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The encapsulation of cells within gel‐phase materials to form bioinks offers distinct advantages for next‐generation 3D bioprinting. 3D bioprinting has emerged as a promising tool for patterning cells, but the technology remains limited in its ability to produce biofunctional, tissue‐like constructs due to a dearth of materials suitable for bioinks. While early demonstrations commonly used viscous polymers optimized for printability, these materials often lacked cell compatibility and biological functionality. In response, advanced materials that exist in the gel phase during the entire printing process are being developed, since hydrogels are uniquely positioned to both protect cells during extrusion and provide biological signals to embedded cells as the construct matures during culture. Here, an overview of the design considerations for gel‐phase materials as bioinks is presented, with a focus on their mechanical, biochemical, and dynamic gel properties. Current challenges and opportunities that arise due to the fact that bioprinted constructs are active, living hydrogels composed of both acellular and cellular components are also evaluated. Engineering hydrogels with consideration of cells as an intrinsic component of the printed bioink will enable control over the evolution of the living construct after printing to achieve greater biofunctionality.

 

Granular, microgel‐based materials have garnered interest as promising tissue engineering scaffolds due to their inherent porosity, which can promote cell infiltration. Adapting these materials for 3D bioprinting, while maintaining sufficient void space to enable cell migration, can be challenging, since the rheological properties that determine printability are strongly influenced by microgel packing and void fraction. In this work, a strategy is proposed to decouple printability and void fraction by blending UV‐crosslinkable gelatin methacryloyl (GelMA) microgels with sacrificial gelatin microgels to form composite inks. It is observed that inks with an apparent viscosity greater than ≈100 Pa s (corresponding to microgel concentrations ≥5 wt%) have rheological properties that enable extrusion‐based printing of multilayered structures in air. By altering the ratio of GelMA to sacrificial gelatin microgels, while holding total concentration constant at 6 wt%, a family of GelMA:gelatin microgel inks is created that allows for tuning of void fraction from 0.20 to 0.57. Furthermore, human umbilical vein endothelial cells (HUVEC) seeded onto printed constructs are observed to migrate into granular inks in a void fraction‐dependent manner. Thus, the family of microgel inks holds promise for use in 3D printing and tissue engineering applications that rely upon cell infiltration.

 

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.

 

Human intestinal organoids from primary human tissues have the potential to revolutionize personalized medicine and preclinical gastrointestinal disease models. A tunable, fully defined, designer matrix, termed hyaluronan elastin‐like protein (HELP) is reported, which enables the formation, differentiation, and passaging of adult primary tissue‐derived, epithelial‐only intestinal organoids. HELP enables the encapsulation of dissociated patient‐derived cells, which then undergo proliferation and formation of enteroids, spherical structures with polarized internal lumens. After 12 rounds of passaging, enteroid growth in HELP materials is found to be statistically similar to that in animal‐derived matrices. HELP materials also support the differentiation of human enteroids into mature intestinal cell subtypes. HELP matrices allow stiffness, stress relaxation rate, and integrin‐ligand concentration to be independently and quantitatively specified, enabling fundamental studies of organoid–matrix interactions and potential patient‐specific optimization. Organoid formation in HELP materials is most robust in gels with stiffer moduli (G’≈ 1 kPa), slower stress relaxation rate (t_1/2≈ 18 h), and higher integrin ligand concentration (0.5 × 10⁻³–1 × 10⁻³mRGD peptide). This material provides a promising in vitro model for further understanding intestinal development and disease in humans and a reproducible, biodegradable, minimal matrix with no animal‐derived products or synthetic polyethylene glycol for potential clinical translation.

 

Neural progenitor cells (NPCs) are a promising cell source to repair damaged nervous tissue. However, expansion of therapeutically relevant numbers of NPCs and their efficient differentiation into desired mature cell types remains a challenge. Material‐based strategies, including culture within 3D hydrogels, have the potential to overcome these current limitations. An ideal material would enable both NPC expansion and subsequent differentiation within a single platform. It has recently been demonstrated that cell‐mediated remodeling of 3D hydrogels is necessary to maintain the stem cell phenotype of NPCs during expansion, but the role of matrix remodeling on NPC differentiation and maturation remains unknown. By culturing NPCs within engineered protein hydrogels susceptible to degradation by NPC‐secreted proteases, it is identified that a critical amount of remodeling is necessary to enable NPC differentiation, even in highly degradable gels. Chemical induction of differentiation after sufficient remodeling time results in differentiation into astrocytes and neurotransmitter‐responsive neurons. Matrix remodeling modulates expression of the transcriptional co‐activator Yes‐associated protein, which drives expression of NPC stemness factors and maintains NPC differentiation capacity, in a cadherin‐dependent manner. Thus, cell‐remodelable hydrogels are an attractive platform to enable expansion of NPCs followed by differentiation of the cells into mature phenotypes for therapeutic use.

 

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.