RESOURCES

Abstract
Abstract In living tissues, cells express their functions following complex signals from their surrounding microenvironment. Capturing both hierarchical architectures at the micro- and macroscale, and anisotropic cell patterning remains a major challenge in bioprinting, and a bottleneck towards creating physiologically-relevant models. Addressing this limitation, we introduced a novel technique, termed Embedded Extrusion-Volumetric Printing (EmVP), converging extrusion-bioprinting and layer-less, ultra-fast volumetric bioprinting, allowing to spatially pattern multiple inks/cell types. Light-responsive microgels were developed for the first time as bioresins (μResins) for light-based volumetric bioprinting, providing a microporous environment permissive for cell homing and self-organization. Tuning the mechanical and optical properties of gelatin-based microparticles enables their use as support bath for suspended extrusion printing, in which features containing high cell densities can be easily introduced. μResins can be sculpted within seconds with tomographic light projections into centimetre-scale, granular hydrogel-based, convoluted constructs. Interstitial microvoids enhanced differentiation of multiple stem/progenitor cells (vascular, mesenchymal, neural), otherwise not possible with conventional bulk hydrogels. As proof-of-concept, EmVP was applied to create complex synthetic biology-inspired intercellular communication models, where adipocyte differentiation is regulated by optogenetic-engineered pancreatic cells. Overall, EmVP offers new avenues for producing regenerative grafts with biological functionality, and for developing engineered living systems and (metabolic) disease models. This article is protected by copyright. All rights reserved

Abstract
Abstract Volumetric Bioprinting (VBP), enables to rapidly build complex, cell-laden hydrogel constructs for tissue engineering and regenerative medicine. Light-based tomographic manufacturing enables spatial-selective polymerization of a bioresin, resulting in higher throughput and resolution than what is achieved using traditional techniques. However, methods for multi-material printing are needed for broad VBP adoption and applicability. Although converging VBP with extrusion bioprinting in support baths offers a novel, promising solution, further knowledge on the engineering of hydrogels as light-responsive, volumetrically printable baths is needed. Therefore, this study investigates the tuning of gelatin macromers, in particular leveraging the effect of molecular weight and degree of modification, to overcome these challenges, creating a library of materials for VBP and Embedded extrusion Volumetric Printing (EmVP). Bioresins with tunable printability and mechanical properties are produced, and a novel subset of gelatins and GelMA exhibiting stable shear-yielding behavior offers a new, single-component, ready-to-use suspension medium for in-bath printing, which is stable over multiple hours without needing temperature control. As a proof-of-concept biological application, bioprinted gels are tested with insulin-producing pancreatic cell lines for 21 days of culture. Leveraging a multi-color printer, complex multi-material and multi-cellular geometries are produced, enhancing the accessibility of volumetric printing for advanced tissue models.

Abstract
Abstract Type 1 Diabetes results from autoimmune response elicited against β-cell antigens. Nowadays, insulin injections remain the leading therapeutic option. However, injection treatment fails to emulate the highly dynamic insulin release that β-cells provide. 3D cell-laden microspheres have been proposed during the last years as a major platform for bioengineering insulin-secreting constructs for tissue graft implantation and a model for in vitro drug screening platforms. Current microsphere fabrication technologies have several drawbacks: the need for an oil phase containing surfactants, diameter inconsistency of the microspheres, and high time-consuming processes. These technologies have widely used alginate for its rapid gelation, high processability, and low cost. However, its low biocompatible properties do not provide effective cell attachment. This study proposes a high-throughput methodology using a 3D bioprinter that employs an ECM-like microenvironment for effective cell-laden microsphere production to overcome these limitations. Crosslinking the resulting microspheres with tannic acid prevents collagenase degradation and enhances spherical structural consistency while allowing the diffusion of nutrients and oxygen. The approach allows customization of microsphere diameter with extremely low variability. In conclusion, a novel bio-printing procedure is developed to fabricate large amounts of reproducible microspheres capable of secreting insulin in response to extracellular glucose stimuli.

Abstract
The importance of 3D printing technologies increased significantly over the recent years. They are considered to have a huge impact in regenerative medicine and tissue engineering, since 3D bioprinting enables the production of cell-laden 3D scaffolds. Transition from academic research to pharmaceutical industry or clinical applications, however, is highly dependent on developing a robust and well-known process, while maintaining critical cell characteristics. Hence, a directed and systematic approach to 3D bioprinting process development is required, which also allows for the monitoring of these cell characteristics. This work presents the development of a flow cytometry-based analytical strategy as a tool for 3D bioprinting research. The development was based on a model process using a commercially available alginate-based bioink, the β-cell line INS-1E, and direct dispensing as 3D bioprinting method. We demonstrated that this set-up enabled viability and proliferation analysis. Additionally, use of an automated sampler facilitated high-throughput screenings. Finally, we showed that each process step, e.g. suspension of cells in bioink or 3D printing, cross-linking of the alginate scaffold after printing, has a crucial impact on INS-1E viability. This reflects the importance of process optimization in 3D bioprinting and the usefulness of the flow cytometry-based analytical strategy described here. The presented strategy has a great potential as a cell characterisation tool for 3D bioprinting and may contribute to a more directed process development.