Single-Step Biofabrication of In Situ Spheroid-Forming Compartmentalized Hydrogel for Clinical-Sized Cartilage Tissue Formation.

3D cellular spheroids offer more biomimetic microenvironments than conventional 2D cell culture technologies, which has proven value for many tissue engineering applications. Despite beneficiary effects of 3D cell culture, clinical translation of spheroid tissue engineering is challenged by limited scalability of current spheroid formation methods. Although recent adoption of droplet microfluidics can provide a continuous production process, use of oils and surfactants, generally low throughput, and requirement of additional biofabrication steps hinder clinical translation of spheroid culture. Here, the use of clean (e.g., oil-free and surfactant-free), ultra-high throughput (e.g., 8.5 mL min-1 , 10 000 spheroids s-1 ), single-step, in-air microfluidic biofabrication of spheroid forming compartmentalized hydrogels is reported. This novel technique can reliably produce 1D fibers, 2D planes, and 3D volumes compartmentalized hydrogel constructs, which each allows for distinct (an)isotropic orientation of hollow spheroid-forming compartments. Spheroids produced within ink-jet bioprinted compartmentalized hydrogels outperform 2D cell cultures in terms of chondrogenic behavior. Moreover, the cellular spheroids can be harvested from compartmentalized hydrogels and used to build shape-stable centimeter-sized biomaterial-free living tissues in a bottom-up manner. Consequently, it is anticipated that in-air microfluidic production of spheroid-forming compartmentalized hydrogels can advance production and use of cellular spheroids for various biomedical applications.

Photoannealing of Microtissues Creates High-Density Capillary Network Containing Living Matter in a Volumetric-Independent Manner.

The vascular tree is crucial for the survival and function of large living tissues. Despite breakthroughs in 3D bioprinting to endow engineered tissues with large blood vessels, there is currently no approach to engineer high-density capillary networks into living tissues in a scalable manner. Here, photoannealing of living microtissue (PALM) is presented as a scalable strategy to engineer capillary-rich tissues. Specifically, in-air microfluidics is used to produce living microtissues composed of cell-laden microgels in ultrahigh throughput, which can be photoannealed into a monolithic living matter. Annealed microtissues inherently give rise to an open and interconnected pore network within the resulting living matter. Interestingly, utilizing soft microgels enables microgel deformation, which leads to the uniform formation of capillary-sized pores. Importantly, the ultrahigh throughput nature underlying the microtissue formation uniquely facilitates scalable production of living tissues of clinically relevant sizes (>1 cm3 ) with an integrated high-density capillary network. In short, PALM generates monolithic, microporous, modular tissues that meet the previously unsolved need for large engineered tissues containing high-density vascular networks, which is anticipated to advance the fields of engineered organs, regenerative medicine, and drug screening.

Mass production of lumenogenic human embryoid bodies and functional cardiospheres using in-air-generated microcapsules.

Organoids are engineered 3D miniature tissues that are defined by their organ-like structures, which drive a fundamental understanding of human development. However, current organoid generation methods are associated with low production throughputs and poor control over size and function including due to organoid merging, which limits their clinical and industrial translation. Here, we present a microfluidic platform for the mass production of lumenogenic embryoid bodies and functional cardiospheres. Specifically, we apply triple-jet in-air microfluidics for the ultra-high-throughput generation of hollow, thin-shelled, hydrogel microcapsules that can act as spheroid-forming bioreactors in a cytocompatible, oil-free, surfactant-free, and size-controlled manner. Uniquely, we show that microcapsules generated by in-air microfluidics provide a lumenogenic microenvironment with near 100% efficient cavitation of spheroids. We demonstrate that upon chemical stimulation, human pluripotent stem cell-derived spheroids undergo cardiomyogenic differentiation, effectively resulting in the mass production of homogeneous and functional cardiospheres that are responsive to external electrical stimulation. These findings drive clinical and industrial adaption of stem cell technology in tissue engineering and drug testing.

Scalable fabrication, compartmentalization and applications of living microtissues.

Living microtissues are used in a multitude of applications as they more closely resemble native tissue physiology, as compared to 2D cultures. Microtissues are typically composed of a combination of cells and materials in varying combinations, which are dictated by the applications' design requirements. Their applications range wide, from fundamental biological research such as differentiation studies to industrial applications such as cruelty-free meat production. However, their translation to industrial and clinical settings has been hindered due to the lack of scalability of microtissue production techniques. Continuous microfluidic processes provide an opportunity to overcome this limitation as they offer higher throughput production rates as compared to traditional batch techniques, while maintaining reproducible control over microtissue composition and size. In this review, we provide a comprehensive overview of the current approaches to engineer microtissues with a focus on the advantages of, and need for, the use of continuous processes to produce microtissues in large quantities. Finally, an outlook is provided that outlines the required developments to enable large-scale microtissue fabrication using continuous processes.

Aqueous Two-Phase Enabled Low Viscosity 3D (LoV3D) Bioprinting of Living Matter.

Embedded 3D bioprinting has great value for the freeform fabrication of living matter. However, embedded 3D bioprinting is currently limited to highly viscous liquid baths or liquid-like solid baths. In contrast, prior to crosslinking, most hydrogels are formulated as low-viscosity solutions and are therefore not directly compatible with bioprinting due to low shape fidelity and poor print stability. The authors here present a method to enable low-viscosity ink 3D (LoV3D) bioprinting, based on aqueous two-phase stabilization of the ink-bath interface. LoV3D allows for the printing of living constructs at high extrusion speeds (up to 1.8 m s-1 ) with high viability due to its exceedingly low-viscosity. Moreover, LoV3D liquid/liquid interfaces offer unique advantages for fusing printed structures, creating intricate vasculature, and modifying surfaces at higher efficiencies than traditional systems. Furthermore, the low interfacial tension of LoV3D bioprinting offers unprecedented nozzle-independent control over filament diameter via large-dimension strand-thinning, which allows for the printing of an exceptionally wide range of diameters down to the width of a single cell. Overall, LoV3D bioprinting is a unique all-aqueous approach with broad material compatibility without the need for rheological ink adaption, which opens new avenues of application in cell patterning, drug screening, engineered meat, and organ fabrication.

Drug delivery systems and materials for wound healing applications.

Chronic, non-healing wounds place a significant burden on patients and healthcare systems, resulting in impaired mobility, limb amputation, or even death. Chronic wounds result from a disruption in the highly orchestrated cascade of events involved in wound closure. Significant advances in our understanding of the pathophysiology of chronic wounds have resulted in the development of drugs designed to target different aspects of the impaired processes. However, the hostility of the wound environment rich in degradative enzymes and its elevated pH, combined with differences in the time scales of different physiological processes involved in tissue regeneration require the use of effective drug delivery systems. In this review, we will first discuss the pathophysiology of chronic wounds and then the materials used for engineering drug delivery systems. Different passive and active drug delivery systems used in wound care will be reviewed. In addition, the architecture of the delivery platform and its ability to modulate drug delivery are discussed. Emerging technologies and the opportunities for engineering more effective wound care devices are also highlighted.