Biofabrication

Allogeneic cell transplantation such as beta-cell replacement for treatment of type 1 diabetes (T1D) is constrained by poor graft survival and functionality, immune rejection, and the lack of scalable biomanufacturing processes. Here, we engineered functional human islet constructs that replicate the physiomimetic human pancreatic microenvironment by employing a clinically-scalable 3D bioprinting system. To support human islet viability and function, we developed alginate-based bioinks incorporating human pancreatic decellularized extracellular matrix (dECM). These bioink formulations were optimized for shear-thinning properties for extrusion of human islets, as well as selective permeability that supports nutrient and therapeutic molecule exchange. Extrusion-based printing parameters were refined to minimize shear stress-induced damage to human islets. The resulting bioprinted pancreatic constructs demonstrated robust structural integrity, high human islet viability (>85%), and long-term glucose-stimulated insulin secretion (GSIS) over a 21-days in vitro culture period, even at a high islet packing density (10,000 islet equivalent/mL) while free islet controls displayed a significant functional decline. The higher performance of bioprinted islets maybe attributable to the supportive 3D dECM-rich microenvironment mitigating culture-induced stress by recapitulating the islet pancreatic niche. This scalable 3D dECM-alginate bioprinted platform represents a new advanced functional material for advancing clinically translatable bio-artificial pancreas therapies for T1D.

Pluripotent stem cells represent a potentially unlimited cell source for the fabrication of human bioartificial tissues to study and treat degenerative conditions such as type 1 diabetes. Alginate is widely used for mammalian cell immobilization and the primary hydrogel studied for pancreatic islet encapsulation. Rheological properties of alginate solutions or fully gelled forms are unsuitable as support matrix for embedded 3D printing. We describe partially gelled self-healing alginate formulations tuned for embedded 3D printing. Perfusable multi-plane hierarchical networks branching into 10 parallel channels, obtained by 3D printing of Pluronic F127 into the alginate support, show high fidelity to computer-assisted models. Therapeutic {beta}-cell doses (40x106 cells/mL) within centimeter-thick perfusable constructs remained viable for at least 1 week of culture under flow, with rapid insulin secretion detected upon glucose challenges. Stem cell-derived islet clusters cultured in 5-channel contructs for 25 days differentiated towards functional insulin-expressing cells. We describe a novel approach to generate cm-scale perfusable endocrine pancreatic constructs using sacrificial embedded 3D printing into alginate. This approach offers an adaptable platform to engineer perfusable cm-scale functional endocrine pancreatic tissues and potentially other vascularized bioartificial tissues.

Indirect bioprinting for cell culture requires the use of several technologies and techniques which currently prevent many researchers not specialized in electrical engineering or materials science from accessing these new tools. In this paper, a printer and all necessary associated hardware was developed and tested for the purpose of seeding human induced Pluripotent Stem Cell (iPSC)-derived endothelial cells (iECs) onto all surfaces of a fibringelatin channel. Immature iECs were seeded onto all channel surfaces and completed differentiation along channel walls. All required tools and methods, including engineering drawing, printable files, code, and hand-tool templates, have been provided with sufficient clarity to enable full, open-source replication of all technique employed.

Three-dimensional (3D) cell culture systems rely on the manipulation of a biologically derived matrix, typically soluble Basement Membrane Extract (sBME), in which cells or cellular aggregates, such as organoids, are suspended. This matrix provides mechanobiological support, promoting cellular processes. However, the handling of sBME-based matrices containing cellular constructs poses significant challenges due to their rheological properties. We developed an integrated bioprinting system to surpass the conventional pipetting, seeding and culture in multiwell plates. The system combines a fluidic cartridge with innovative 3D-printed biocompatible culture tools designed to host and preserve high-throughput microcultures of Patient-Derived Organoids (PDOs) in sBME. The miniaturized hanging-drop configuration enables extended culture periods and high-throughput imaging screenings. This comprehensive approach overcomes common issues associated with sBME, including sedimentation of cellular aggregates, premature gelation, and structural collapse, which negatively impact culture quality and reproducibility throughout the entire 3D culture workflow, from seeding to culture maintenance, and post-culture analyses. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/678315v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@19f976eorg.highwire.dtl.DTLVardef@8eeefcorg.highwire.dtl.DTLVardef@1ebe6c7org.highwire.dtl.DTLVardef@7c4403_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights- Miniaturized 3D hanging-drop matrix-embedded organoid culture in a 384-well plate - Custom cartridge enables homogeneous bioprinting of organoids in sBME-based matrix - 3D-printed tools support compact, scalable multiwell culture systems - System suited for miniaturized culture organoids for high-throughput drug screening - Scalable miniaturized culture system for extended periods of time

There is a notable gap in the scientific understanding of the cellular role in cultured cell-based foods. Unravelling the effects of the interactions between ingredient micro/nanostructure and cells and their significance on nutrition and texture is of great importance. In addition, bioprinting methods face notable limitations in animal-free formulations and scale. Herein, we introduce a proof-of-concept bioprinting method based on the in situ integration of self-assembling events, allowing printing without supporting baths. Our approach enabled a food-grade 3D bioprinted model with 8.5 mm height and a hardness of 284 mN, supporting the early differentiation of myoblasts producing embryonic myosin heavy chain, after 7 days of differentiation. Cellular protein content increased up to 18-fold per initial cell without changes in construct texture. The method provides a novel concept to produce robust, cell-dense platforms for further research on food-grade bioprinted foods.

Traditional livestock farming is resource-intensive and environmentally unsustainable, necessitating alternative methods for meat production. Cell cultured meat, produced by expanding and differentiating animal cells, offers great potential for substituting for conventional animal meat. Nevertheless, it is still limited by the scalability and efficiency of current cell culture technologies. In this study, we developed an RGD peptide-modified alginate hydrogel microtube microbioreactor (AlgTubes) to support the scalable culture of anchor-dependent cells, such as myoblasts and adipocytes, for cell-cultured meat production. AlgTubes provide a cell-friendly 3D microenvironment that enhances cell viability, growth, and yield while overcoming limitations of conventional bioreactors, such as shear stress, aggregation, and diffusion constraints. We successfully expanded mouse (C2C12) and quail (QM7) myoblasts in AlgTubes, achieving cell densities exceeding 1.0 x 10 cells/mL, far surpassing traditional stirred-tank bioreactors. Differentiation resulted in the formation of mature myotubes. Co-culturing myoblasts with mesenchymal stem cells or fibroblasts further improved yield and viability, particularly under differentiation conditions. By significantly increasing cell culture density, AlgTubes can substantially reduce culture volume, lowering labor requirements, reagent costs, equipment needs, facility space, and manufacturing expenses.

Vascularization remains a major obstacle in tissue engineering. Here, we introduce a developmentally inspired bioprinting strategy to generate centimeter-scale, self-organising "mother vessel" constructs from iPSC-derived human mesodermal progenitor cells (hiMPCs). By systematically optimizing the bioink composition, we identified a formulation that combines high print fidelity, mechanical stability and cell compatibility within a single-step bioprinting process. Within the first week after printing, hiMPCs in the "mother vessel" constructs underwent spontaneous differentiation and morphogenesis, forming intima-, media-, and adventitia-like layers containing CD31 endothelial, SMA mural and CD34/CD150 progenitor cells. Remarkably, Iba1 macrophage-like cells appeared despite their absence in the initial population, indicating intrinsic differentiation into both vascular and non-vascular lineages essential for angiogenesis, remodeling and tissue homeostasis. Surrounding the newly formed vessel wall-like structure was a broad, vascularized mesodermal tissue compartment that also contained the above-mentioned progenitors. Co-culture with prevascularized mesodermal organoids resulted in early structural interconnection of microvessels with the printed wall, representing a prerequisite for subsequent hierarchical vascular network formation. As a proof-of-concept, the mother vessel withstood controlled flow conditions in a bioreactor without detectable leakage, demonstrating its principal suitability for perfusion analyses. Together, these findings establish a biologically driven platform that bridges macro- and microvascularization. This may pave the way toward perfusable, vascularized larger tissue constructs, a major bottleneck in regenerative biofabrication.

The vascularization of engineered tissues and organoids has remained a major unresolved challenge in regenerative medicine. While multiple approaches have been developed to vascularize in vitro tissues, it has thus far not been possible to generate sufficiently dense networks of small-scale vessels to perfuse large de novo tissues. Here, we achieve the perfusion of multi-mm3 tissue constructs by generating networks of synthetic capillary-scale 3D vessels. Our 3D soft microfluidic strategy is uniquely enabled by a 3D-printable 2-photon-polymerizable hydrogel formulation, which allows for precise microvessel printing at scales below the diffusion limit of living tissues. We demonstrate that these large-scale engineered tissues are viable, proliferative and exhibit complex morphogenesis during long-term in-vitro culture, while avoiding hypoxia and necrosis. We show by scRNAseq and immunohistochemistry that neural differentiation is significantly accelerated in perfused neural constructs. Additionally, we illustrate the versatility of this platform by demonstrating long-term perfusion of developing liver tissue. This fully synthetic vascularization platform opens the door to the generation of human tissue models at unprecedented scale and complexity.

Endocrine disruptors (EDs) are an exogenous group of compounds associated with thyroid malfunctioning in the human body. Nonetheless, there are currently no adequate in vivo or in vitro models for the preclinical testing of these compounds since both animal and two-dimensional (2D) cell-based models are not able to mimic thyroid physiological conditions from both functional and three-dimensional (3D) organization perspective. Recently, bioprinting technologies emerged as an innovative tool in the field of regenerative medicine and advanced 3D in vitro models that allow the creation of 3D well-organized structures able to mirror physiologically relevant tissue and organ architectures. In this study, we evaluated microfluidic bioprinting as a biofabrication technology to develop a 3D in vitro model of the thyroid gland. We studied the fundamental parameters to obtain a fine control over the bioprinted fibres for different biomaterials. Then, we assessed the possibility to bioprint single thyroid cells, thyroid spheroids and finally mouse embryonic stem cell-derived thyroid follicles. The different cell types maintained high viability and metabolic activity. The bioprinted thyroid model showed high expression of different early and late functional markers and to be responsive to ED exposure. These bioprinted thyroid constructs could provide a new set of advanced 3D in vitro models to test potential EDs and possible adverse outcomes that may be associated with their administration or exposure.

Efficient, scalable, and cost-effective production of mammalian cells and biotherapeutic particles remains a major challenge for both research and clinical applications. Conventional 2D and 3D culture systems suffer from low volumetric yields, poor scalability, and high costs. Previously, we developed collagen hydrogel tube microbioreactors (ColTubes) that support high-density, high-viability cell culture by preventing excessive cell aggregation and minimizing hydrodynamic stress. However, ColTubes exhibit adhesion to culture vessels and to each other, and leaked cells frequently attach to outer tube surfaces -- behaviors that would limit scalability. Here we introduce AlgColTubes: collagen hydrogel tubes coated with a thin, ionically crosslinked alginate layer to overcome these limitations. Scanning electron microscopy confirms that alginate penetrates the collagen wall and forms a stable interpenetrating hydrogel network, whose depth can be tuned by coating concentration and duration. The alginate coating remains structurally intact under static and dynamic culture conditions without impairing nutrient transport or cell growth. AlgColTubes eliminate tube-tube and tube-vessel adhesion, and prevent exogenous cell attachment to the outer surface, while maintaining cell viability and proliferation comparable to uncoated ColTubes. Their unique architecture -- an adhesive collagen interior and non-adhesive alginate exterior -- further enables a truncated-tube format for continuous release of biotherapeutic particles through open tube ends. We demonstrate that lentivirus is released from truncated AlgColTubes in a segment length-dependent manner, reaching ~100% release efficiency at 1-mm segment lengths. AlgColTubes provide a scalable, cost-effective platform for high-yield cell and particle manufacturing, with broad potential across basic research, translational studies, and industrial bioprocessing.

Embedded extrusion printing can process collagen-rich bioinks, but their low viscosity and slow fibrillogenesis compromise print fidelity and post-deposition stability. Here, we developed a collagen fibril-inducing support bath (FIB) that combines mechanical support for embedded printing with biochemical induction of collagen assembly. Microfibrillated or nanofibrillated cellulose was incorporated into a fibril-inducing buffer, and formulations were screened at 37 degrees Celsius for rheological behaviour and optical transparency. The selected FIB was evaluated by printing 1% and 5% (w/v) articular cartilage-derived extracellular matrix (ECM) inks at 10-20 mm/s and compared with a cellulose-only control bath. FIB exhibited yield-stress, shear-thinning and rapid recovery behaviour that supported reproducible filament deposition. Unlike the control bath, FIB enabled intact construct retrieval following stabilisation and promoted the formation of fibrillar collagen within the printed strands. Scanning electron microscopy revealed D-banded collagen fibrils preferentially oriented along the deposition direction, with dominant orientation peaks within +/- 10-15 degrees. The platform supported the fabrication of 15 x 15 x 1.5 mm sheets and 6 x 6 x 6 mm scaffolds whose macroscopic dimensions were retained after processing. Constructs produced from 5% ECM inks exhibited approximately fourfold higher ramp and relaxation moduli than those produced from 1% ECM inks. Extracts from both formulations caused no detectable reduction in cell metabolic activity after 24 h or 72 h. Mesenchymal stem/stromal cells (MSCs) seeded onto printed sheets became markedly elongated and aligned by day 3, with approximately 80% of cells having an aspect ratio exceeding 1.5, significantly greater than cells seeded onto casted ECM controls, with a mean deviation of ~9 degrees from the filament print direction. These findings establish FIB as a bioactive support bath that couples embedded printability with collagen fibrillogenesis, enabling recoverable collagen-rich constructs with aligned fibrillar architecture that directs early cellular organisation.

Micropatterning technologies enable biomimetic organoid generation with controlled in vitro morphogenesis but current approaches lack scalability. Here, we develop a mask-based photopatterning platform to enhance organoid culture reproducibility and scalability on diverse substrate formats. We demonstrate its use in generating architecturally defined neural organoids of varying geometries with reproducible morphogenesis. This technology provide a versatile and low-cost strategy for generating geometrically controlled organoids.

Open-source designs represent an attractive and new tool for research as it provides both affordable and accessible options to the lab environment. In particular, with the advent of new and cheap additive manufacturing technologies, the open-source design of lab hardware enables others to perform research that would be difficult otherwise. This manuscript describes an air-jet system designed to be open-source and simple to produce with 3D printing. The fully 3D printed air-jet was designed for the generation of hydrogel microbeads of a controllable size. Alginate microbeads were used as a working model, given that it has many promising research applications due to their injectability and highly reproducible properties. A fit definitive design of experiments was performed to determine critical factors affecting diameter, index of dispersity, and circularity of microbeads from this air-jet design. By regulating alginate concentration, air pressure, pump speed, and needle diameter could achieve control over microbeads size from 200-800 {micro}m with low variance. Furthermore, we also demonstrate the potential probiotic research applications of the open-source air-jet through the encapsulation of bacteria in alginate microbeads with controllable degradation. The results of this study exhibit an open-source platform for making microscale biomaterials with controllable properties that can be achieved through budget 3D printers.

3D bioprinting has revolutionized tissue engineering, enabling intricate, physiologically relevant constructs unattainable with conventional techniques, yet it remains limited in integrating soft and rigid multifunctional components for complex multi-tissue applications. In this study, we introduce a 3D hybrid bioprinting approach implementing the Hybprinter platform, which integrates multiple 3D printing modules under optimized conditions for a continuous bioprinting process with multiple soft and hard biomaterials. This approach demonstrates robust biocompatibility and broad tissue engineering potential for modeling and therapeutic applications. The capacity to fabricate multi-hydrogel hybrid constructs is illustrated by representative examples highlighting vascularization, multifunctionality, mechanical robustness, and implant suturability. Notably, compared with commonly fabricated hydrogel-only constructs, the resulting hybrid constructs achieve over a 1000-fold increase in mechanical strength, and demonstrated enhanced osteogenic differentiation, underscoring their suitability for load-bearing musculoskeletal and orthopedic tissue engineering. Additionally, cell-laden hydrogel constructs demonstrated robust chondrogenic differentiation, highlighting the capacity for lineage-specific tissue development in vitro. Beyond these outcomes, the presented hybrid bioprinting approach integrates essential tissue engineering attributes that unites mechanical robustness and suturable capacity with multi-material integration, gradient property design, incorporation of bioactive agents, and support for multi-cell loading. This versatile platform advances complex tissue engineering and holds promise for patient specific, organ-on-demand applications.

Advances in 3D bioprinting have opened new possibilities in the development of bioengineered muscle models that mimic the structure and functionality of native tissues. The combination of skeletal muscle tissue and artificial elements promotes diverse innovative solutions of interest in both the biomedical field and the development of biohybrid actuators. However, current bioengineering approaches do not fully recreate the complex fascicle-like hierarchical organization of skeletal muscle, impacting on the muscle maturation process due to a lack of oxygen and nutrients supply in the scaffold inner regions. Here we explored co-axial 3D bioprinting as a strategy towards overcoming this challenge, creating individual/non-fused filaments with controlled thickness that present a fascicle-like organization. Compared to conventional 3D-bioprinting, where cell-laden bioink is disposed by a single syringe, our Pluronic-assisted co-axial 3D-bioprinting system (PACA-3D) creates a physical confinement of the bioink during the extrusion process, effectively obtaining thin and independent printed fibers with controlled shape. Fabrication of skeletal muscle-based actuators with PACA-3D resulted in improved cell differentiation, obtaining stronger bioactuators with increased force output when compared to bioactuators fabricated by conventional 3D bioprinting. The versatility of our technology has been demonstrated using different biomaterials, showing its potential to develop more complex biohybrid tissue-based architectures with improved functionality.

Large scale cell-laden hydrogel models hold great promise for tissue repair and organ transplantation, but their fabrication is faced with challenges in achieving clinically-relevant size and hierarchical structures. 3D bioprinting is an emerging technology, but its application in large, solid hydrogel fabrication has been limited by the slow printing speed that can affect the part quality and the biological activity of the encapsulated cells. Here we present a Fast hydrogeL prOjection stereolithogrAphy Technology (FLOAT) that allows the creation of a centimeter-sized, multiscale solid hydrogel model within minutes. Through precisely controlling the photopolymerization condition, we established low suction force-driven, high-velocity flow of the hydrogel prepolymer that supports the continuous replenishment of the prepolymer solution below the curing part and the nonstop part growth. We showed that this process is unique to the hydrogel prepolymer without externally supplemented oxygen. The rapid printing of centimeter-sized hydrogel models using FLOAT was shown to significantly reduce the part deformation and cellular injury caused by the prolonged exposure to the environmental stresses in layer-by-layer based printing methods. Media perfusion in the printed vessel network was shown to promote cell survival and metabolic function in the deep core of the large-sized hydrogel model over long term. The FLOAT is compatible with multiple photocurable hydrogel materials and the printed scaffold supports the endothelialization of prefabricated vascular channels. Together, these studies demonstrate a rapid 3D hydrogel printing method and highlight the potential of this method in the fabrication of large-sized engineered tissue models.

The intestine is a complex tissue with a characteristic three-dimensional (3D) crypt-villous architecture, which plays a key role in the intestinal function. This function is also regulated by the intestinal stroma that actively supports the intestinal epithelium, maintaining homeostasis. Efforts to account for the 3D complex structure of the intestinal tissue have been focused mainly in mimicking the epithelial barrier, while solutions to include the stromal compartment are scarce and unpractical to be used in routine experiments. Here we demonstrate that by employing an optimized bioink formulation and the suitable printing parameters it is possible to produce fibroblast-laden crypt-villous structures by means of digital light processing (DLP) stereolithography. This process provides excellent cell viability, accurate spatial resolution and high printing throughput, resulting in a robust biofabrication approach that yields functional gut mucosa tissues compatible with conventional testing techniques. Teaser3D bioprinting approach for the direct fabrication of advanced cell-laden tissue constructs by means of visible-light photopolymerization.

Adipocyte spheroids are a promising three-dimensional (3D) cell culture model for obesity research because they reproduce 3D adipose tissue structures and cell-cell interactions better than 2D cultures. However, current methods fail to produce uniformly sized, small adipocyte spheroids at large scales, significantly limiting their use in analysis such as large-scale drug screening. Here, we develop a scalable method that combines simple microfluidics with templated emulsification to generate small, uniformly sized adipocyte spheroids. By encapsulating preadipocytes in numerous hollow agarose microcapsules and incubating them for two days, we reproducibly produced more than 100,000 uniform spheroids with diameters of approximately 50 {micro}m (CV: <13%); we then differentiated preadipocyte spheroids into adipocyte spheroids after an 8-day induction period. Our platform enhances large-scale 3D analysis using adipocyte spheroids for obesity research and can be adapted to generate various spheroid and organoid models, advancing biomedical research across diverse fields.

Automation of organoid and cell culture processes is essential for achieving scalable and standardized experimentation in regenerative medicine and stem cell research. However, existing microfluidic platforms often rely on complex setups, limiting their integration within standard incubator environments. To address these challenges, we developed a compact, scalable multi-well platform featuring 3D-printed, servo-actuated disposable microvalves for fully automated media and drug exchange. This design eliminates the need for external pressure sources and control channels, providing a simplified and cost-effective solution for organoid culture. The platform integrates an internet-connected microscopy module with a motorized XYZ stage, allowing continuous, real-time imaging of individual wells directly within the incubator. It supports precise and reliable fluid handling under physiological conditions, improving throughput, reproducibility, and accessibility. We validate the platform through bench-top testing and in both mouse and human organoid models. Morphological analysis, immunohistochemistry (IHC), and qPCR demonstrate comparable viability, growth, and gene expression profiles between automated and manual culture conditions. These results establish a robust and scalable framework for fully automated organoid culture, offering a simplified and accessible alternative to conventional microfluidic systems with broad applications in regenerative medicine, drug discovery, and scalable biological screening. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/732526v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@5efd07org.highwire.dtl.DTLVardef@3600d0org.highwire.dtl.DTLVardef@16f85f5org.highwire.dtl.DTLVardef@c39fbd_HPS_FORMAT_FIGEXP M_FIG C_FIG

Pathological angiogenesis, such as that observed in wet age-related macular degeneration (AMD), is challenging to reproduce in vitro. While previous organ-on-chip approaches have incorporated retinal pigment epithelium (RPE) and endothelial barriers, models integrating human retinal organoids with vascular networks remain limited. Here, we report the development of a fully 3D-printed microfluidic device for co-culture of human induced pluripotent stem cell (hiPSC)-derived retinal organoids containing RPE regions with endothelial cells. The device, fabricated from flexible thermoplastic polyurethane (TPU) on a transparent polyvinyl chloride (PVC) substrate, enables direct organoid-endothelial interaction within a fibrin-Matrigel matrix without physical barriers. In this system, endothelial cells formed choroid-like networks that integrated with retinal organoids. Vascular network density and invasion into RPE regions were enhanced by VEGF stimulation, recapitulating features of wet AMD. Furthermore, fluorescent liposomes distributed along endothelial structures and accumulated at the organoid interface, supporting the application of this model for nanoparticle delivery studies. This 3D-printed retinal organoid-on-chip provides a simple, reproducible, and physiologically relevant platform that complements existing retinal models for investigating angiogenesis and evaluating therapeutic strategies.