel scaffold that provides the advantages of conventional bulk PEG hydrogels for engineering cellular microenvironments and allows for rapid cell migration. PEG microgels were used to assemble a densely packed granular system with an intrinsic interstitium-like negative space. In this material, guest-host molecular interactions provide reversible non-covalent linkages between discrete PEG microgel particles to form a cohesive bulk material. In guest-host chemistry, different guest molecules reversibly and non-covalently interact with their cyclic host molecules. Two species of PEG microgels were made, each with one functional group at the end of the four arm PEG-MAL functionalized using thiol click chemistry. The first was functionalized with the host molecule ß-cyclodextrin, a cyclic oligosaccharide of repeating d-glucose units, and the other functionalized with the guest molecule adamantane. These two species provide a reversible guest-host interaction between microgel particles when mixed, generating an interlinked network with a percolated interstitium. We showed that this granular configuration, unlike conventional bulk PEG hydrogels, enabled the rapid migration of THP-1 monocyte cells. The guest-host microgels also exhibited shear-thinning behavior, providing a unique advantage over current bulk PEG hydrogels.