Support Bath Research Articles

Soft-tissue-mimicking using silicones for the manufacturing of soft phantoms by fresh 3D printing

PurposeThe purpose of this study is to use the Freeform Reversible Embedding of Suspended Hydrogels (FRESH) additive manufacturing (AM) technique for manufacturing a liver phantom which can mimic the corresponding soft living tissue. One of the possible applications is surgical planning.Design/methodology/approachA thermo-reversible Pluronic® F-127-based support bath is used for the FRESH technique. To verify how three-dimensional (3D)-printed new materials can mimic liver tissue, dynamic mechanical analysis and oscillation shear rheometry tests are carried out to identify mechanical characteristics of different 3D printed silicone samples. Additionally, the differential scanning calorimetry was done on the silicone samples. Then, a validation of a 3D printed silicone liver phantom is performed with a 3D scanner. Finally, the surface topography of the 3D printed liver phantom was fulfiled and microscopy analysis of its surface.FindingsSilicone samples were able to mimic the liver, therefore obtaining the first soft phantom of the liver using the FRESH technique.Practical implicationsBecause of the use of soft silicones, surgeons could practice over these improved phantoms which have an unprecedented degree of living tissue mimicking, enhancing their rehearsal experience before surgery.Social implicationsAn improvement in surgeons surgery skills would lead to a bettering in the patient outcome.Originality/valueThe first research study was carried out to mimic soft tissue and apply it to the 3D printing of organ phantoms using AM FRESH technique.

Decellularized kidney extracellular matrix bioinks recapitulate renal 3D microenvironment in vitro

Decellularized extracellular matrices (ECMs) are able to provide the necessary and specific cues for remodeling and maturation of tissue-specific cells. Nevertheless, their use for typical biofabrication applications requires chemical modification or mixing with other polymers, mainly due to the limited viscoelastic properties. In this study, we hypothesize that a bioink exclusively based on decellularized kidney ECM (dKECM) could be used to bioprint renal progenitor cells. To address these aims, porcine kidneys were decellularized, lyophilized and digested to yield a viscous solution. Then, the bioprinting process was optimized using an agarose microparticle support bath containing transglutaminase for enzymatic crosslinking of the dKECM. This methodology was highly effective to obtain constructs with good printing resolution and high structural integrity. Moreover, the encapsulation of primary renal progenitor cells resulted in high cell viability, with creation of 3D complex structures over time. More importantly, this tissue-specific matrix was also able to influence cellular growth and differentiation over time. Taken together, these results demonstrate that unmodified dKECM bioinks have great potential for bioengineering renal tissue analogs with promising translational applications and/or for in vitro model systems. Ultimately, this strategy may have greater implications on the biomedical field for the development of bioengineered substitutes using decellularized matrices from other tissues.

Nanoclay Suspension-Enabled Extrusion Bioprinting of Three-Dimensional Soft Structures

Abstract Three-dimensional (3D) extrusion printing of cellular/acellular structures with biocompatible materials has been widely investigated in recent years. However, the requirement of a suitable solidification rate of printable ink materials constrains the utilization of extrusion-based 3D printing techniques. In this study, the nanoclay yield-stress suspension-enabled extrusion-based 3D printing system has been investigated and demonstrated to overcome solidification rate constraints during printing. Utilizing the liquid–solid transition property of nanoclay suspension, two fabrication approaches, including nanoclay support bath-enabled printing and nanoclay-enabled direct printing, have been proposed. For the former approach, nanoclay (Laponite® EP) has been used as a support bath material to fabricate alginate-based tympanic membrane patches. The constituents of alginate-based ink have been investigated to have the desired mechanical property of alginate-based tympanic membrane patches and facilitate the printing process. For the latter approach, nanoclay (Laponite® XLG) has been used as an internal scaffold material to help print poly (ethylene glycol) diacrylate (PEGDA)-based neural chambers, which can be further cross-linked in air. Mechanical stress analysis has been performed to explore the geometric limitation of printable Laponite® XLG-PEGDA neural chambers.

Optimization of Freeze-FRESH Methodology for 3D Printing of Microporous Collagen Constructs.

Freeform reversible embedding of suspended hydrogels (FRESH) is a layer-by-layer extrusion-based technique to enable three-dimensional (3D) printing of soft tissue constructs by using a thermo-reversible gelatin support bath. Suboptimal resolution of extrusion-based printing limits its use for the creation of microscopic features in the 3D construct. These microscopic features (e.g., pore size) are known to have a profound effect on cell migration, cell-cell interaction, proliferation, and differentiation. In a recent study, FRESH-based 3D printing was combined with freeze-casting in the Freeze-FRESH (FF) method, which yielded alginate constructs with hierarchical porosity. However, use of the FF approach allowed little control of micropore size in the printed alginate constructs. Herein, the FF methodology was optimized for 3D printing of collagen constructs with greater control of microporosity. Modifications to the FF method entailed melting of the FRESH bath before freezing to allow more efficient heat transport, achieve greater control on microporosity, and permit polymerization of collagen molecules to enable 3D printing of stable microporous collagen constructs. The effects of different freezing temperatures on microporosity and physical properties of the 3D-printed collagen constructs were assessed. In addition, finite element (FE) models were generated to predict the mechanical properties of the microporous constructs. Further, the impact of different micropore sizes on cellular response was evaluated. Results showed that the microporosity of 3D-printed collagen constructs can be tailored by customizing the FF approach. Compressive modulus of microporous constructs was significantly lower than the non-porous control, and the FE model verified these findings. Constructs with larger micropore size were more stable and showed significantly greater cell infiltration and metabolic activity. Together, these results suggest that the FF method can be customized to guide the design of 3D-printed microporous collagen constructs.

3D Printing Unique Nanoclay-Incorporated Double-Network Hydrogels for Construction of Complex Tissue Engineering Scaffolds.

The development of new biomaterial inks with good structural formability and mechanical strength is critical to the fabrication of 3D tissue engineering scaffolds. For extrusion-based 3D printing, the resulting 3D constructs are essentially a sequential assembly of 1D filaments into 3D constructs. Inspired by this process, this paper reports the recent study on 3D printing of nanoclay-incorporated double-network (NIDN) hydrogels for the fabrication of 1D filaments and 3D constructs without extra assistance of support bath. The frequently used "house-of-cards" architectures formed by nanoclay are disintegrated in the NIDN hydrogels. However, nanoclay can act as physical crosslinkers to interact with polymer chains of methacrylated hyaluronic acid (HAMA) and alginate (Alg), which endows the hydrogel precursors with good structural formability. Various straight filaments, spring-like loops, and complex 3D constructs with high shape-fidelity and good mechanical strength are fabricated successfully. In addition, the NIDN hydrogel system can easily be transformed into a new type of magnetic responsive hydrogel used for 3D printing. The NIDN hydrogels also supported the growth of bone marrow mesenchymal stem cells and displayed potential calvarial defect repair functions.

Poloxamer/Poly(ethylene glycol) Self-Healing Hydrogel for High-Precision Freeform Reversible Embedding of Suspended Hydrogel.

Freeform reversible embedding of suspended hydrogel (FRESH) is an additive manufacturing technique enabling the 3D printing of soft materials with low or no yield stress. The printed material is embedded during the process until its solidification. From the literature, FRESH abilities are self-healing, reusability, suspending, thermal stability, and high-precision printing. This study proposes a new support hydrogel bath formulation for FRESH 3D printing. To do so, a poloxamer micellar thermoreversible hydrogel is tuned through the addition of poly(ethylene glycol) (PEG) to adapt rheological properties. PEG macromolecules interact with poly(ethylene oxide) blocks of poloxamer and favor micelle dehydration, and then decreasing the gelation temperature, the yield stress, and the viscosity. Parameters such as the Oldroyd number and the Rayleigh-Plateau instability, both dependent on yield stress, were studied to determine their impact on the FRESH 3D printing resolution and accuracy. It was found that print accuracy of embedded parts increases with increasing yield stress but then the self-healing property gets limited, leading to crevasse formation. The usefulness of this approach is distinctly demonstrated through a six-axis printing of a highly complex silicone anatomical model. Printing fidelity of 96.0 ± 3.58% (5-40 mm printed parts) is thus achieved using the newly formulated FRESH material, while only 56.0 ± 0.76% fidelity is obtained using the standard formulation. The present study thus showed that complex FRESH 3D printing of soft materials is possible in this tunable hydrogel and that parts can be manufactured on an industrial scale, thanks to the reusability of the support bath.

Emergence of FRESH 3D printing as a platform for advanced tissue biofabrication.

In tissue engineering, an unresolved challenge is how to build complex 3D scaffolds in order to recreate the structure and function of human tissues and organs. Additive manufacturing techniques, such as 3D bioprinting, have the potential to build biological material with unprecedented spatial control; however, printing soft biological materials in air often results in poor fidelity. Freeform Reversible Embedding of Suspended Hydrogels (FRESH) is an embedded printing approach that solves this problem by extruding bioinks within a yield-stress support bath that holds the bioinks in place until cured. In this Perspective, we discuss the challenges of 3D printing soft and liquid-like bioinks and the emergence for FRESH and related embedded printing techniques as a solution. This includes the development of FRESH and embedded 3D printing within the bioprinting field and the rapid growth in adoption, as well as the advantages of FRESH printing for biofabrication and the new research results this has enabled. Specific focus is on the customizability of the FRESH printing technique where the chemical composition of the yield-stress support bath and aqueous phase crosslinker can all be tailored for printing a wide range of bioinks in complex 3D structures. Finally, we look ahead at the future of FRESH printing, discussing both the challenges and the opportunities that we see as the biofabrication field develops.

Simulated filament shapes in embedded 3D printing.

Embedded 3D printing, wherein fluid inks are extruded into support baths, has enabled the manufacture of complex, custom structures ranging from cell-laden tissue analogues to soft robots. This method encompasses two techniques: embedded ink writing (EIW), where filaments are extruded, and embedded droplet printing (EDP), where droplets are suspended. Materials for embedded 3D printing can be Newtonian, but often both the ink and the support bath are yield stress fluids, following elastic behavior below the yield stress and shear-thinning, viscous behavior above the yield stress. The effect of surface tension on print quality has been debated, as inks have been printed into supports at high and low surface tensions. In order to guide material selection for embedded 3D printing and identify key scaling relationships that influence print quality, this study investigates the role of ink rheology, support rheology, and surface tension on the morphology of single filaments. Numerical simulations in OpenFOAM demonstrate that at low viscosities, surface tension controls the filament morphology. Where capillarity is suppressed, the ratio of the local ink and support viscosities and the shape of the yield surface in the support control the filament shape. Herschel-Bulkley support fluids (yield stress fluids) produce more stable, accurately positioned filaments than Newtonian supports. In the short term, non-zero surface tensions can suppress filament shape defects in EIW and are essential for producing droplets in EDP.

3D Bioprinting using UNIversal Orthogonal Network (UNION) Bioinks.

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, we developed a versatile, bioorthogonal bioink crosslinking mechanism that is cell compatible and works with a range of polymers. We term this family of materials UNIversal, Orthogonal Network (UNION) bioinks. As demonstration of UNION bioink versatility, gelatin, hyaluronic acid (HA), recombinant elastin-like protein (ELP), and polyethylene glycol (PEG) were each used as backbone polymers to create inks with storage moduli spanning 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 were 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.

Toward a neurospheroid niche model: optimizing embedded 3D bioprinting for fabrication of neurospheroid brain-like co-culture constructs

A crucial step in creating reliable in vitro platforms for neural development and disorder studies is the reproduction of the multicellular three-dimensional (3D) brain microenvironment and the capturing of cell-cell interactions within the model. The power of self-organization of diverse cell types into brain spheroids could be harnessed to study mechanisms underlying brain development trajectory and diseases. A challenge of current 3D organoid and spheroid models grown in petri-dishes is the lack of control over cellular localization and diversity. To overcome this limitation, neural spheroids can be patterned into customizable 3D structures using microfabrication. We developed a 3D brain-like co-culture construct using embedded 3D bioprinting as a flexible solution for composing heterogenous neural populations with neurospheroids and glia. Specifically, neurospheroid-laden free-standing 3D structures were fabricated in an engineered astrocyte-laden support bath resembling a neural stem cell niche environment. A photo-crosslinkable bioink and a thermal-healing supporting bath were engineered to mimic the mechanical modulus of soft tissue while supporting the formation of self-organizing neurospheroids within elaborate 3D networks. Moreover, bioprinted neurospheroid-laden structures exhibited the capability to differentiate into neuronal cells. These brain-like co-cultures could provide a reproducible platform for modeling neurological diseases, neural regeneration, and drug development and repurposing.

FRESH 3D Bioprinting a Full-Size Model of the Human Heart.

Recent advances in embedded three-dimensional (3D) bioprinting have expanded the design space for fabricating geometrically complex tissue scaffolds using hydrogels with mechanical properties comparable to native tissues and organs in the human body. The advantage of approaches such as Freeform Reversible Embedding of Suspended Hydrogels (FRESH) printing is the ability to embed soft biomaterials in a thermoreversible support bath at sizes ranging from a few millimeters to centimeters. In this study, we were able to expand this printable size range by FRESH bioprinting a full-size model of an adult human heart from patient-derived magnetic resonance imaging (MRI) data sets. We used alginate as the printing biomaterial to mimic the elastic modulus of cardiac tissue. In addition to achieving high print fidelity on a low-cost printer platform, FRESH-printed alginate proved to create mechanically tunable and suturable models. This demonstrates that large-scale 3D bioprinting of soft hydrogels is possible using FRESH and that cardiac tissue constructs can be produced with potential future applications in surgical training and planning.

Embedded 3D Bioprinting of Gelatin Methacryloyl-Based Constructs with Highly Tunable Structural Fidelity.

Three-dimensional (3D) bioprinting of hydrogel-based constructs at adequate consistency and reproducibility can be obtained through a compromise between the hydrogel's inherent instability and printing fidelity. There is an increasing demand to develop bioprinting modalities that enable high-fidelity fabrication of 3D hydrogel structures that closely correspond to the envisioned design. In this work, we performed a systematic, in-depth characterization and optimization of embedded 3D bioprinting to create 3D gelatin-methacryloyl (gelMA) structures with highly controlled fidelity using Carbopol as suspension bath. The role of various embedded printing process parameters in bioprinting fidelity was investigated using a combination of experimental and theoretical approaches. We examined the effect of rheological properties of gelMA and Carbopol at varying concentrations, as well as printing conditions on the volumetric flow rate of gelMA bioink. Printing speed was examined and optimized to successfully print gelMA into the support bath at varying Carbopol concentrations. Printing fidelity was characterized in terms of printed strand diameter, uniformity, angle, and area. The optimal Carbopol solution that retained filament shape at highest fidelity was determined. The efficacy of developed bioprinting approach was then demonstrated by fabricating 3D hydrogel constructs with varying geometries and visualized using an advanced synchrotron-based imaging technique. We also investigated the influence of the Carbopol medium on cross-linking and the resulting stiffness of gelMA constructs. Finally, in vitro cytotoxicity of the developed bioprinting approach was assessed by printing human umbilical vein endothelial cells encapsulated in the gelMA bioink. These results demonstrate the significance of the close interplay between bioink-support bath rheology and printing parameters and help to establish an optimized workflow for creating 3D hydrogel structures with high fidelity and cytocompatibility via embedded bioprinting techniques. This robust platform could further expand the application of bioprinted soft tissue constructs in a wide variety of biomedical applications.

Bioprinting Organs-Progress Toward a Moonshot Idea.

Bioprinting Organs-Progress Toward a Moonshot Idea.

Corner accuracy in direct ink writing with support material

3D printing methods which enable control over the position and orientation of embedded particles have promising applications in cell patterning and composite scaffolds. Extrusion-based additive manufacturing techniques such as fused deposition modeling and direct ink writing can experience particle patterning defects at corners which could hinder cell survival at corners and create unintended property gradients. Here, we propose models which predict the behavior of deposited lines at corners for moderate viscosity inks which are impacted by both capillarity and viscous dissipation. Using direct ink writing with acoustophoresis and a Carbopol-based support gel, we write polygons out of dental resin-based composite inks containing a narrow distribution of microparticles at the center of the filament. A Laplace pressure differential between the inner and outer surfaces of the corner drives corner smoothing, wherein the inner radius of the corner increases. Double deposition, or printing on the same area twice, drives corner swelling, wherein excess ink is diverted to the outer edge of the corner. Fast turns at corners produce ringing, wherein vibrations in the stage manifest in oscillations in the print path. Swelling and ringing effects are apparent in the particle distributions at corners immediately after deposition, while smoothing effects are apparent after the printed structure has had time to relax. When the nozzle returns to write a neighboring line, it imposes shear stresses which mitigate inconsistencies in microstructure at corners by erasing defects which appeared during relaxation. Using a support bath instead of layer-by-layer support suppresses microstructural corner defects.

3D Bioprinting of Carbohydrazide-Modified Gelatin into Microparticle-Suspended Oxidized Alginate for the Fabrication of Complex-Shaped Tissue Constructs.

Extrusion-based bioprinting of hydrogels in a granular secondary gel enables the fabrication of cell-laden three-dimensional (3D) constructs in an anatomically accurate manner, which is challenging using conventional extrusion-based bioprinting processes. In this study, carbohydrazide-modified gelatin (Gel-CDH) was synthesized and deposited into a new multifunctional support bath consisting of gelatin microparticles suspended in an oxidized alginate (OAlg) solution. During extrusion, Gel-CDH and OAlg were rapidly cross-linked because of the Schiff base formation between aldehyde groups of OAlg and amino groups of Gel-CDH, which has not been demonstrated in the domain of 3D bioprinting before. Rheological results indicated that hydrogels with lower OAlg to Gel-CDH ratios possessed superior mechanical rigidity. Different 3D geometrically intricate constructs were successfully created upon the determination of optimal bioprinting parameters. Human mesenchymal stem cells and human umbilical vein endothelial cells were also bioprinted at physiologically relevant cell densities. The presented study has offered a novel strategy for bioprinting of natural polymer-based hydrogels into 3D complex-shaped biomimetic constructs, which eliminated the need for cytotoxic supplements as external cross-linkers or additional cross-linking processes, therefore expanding the availability of bioinks.

Freeze-FRESH: A 3D Printing Technique to Produce Biomaterial Scaffolds with Hierarchical Porosity.

Tissues are organized in hierarchical structures comprised of nanoscale, microscale, and macroscale features. Incorporating hierarchical structures into biomaterial scaffolds may enable better resemblance of native tissue structures and improve cell interaction, but it is challenging to produce such scaffolds using a single conventional scaffold production technique. We developed the Freeze-FRESH (FF) technique that combines FRESH 3D printing (3DP) and freeze-casting to produce 3D printed scaffolds with microscale pores in the struts. FF scaffolds were produced by extrusion 3DP using a support bath at room temperature, followed by freezing and lyophilization, then the FF scaffolds were recovered from the bath and crosslinked. The FF scaffolds had a hierarchical pore structure from the combination of microscale pores throughout the scaffold struts and macroscale pores in the printed design, while control scaffolds had only macroscale pores. FF scaffolds frozen at −20 °C and −80 °C had similar pore sizes, due to freezing in the support bath. The −20 °C and −80 °C FF scaffolds had porous struts with 63.55% ± 2.59% and 56.72% ± 13.17% strut porosity, respectively, while control scaffolds had a strut porosity of 3.15% ± 2.20%. The −20 °C and −80 °C FF scaffolds were softer than control scaffolds: they had pore wall stiffness of 0.17 ± 0.06 MPa and 0.23 ± 0.05 MPa, respectively, compared to 1.31 ± 0.39 MPa for the control. The FF scaffolds had increased resilience in bending compared with control. FF scaffolds supported MDA-MB-231 cell growth and had significantly greater cell numbers than control scaffolds. Cells formed clusters on the porous struts of FF scaffolds and had similar morphologies as the freeze cast scaffolds. The FF technique successfully introduced microscale porosity into the 3DP scaffold struts to produce hierarchical pore structures that enhanced MDA-MB-231 growth.

Optimization of Silicone 3D Printing with Hierarchical Machine Learning

Additive manufacturing of soft materials requires optimization of printable inks, formulations of these feedstocks, and complex printing processes that must balance a large number of disparate but highly correlated variables. Here, hierarchical machine learning (HML) is applied to 3D printing of silicone elastomer via freeform reversible embedding (FRE), which is challenging because it involves depositing a Newtonian prepolymer liquid phase within a Bingham plastic support bath. The advantage of the HML algorithm is that it can predict the behavior of complex physical systems using sparse data sets through integration of physical modeling in a framework of statistical learning. Here, it is shown that this algorithm can be used to simultaneously optimize material, formulation, and processing variables. The FRE method for 3D printing silicone parts was optimized based on a training set with 38 trial runs. Compared with the previous results from iterative optimization approaches using design-of-experiment and steepest-ascent methods, HML increased printing speed by up to 2.5 × while retaining print fidelity and also identified a unique silicone formulation and printing parameters that had not been found previously through trial-and-error approaches. These results indicate that HML is an effective tool with the potential for broad application for planning and optimizing in additive manufacturing of soft materials via the FRE method.

Printing of Hydrophobic Materials in Fumed Silica Nanoparticle Suspension.

Freeform three-dimensional (3D) printing of functional structures from liquid hydrophobic build materials is of great significance and widely used in various fields such as soft robotics and microfluidics. In particular, a yield-stress support bath-enabled 3D-printing methodology has been emerging to fabricate complex 3D structures. Unfortunately, the reported support bath materials are either hydrophobic or not versatile enough for the printing of a wide range of hydrophobic materials. The objective of this study is to propose a fumed silica nanoparticle-based yield-stress suspension as a hydrophobic support bath to enable 3D extrusion printing of various hydrophobic ink materials in a printing-then-solidification fashion. Hydrophobic ink is freeform-deposited in a hydrophobic fumed silica-mineral oil suspension and maintains its shape during printing; it is not cured until the whole structure is complete. Various hydrophobic inks including poly(dimethylsiloxane) (PDMS), SU-8 resin, and epoxy-based conductive ink are printed into complex 3D structures in the fumed silica-mineral oil bath and then cured using relevant cross-linking mechanisms, even at a temperature as high as 90 °C, to prove the feasibility and versatility of the proposed printing approach. In addition, the deposited feature can easily reach a much better resolution such as 30 μm for PDMS filaments due to the negligible interfacial tension effect.

Nydus One Syringe Extruder (NOSE): A Prusa i3 3D printer conversion for bioprinting applications utilizing the FRESH-method

<h2>Abstract</h2> Bioprinting, combined with other tissue engineering techniques, is a promising method to create engineered tissues or standardized 3D cell culturing models. It has potential applications in the development of animal free models for drug toxicity screenings or for personalized medicine approaches. Here we report the conversion of the worldwide used open source 3D printer (RepRap, Prusa i3) to a functional and affordable bioprinter by substituting the common plastic-extruder with the <b>N</b>ydus <b>O</b>ne <b>S</b>yringe <b>E</b>xtruder (<b>NOSE</b>). The <b>NOSE</b> modification enables mechanical hydrogel extrusion as well as a tunable deposition precision and volume by featuring a modular syringe-holder concept. A cost effective and simple hardware design including a custom software (termed ‘composer') was developed to prove that this technique can be made accessible for a broader public. Furthermore, the printing procedure was optimized for the peer-reviewed FRESH-method by Hinton et al. which allows to print geometrical complex cell-laden constructs. We provide a detailed protocol on the creation of the FRESH-gel to ensure the reproducibility of this state of the art method. As a proof of concept HEK293 cells as well as mouse embryonic stem cells (mESC) were utilized for cell-laden printing. Depending on the cellular source the survival rates range from 60% up to 95%. Automatized quantitative viability analysis was performed using the open source image analysis tool Icy, in particular the "spot detector" plugin. A detailed protocol allows the recreation of the assay. Further data utilizing a support bath printing technique reveal limitations regarding the printing and residential time of cell-laden constructs.

Direct writing alginate bioink inside pre-polymers of hydrogels to create patterned vascular networks

We describe a strategy to fabricate a hydrogel-based microvascular construct by direct writing alginate bioink inside the viscous pre-polymer of hydrogels, which acts as a support bath. As the print needle translates through the polymers, the extruded alginate instantaneously forms calcium alginate hydrogel (Ca-Alg) templates deposited within the bath. This phase change allows the formed templates to be anchored within the pre-polymers, while maintaining their structure. After the printing process, the pre-polymers are solidified to form a mechanically robust hydrogel. Finally, a hydrogel construct with embedded microchannels is generated by liquefying and removing the Ca-Alg templates. Using this method, not only the alginate ink alone can be directly printed within the engineered constructs, but also the size and shape of the formed microchannels are controllable. Furthermore, a confluent endothelial layer for the generation of vascular networks can be constructed by adhering and proliferating endothelial cells on the channel linings. This strategy demonstrates a promising technique for rapid construction of in vitro vasculatures, which would provide a versatile platform for a wide array of applications such as tissue engineering, organ-on-a-chip and drug screening.