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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.

 

Three‐dimensional cell encapsulation has rendered itself a staple in the tissue engineering field. Using recombinantly engineered, biopolymer‐based hydrogels to encapsulate cells is especially promising due to the enhanced control and tunability it affords. Here, we describe in detail the synthesis of our hyaluronan (i.e., hyaluronic acid) and elastin‐like protein (HELP) hydrogel system. In addition to validating the efficacy of our synthetic process, we also demonstrate the modularity of the HELP system. Finally, we show that cells can be encapsulated within HELP gels over a range of stiffnesses, exhibit strong viability, and respond to stiffness cues. © 2023 Wiley Periodicals LLC.

Basic Protocol 1: Elastin‐like protein modification with hydrazine

Basic Protocol 2: Nuclear magnetic resonance quantification of elastin‐like protein modification with hydrazine

Basic Protocol 3: Hyaluronic acid–benzaldehyde synthesis

Basic Protocol 4: Nuclear magnetic resonance quantification of hyaluronic acid–benzaldehyde

Basic Protocol 5: 3D cell encapsulation in hyaluronan elastin‐like protein gels

 

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.

 

Dynamic covalent chemistry (DCC) crosslinks can form hydrogels with tunable mechanical properties permissive to injectability and self‐healing. However, not all hydrogels with transient crosslinks are easily extrudable. For this reason, two additional design parameters must be considered when formulating DCC‐crosslinked hydrogels: 1) degree of functionalization (DoF) and 2) polymer molecular weight (MW). To investigate these parameters, hydrogels comprised of two recombinant biopolymers: 1) a hyaluronic acid (HA) modified with benzaldehyde and 2) an elastin‐like protein (ELP) modified with hydrazine (ELP‐HYD), are formulated. Several hydrogel families are synthesized with distinct HA MW and DoF while keeping the ELP‐HYD component constant. The resulting hydrogels have a range of stiffnesses,G′ ≈ 10–1000 Pa, and extrudability, which is attributed to the combined effects of DCC crosslinks and polymer entanglements. In general, lower MW formulations require lower forces for injectability, regardless of stiffness. Higher DoF formulations exhibit more rapid self‐healing. Gel extrusion through a cannula (2 m length, 0.25 mm diameter) demonstrates the potential for minimally invasive delivery for future biomedical applications. In summary, this work highlights additional parameters that influence the injectability and network formation of DCC‐crosslinked hydrogels and aims to guide future design of injectable hydrogels.

 

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.

 

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.

 

Blending sacrificial, cell-laden microgels with structural, UV-crosslinkable microgels produces a family of modular bioinks with tunable void fractions that influence cellular morphology while maintaining a depth-independent cell distribution.

 

Hydrogels cross-linked by dynamic covalent chemistry (DCC) are stiff and remodelable, making them ideal biomimetics for tissue engineering applications. Due to the reversibility of DCC cross-links, the opportunity exists to transiently control hydrogel network formation through the use of small molecule competitors. Specifically, we incorporate low molecular weight competitors that reversibly disrupt the formation of hydrazone cross-links as they diffuse through a recombinant hydrogel. Using complementary experimental, computational, and theoretical polymer physics approaches, we present a family of competitors that predictably alter hydrogel gelation time and mechanics. By changing the competitor chemistry, we connect key reaction parameters (forward and reverse reactions rates and thermodynamic equilibrium constants) to the delayed onset of a percolated network, increased hydrogel gelation time, and transient control of hydrogel stiffness. Using human intestinal organoids as a model system, we demonstrate the ability to tune gelation kinetics of a recombinant hydrogel for uniform encapsulation of individual, patient-derived stem cells and their proliferation into three-dimensional structures. Taken together, our data establish a validated framework to relate molecular-level parameters of transient competitors to predicted macromolecular-network properties. As interest in biomimetic, DCC-cross-linked hydrogels continues to grow, these results will enable the rationale design of bespoke, dynamic biomaterials for tissue engineering. 

Three-dimensional bioprinting has emerged as a promising tool for spatially patterning cells to fabricate models of human tissue. Here, we present an engineered bioink material designed to have viscoelastic mechanical behavior, similar to that of living tissue. This viscoelastic bioink is cross-linked through dynamic covalent bonds, a reversible bond type that allows for cellular remodeling over time. Viscoelastic materials are challenging to use as inks, as one must tune the kinetics of the dynamic cross-links to allow for both extrudability and long-term stability. We overcome this challenge through the use of small molecule catalysts and competitors that temporarily modulate the cross-linking kinetics and degree of network formation. These inks were then used to print a model of breast cancer cell invasion, where the inclusion of dynamic cross-links was found to be required for the formation of invasive protrusions. Together, we demonstrate the power of engineered, dynamic bioinks to recapitulate the native cellular microenvironment for disease modeling.