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Creators/Authors contains: "Roth, Julien G."

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  1. Abstract

    The biofabrication of three-dimensional (3D) tissues that recapitulate organ-specific architecture and function would benefit from temporal and spatial control of cell-cell interactions. Bioprinting, while potentially capable of achieving such control, is poorly suited to organoids with conserved cytoarchitectures that are susceptible to plastic deformation. Here, we develop a platform, termed Spatially Patterned Organoid Transfer (SPOT), consisting of an iron-oxide nanoparticle laden hydrogel and magnetized 3D printer to enable the controlled lifting, transport, and deposition of organoids. We identify cellulose nanofibers as both an ideal biomaterial for encasing organoids with magnetic nanoparticles and a shear-thinning, self-healing support hydrogel for maintaining the spatial positioning of organoids to facilitate the generation of assembloids. We leverage SPOT to create precisely arranged assembloids composed of human pluripotent stem cell-derived neural organoids and patient-derived glioma organoids. In doing so, we demonstrate the potential for the SPOT platform to construct assembloids which recapitulate key developmental processes and disease etiologies.

     
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    Free, publicly-accessible full text available December 1, 2024
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  3. Abstract

    While the human body has many different examples of perfusable structures with complex geometries, biofabrication methods to replicate this complexity are still lacking. Specifically, the fabrication of self‐supporting, branched networks with multiple channel diameters is particularly challenging. Herein, the Gelation of Uniform Interfacial Diffusant in Embedded 3D Printing (GUIDE‐3DP) approach for constructing perfusable networks of interconnected channels with precise control over branching geometries and vessel sizes is presented. To achieve user‐specified channel dimensions, this technique leverages the predictable diffusion of cross‐linking reaction‐initiators released from sacrificial inks printed within a hydrogel precursor. The versatility of GUIDE‐3DP to be adapted for use with diverse physicochemical cross‐linking mechanisms is demonstrated by designing seven printable material systems. Importantly, GUIDE‐3DP allows for the independent tunability of both the inner and outer diameters of the printed channels and the ability to fabricate seamless junctions at branch points. This 3D bioprinting platform is uniquely suited for fabricating lumenized structures with complex shapes characteristic of multiple hollow vessels throughout the body. As an exemplary application, the fabrication of vasculature‐like networks lined with endothelial cells is demonstrated. GUIDE‐3DP represents an important advance toward the fabrication of self‐supporting, physiologically relevant networks with intricate and perfusable geometries.

     
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  5. Abstract

    Mechanically tunable hydrogels are attractive platforms for 3D cell culture, as hydrogel stiffness plays an important role in cell behavior. Traditionally, hydrogel stiffness has been controlled through altering either the polymer concentration or the stoichiometry between crosslinker reactive groups. Here, an alternative strategy based upon tuning the hydrophilicity of an elastin‐like protein (ELP) is presented. ELPs undergo a phase transition that leads to protein aggregation at increasing temperatures. It is hypothesized that increasing this transition temperature through bioconjugation with azide‐containing molecules of increasing hydrophilicity will allow direct control of the resulting gel stiffness by making the crosslinking groups more accessible. These azide‐modified ELPs are crosslinked into hydrogels with bicyclononyne‐modified hyaluronic acid (HA‐BCN) using bioorthogonal, click chemistry, resulting in hydrogels with tunable storage moduli (100–1000 Pa). Human mesenchymal stromal cells (hMSCs), human umbilical vein endothelial cells (HUVECs), and human neural progenitor cells (hNPCs) are all observed to alter their cell morphology when encapsulated within hydrogels of varying stiffness. Taken together, the use of protein hydrophilicity as a lever to tune hydrogel mechanical properties is demonstrated. These hydrogels have tunable moduli over a stiffness range relevant to soft tissues, support the viability of encapsulated cells, and modify cell spreading as a consequence of gel stiffness.

     
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  6. Abstract

    Mechanical cues from the extracellular matrix (ECM) regulate vascular endothelial cell (EC) morphology and function. Since naturally derived ECMs are viscoelastic, cells respond to viscoelastic matrices that exhibit stress relaxation, in which a cell‐applied force results in matrix remodeling. To decouple the effects of stress relaxation rate from substrate stiffness on EC behavior, we engineered elastin‐like protein (ELP) hydrogels in which dynamic covalent chemistry (DCC) was used to crosslink hydrazine‐modified ELP (ELP‐HYD) and aldehyde/benzaldehyde‐modified polyethylene glycol (PEG‐ALD/PEG‐BZA). The reversible DCC crosslinks in ELP‐PEG hydrogels create a matrix with independently tunable stiffness and stress relaxation rate. By formulating fast‐relaxing or slow‐relaxing hydrogels with a range of stiffness (500–3300 Pa), we examined the effect of these mechanical properties on EC spreading, proliferation, vascular sprouting, and vascularization. The results show that both stress relaxation rate and stiffness modulate endothelial spreading on two‐dimensional substrates, on which ECs exhibited greater cell spreading on fast‐relaxing hydrogels up through 3 days, compared with slow‐relaxing hydrogels at the same stiffness. In three‐dimensional hydrogels encapsulating ECs and fibroblasts in coculture, the fast‐relaxing, low‐stiffness hydrogels produced the widest vascular sprouts, a measure of vessel maturity. This finding was validated in a murine subcutaneous implantation model, in which the fast‐relaxing, low‐stiffness hydrogel produced significantly more vascularization compared with the slow‐relaxing, low‐stiffness hydrogel. Together, these results suggest that both stress relaxation rate and stiffness modulate endothelial behavior, and that the fast‐relaxing, low‐stiffness hydrogels supported the highest capillary density in vivo.

     
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  7. Abstract

    Three‐dimensional (3D) bioprinting is a promising technology to produce tissue‐like structures, but a lack of diversity in bioinks is a major limitation. Ideally each cell type would be printed in its own customizable bioink. To fulfill this need for a universally applicable bioink strategy, a versatile bioorthogonal bioink crosslinking mechanism that is cell compatible and works with a range of polymers is developed. This family of materials is termed UNIversal, Orthogonal Network (UNION) bioinks. As demonstration of UNION bioink versatility, gelatin, hyaluronic acid (HA), recombinant elastin‐like protein (ELP), and polyethylene glycol (PEG) are each used as backbone polymers to create inks with storage moduli spanning from 200 to 10 000 Pa. Because UNION bioinks are crosslinked by a common chemistry, multiple materials can be printed together to form a unified, cohesive structure. This approach is compatible with any support bath that enables diffusion of UNION crosslinkers. Both matrix‐adherent human corneal mesenchymal stromal cells and non‐matrix‐adherent human induced pluripotent stem cell‐derived neural progenitor spheroids are printed with UNION bioinks. The cells retained high viability and expressed characteristic phenotypic markers after printing. Thus, UNION bioinks are a versatile strategy to expand the toolkit of customizable materials available for 3D bioprinting.

     
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