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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. 

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

 

Radiation therapy, one of the most effective therapies to treat cancer, is highly toxic to healthy tissue. The delivery of radiation at ultra-high dose rates, FLASH radiation therapy (FLASH), has been shown to maintain therapeutic anti-tumor efficacy while sparing normal tissues compared to conventional dose rate irradiation (CONV). Though promising, these studies have been limited mainly to murine models. Here, we leveraged enteroids, three-dimensional cell clusters that mimic the intestine, to study human-specific tissue response to radiation. We observed enteroids have a greater colony growth potential following FLASH compared with CONV. In addition, the enteroids that reformed following FLASH more frequently exhibited proper intestinal polarity. While we did not observe differences in enteroid damage across groups, we did see distinct transcriptomic changes. Specifically, the FLASH enteroids upregulated the expression of genes associated with the WNT-family, cell-cell adhesion, and hypoxia response. These studies validate human enteroids as a model to investigate FLASH and provide further evidence supporting clinical study of this therapy.

Insight Box Promising work has been done to demonstrate the potential of ultra-high dose rate radiation (FLASH) to ablate cancerous tissue, while preserving healthy tissue. While encouraging, these findings have been primarily observed using pre-clinical murine and traditional two-dimensional cell culture. This study validates the use of human enteroids as a tool to investigate human-specific tissue response to FLASH. Specifically, the work described demonstrates the ability of enteroids to recapitulate previous in vivo findings, while also providing a lens through which to probe cellular and molecular-level responses to FLASH. The human enteroids described herein offer a powerful model that can be used to probe the underlying mechanisms of FLASH in future studies.

 

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.

 

Microextrusion‐based 3D bioprinting into support baths has emerged as a promising technique to pattern soft biomaterials into complex, macroscopic structures. It is hypothesized that interactions between inks and support baths, which are often composed of granular microgels, can be modulated to control the microscopic structure within these macroscopic‐printed constructs. Using printed collagen bioinks crosslinked either through physical self‐assembly or bioorthogonal covalent chemistry, it is demonstrated that microscopic porosity is introduced into collagen inks printed into microgel support baths but not bulk gel support baths. The overall porosity is governed by the ratio between the ink's shear viscosity and the microgel support bath's zero‐shear viscosity. By adjusting the flow rate during extrusion, the ink's shear viscosity is modulated, thus controlling the extent of microscopic porosity independent of the ink composition. For covalently crosslinked collagen, printing into support baths comprised of gelatin microgels (15‐50 µm) results in large pores (≈40 µm) that allow human corneal mesenchymal stromal cells (MSCs) to readily spread, while control samples of cast collagen or collagen printed in non‐granular support baths do not allow cell spreading. Taken together, these data demonstrate a new method to impart controlled microscale porosity into 3D printed hydrogels using granular microgel support baths.

 

3D bioprinting has enabled the fabrication of tissue‐mimetic constructs with freeform designs that include living cells. In the development of new bioprinting techniques, the controlled use of diffusion has become an emerging strategy to tailor the properties and geometry of printed constructs. Specifically, the diffusion of molecules with specialized functions, including crosslinkers, catalysts, growth factors, or viscosity‐modulating agents, across the interface of printed constructs will directly affect material properties such as microstructure, stiffness, and biochemistry, all of which can impact cell phenotype. For example, diffusion‐induced gelation is employed to generate constructs with multiple materials, dynamic mechanical properties, and perfusable geometries. In general, these diffusion‐based bioprinting strategies can be categorized into those based on inward diffusion (i.e., into the printed ink from the surrounding air, solution, or support bath), outward diffusion (i.e., from the printed ink into the surroundings), or diffusion within the printed construct (i.e., from one zone to another). This review provides an overview of recent advances in diffusion‐based bioprinting strategies, discusses emerging methods to characterize and predict diffusion in bioprinting, and highlights promising next steps in applying diffusion‐based strategies to overcome current limitations in biofabrication.

 

Abstract Three-dimensional (3D) bioprinting is a promising technique for spatially patterning cells and materials into constructs that mimic native tissues and organs. However, a trade-off exists between printability and biological function, where weak materials are typically more suited for 3D cell culture but exhibit poor shape fidelity when printed in air. Recently, a new class of assistive materials has emerged to overcome this limitation and enable fabrication of more complex, biologically relevant geometries, even when using soft materials as bioinks. These materials include support baths, which bioinks are printed into, and sacrificial inks, which are printed themselves and then later removed. Support baths are commonly yield-stress materials that provide physical confinement during the printing process to improve resolution and shape fidelity. Sacrificial inks have primarily been used to create void spaces and pattern perfusable networks, but they can also be combined directly with the bioink to change its mechanical properties for improved printability or increased porosity. Here, we outline the advantages of using such assistive materials in 3D bioprinting, define their material property requirements, and offer case study examples of how these materials are used in practice. Finally, we discuss the remaining challenges and future opportunities in the development of assistive materials that will propel the bioprinting field forward toward creating full-scale, biomimetic tissues and organs. 

Abstract Three-dimensional (3D) bioprinting seeks to unlock the rapid generation of complex tissue constructs, but long-standing challenges with efficient in vitro microvascularization must be solved before this can become a reality. Microvasculature is particularly challenging to biofabricate due to the presence of a hollow lumen, a hierarchically branched network topology, and a complex signaling milieu. All of these characteristics are required for proper microvascular—and, thus, tissue—function. While several techniques have been developed to address distinct portions of this microvascularization challenge, no single approach is capable of simultaneously recreating all three microvascular characteristics. In this review, we present a three-part framework that proposes integration of existing techniques to generate mature microvascular constructs. First, extrusion-based 3D bioprinting creates a mesoscale foundation of hollow, endothelialized channels. Second, biochemical and biophysical cues induce endothelial sprouting to create a capillary-mimetic network. Third, the construct is conditioned to enhance network maturity. Across all three of these stages, we highlight the potential for extrusion-based bioprinting to become a central technique for engineering hierarchical microvasculature. We envision that the successful biofabrication of functionally engineered microvasculature will address a critical need in tissue engineering, and propel further advances in regenerative medicine and ex vivo human tissue modeling.