This research was supported by Ningbo Natural Science Foundation (2022J121, 2023J159), Key project of Natural Science Foundation of Ningbo City (2021J256), Open Foundation of the State Key Laboratory of Molecular Engineering of Polymers (Fudan University) (K2024-35), and Key Laboratory of Precision Medicine for Atherosclerotic Diseases of Zhejiang Province, China (2022E10026). Thanks for the technical support by the Core Facilities, Health Science Center of Ningbo University.

1. Preparation of the &#954; -carrageenan sub-microgel suspension bath
<ol>
	<li>Prepare 500 mL of &#954;-carrageenan suspension bath (0.35% wt/vol) by adding 1.75 g of &#954; -carrageenan powder into 500 mL of phosphate-buffered saline (PBS, pH 7.4) solution within a 1,000 mL glass bottle.</li>
	<li>Introduce a 70 mm magnetic stirrer bar into the glass bottle to stir the aqueous mixture. Tighten the glass bottle cap and then loosen it by half a turn.</li>
	<li>Place the glass bottle in a 70 &#176;C water bath and heat it. Turn on the magnetic stirrer at a speed of 300 rpm, place the bottle, and stir until the polymer is completely dissolved.</li>
	<li>Take another glass bottle and place it in an autoclave for sterilization, running at 121 &#176;C for 30 min.</li>
	<li>After the high-pressure sterilizer cools to 80 &#176;C, remove the bottle from the sterilizer and transfer it to the ultra-clean workbench for further handling.</li>
	<li>Filter the completely dissolved &#954;-carrageenan solution into the sterilized glass bottle using a 0.22 &#181;m filter. 
	NOTE: Perform filtration swiftly while the solution is hot to prevent the &#954;-carrageenan from cooling and obstructing the filter.</li>
	<li>Store the &#954;-carrageenan solution at 4 &#176;C to induce a cation-crosslinked gelation for 12 h.</li>
	<li>Mechanically grind the &#954;-carrageenan hydrogels using a 60 mm magnetic stirrer with a stirring speed of 1200 rpm at 25 &#176;C until it successfully transforms into a liquid state, taking approximately 60 min. 
	NOTE: Store the &#954;-carrageenan hydrogels between 25-37 &#176;C to avoid gelation at low temperatures or dissolution at high temperatures.</li>
</ol>
2. Preparation of gelatin methacrylate/silk fibroin methacrylate composite bioinks
<ol>
	<li>Prepare composite bioinks by combining 10% (wt/vol) gelatin methacrylate (GelMA) and 6% (wt/vol) silk fibroin methacrylate (SFMA) in PBS solution (pH 7.4). Weigh out 2.0 g of GelMA and 1.2 g of SFMA powders separately.</li>
	<li>Slowly add the powders into two 50 mL centrifuge tubes. Add 10 mL of PBS solution separately into the 50 mL centrifuge tubes.</li>
	<li>Add a 10 mm magnetic stirrer to each and constantly stir and heat in a water bath at 45 &#176;C. Allow the GelMA and SFMA powders to completely dissolve, taking approximately 30 min.</li>
	<li>Mix the GelMA and SFMA solutions in an equal volume under continuous stirring at 45 &#176;C. Filter the composite bioinks using a 100 &#181;m pore size filter.</li>
	<li>Sterilize the composite GelMA/SFMA bioinks through UV irradiation in a biosafety cabinet for 12 h. 
	NOTE: Prepare and use the bioinks immediately to prevent premature gelation of the SFMA through self-assembly.</li>
</ol>
3. Rheological characterization of the &#954; -carrageenan sub-microgel suspension bath
<ol>
	<li>Start up and prepare rheology-related equipment.
	<ol>
		<li>Switch on the air compressor for 30 min and ensure that the pressure reaches 30 psi. Remove the bearing clamp by turning the draw rod anti-clockwise. Fix the black cover below and rotate the knob axis on the top of the instrument clockwise.</li>
		<li>Power on the rheometer and allow it to initialize. Turn on the circulation water switch and allow the temperature to reach 15 &#176;C.</li>
		<li>Open the rheometer control software to establish the online connection.</li>
	</ol>
	</li>
	<li>Calibrate the rheometer and install the parallel plate geometry.
	<ol>
		<li>Perform instrument calibration in the control software, which mainly involves adjusting the calibration instrument inertia path. Fit a 40 mm diameter parallel plate geometry to the end of the draw rod. Hold the Lock button for 3 s to move the motor shaft to the home position for consistent geometry placement.</li>
		<li>Select the Calibrate Manager in the control software and subsequently click the Inertia, Friction Calibration, and Rotational Mapping to calibrate the parallel plate geometry.</li>
	</ol>
	</li>
	<li>Zero the rheometer gap height by conducting a standard gap zeroing procedure to ensure precise measurement.</li>
	<li>Set up the experimental steps, including a flow ramp ranging from 0.01 to 1000 1/s shear rate, an amplitude sweep ranging from 0.1% to 100% strain at 10 rad/s, a multi-step oscillation time sweep lasting for 60 s with alternating strains of 1% and 100% at 10 rad/s, and a multi-step Peak Hold sweep lasting for 120 s with alternating shear rate of 0.1 1/s and 10 1/s.</li>
	<li>Drop 2 mL of 0.35% &#954;-carrageenan suspensions onto the rheometer&#39;s Peltier plate.&#160;</li>
	<li>Position the geometry gap to 510 &#956;m and remove any excess overflow at the edge of the clamp device.&#160;Set the sample gap to 500 &#181;m to allow the sample to extend slightly beyond the clamp edge.</li>
	<li>Conduct a flow experiment by selecting the Flow Ramp from the experimental design options.
	<ol>
		<li>Choose the Flow Ramp test in the experimental design. Set the shear rates from 0.001 to 10 1/s at a temperature of 25 &#176;C.</li>
		<li>Perform step 3.5 and the Flow Ramp experiment on the added sample.</li>
	</ol>
	</li>
	<li>Perform a cyclic Peak Hold test to assess the material&#39;s recovery performance.
	<ol>
		<li>Choose Peak Hold sweep in the experimental design and set 5 consecutive experiments with a time of 120 s at 25 &#176;C.</li>
		<li>Set the shear rate to 0.01 1/s for the first, third, and fifth steps and to 10 1/s for the second and fourth steps.</li>
		<li>Add a new sample of 2 mL. Perform step 3.5 and the cyclic recovery test.</li>
	</ol>
	</li>
	<li>Perform viscoelasticity analysis to evaluate the potential gel-sol transition of the &#954;-carrageenan suspension bath.
	<ol>
		<li>Choose the Amplitude Sweep test in experimental design. Set the oscillation strain from 0.1% to 100% at a temperature of 25 &#176;C. Click Start to perform the Amplitude Sweep experiment on the added sample.</li>
	</ol>
	</li>
	<li>Perform alternative strain analysis to assess the material&#39;s elastic recovery performance.
	<ol>
		<li>Choose Oscillation Time Sweep in the experimental design and set 5 consecutive experiments with a time of 60 s at 25 &#176;C.</li>
		<li>Set the strain to 1% for the first, third, and fifth steps and to 100% for the second and fourth steps.</li>
		<li>Add a new sample of 2 mL. Perform step 3.5 and the continuous cyclic recovery test. 
		NOTE: Clean the geometry plate and Peltier plate after each experiment test and add a new sample of 2 mL.</li>
	</ol>
	</li>
</ol>
4. Printing biomimetic hydrogel scaffolds using a custom-designed micro-extrusion bioprinter
<ol>
	<li>Design STL-format cubes using a 3D graphics software and download tissue-like models from the 3D database (www.thingiverse.com; https://3d. nih.gov/.).&#8203;
	<ol>
		<li>Import the STL-format models into PANGO software and input the printing parameters. Set the specific X, Y, and Z coordinates of the model in 3D space, input the expected scaling size of the model in millimeters (mm), adjust the size and scaling ratio of the object on the X, Y, and Z axes, and adjust the rotational angle of the model in space as needed.</li>
		<li>Input the estimated filament diameter (typically around 200-500 &#181;m for most bioinks) into the layer thickness field to determine the Z-axis thickness of each layer. Set the printing speed according to the expected line thickness (approximately 10-90 mm/s) and set the fill density to 30%-80%.</li>
		<li>Export the final parameters as G-code onto an SD card.</li>
	</ol>
	</li>
	<li>Run the bioprinter and micro-extrusion pump.
	<ol>
		<li>Switch on the micro-extrusion pump controller, select the corresponding volume syringe (5 mL), and set the extrusion rate at 0.08 mL/min and the duration of extrusion in coordination with the desired printing time obtained by dividing the hydrogel volume by the extrusion rate.</li>
		<li>Fit the syringe with a needle featuring an inner diameter of 210 &#181;m into the syringe slot of the bioprinter.</li>
		<li>Adjust the syringe controller to ensure that the plunger of the syringe is in close contact with the screw.</li>
		<li>Place 3 mL of suspension bath into a 35 mm cell culture dish. Position the dish on the platform based on the code settings and confirm that the printing head is 1 mm above the bottom of the dish. 
		NOTE: Conduct a pre-test to ensure the accuracy of dish positions on the printing platform during printing.</li>
		<li>Insert the SD card containing the code into the 3D bioprinter, activate the code file, start the bioprinter, and click the Start button on the controller.</li>
	</ol>
	</li>
	<li>Processing of the constructs
	<ol>
		<li>Expose the printed construct to 405 nm blue light for 1 min to initiate photocrosslinking.</li>
		<li>Remove the &#954;-carrageenan gel using a 1 mL pipette, followed by the addition of 3 mL of PBS (pH 7.4) for washing and subsequent removal. Repeat this process for thorough washing. 
		NOTE: Perform the washing process gently to avoid damaging the printed constructs.</li>
	</ol>
	</li>
</ol>
5. Embedded 3D bioprinting of esophageal muscle-layer analogs
<ol>
	<li>Culture rabbit esophageal smooth muscle cells (eSMCs).
	<ol>
		<li>Initiate the culture in T75 flasks with 2 x 106 cells using 15 mL of DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin.</li>
		<li>Maintain the eSMCs at 37 &#176;C in a humidified atmosphere of 5% CO2 and 95% air until they reach 80% confluency.</li>
	</ol>
	</li>
	<li>Preparation of esophageal muscle layer bioink
	<ol>
		<li>Aspirate the medium upon reaching confluency and wash the eSMCs with 5 mL of 1x PBS.</li>
		<li>Add 2 mL of 0.2% Trypsin-EDTA to the flask and incubate for 2 min to digest the cells. Tap the flask to detach the cells from the wall.</li>
		<li>Terminate digestion by adding 2 mL of DMEM and transfer the cell suspension to a 15 mL centrifuge tube. Measure the cell density using a cell counter with 10 &#181;L of cell-suspension.</li>
		<li>Centrifuge at 675 x g for 3 min, carefully aspirate the supernatant and resuspend the cell pellet in the composite GelMA/SFMA bioinks (as prepared in step 2.1). Adjust the final cell concentration to achieve a density of 10 x 106 cells/mL in the bioink, thereby optimizing conditions for subsequent 3D bioprinting applications.</li>
	</ol>
	</li>
	<li>Preparation of the 3D bioprinter and associated materials
	<ol>
		<li>Autoclave the essential printing instruments, including syringe needles, forceps, a magnetic stirrer, and a 1000 mL silicate glass bottle, under standard sterilization conditions (121 &#176;C for 30 min) to ensure an aseptic environment for the printing process.</li>
		<li>Place the bioprinter inside a biosafety cabinet and sterilize it with ultraviolet (UV) light for a duration of 30 min to maintain sterility.</li>
		<li>Prepare 50 mL of sterile &#954;-carrageenan and 5 mL of sterile GelMA/SFMA composite bioinks, as described in step 1 and step 2, respectively.</li>
		<li>Design the model, set the desired parameters, and prepare the bioprinter as outlined in step 4.1.</li>
	</ol>
	</li>
	<li>Initiation of the bioprinting process
	<ol>
		<li>Perform the procedures described in steps 4.2 and 4.3 to bioprint the 3D esophageal muscle layer, followed by PBS washing and replacement.</li>
		<li>Perform cell printing with sterile techniques throughout the printing process to minimize the risk of cell contamination in the printed structure.</li>
	</ol>
	</li>
	<li>Post-printing cell culture medium replacement
	<ol>
		<li>Carefully aspirate the PBS and replace it with 3 mL of DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Ensure that the printed constructs are entirely submerged to provide optimal cell growth conditions.</li>
		<li>Incubate the cell culture dish containing the printed constructs in an incubator set to 37 &#176;C with 5% CO2. Replace the cell culture medium with fresh medium daily, using 3 mL each time, and monitor the state of the cells within the constructs using light microscopy.</li>
	</ol>
	</li>
</ol>
6. Evaluation of eSMCs viability within the printed constructs via Live/Dead assay staining
<ol>
	<li>Remove the cell culture medium after 5 h of incubation and wash the cell-laden constructs with PBS.</li>
	<li>Add 2 mL of the standard calcein-AM/propidium iodide solution (1:1000) to the printed cell-laden constructs and incubate.</li>
	<li>Wash the constructs 3x with PBS after a 45 min incubation and then add fresh cell culture medium.</li>
	<li>Observe live and dead cells and capture fluorescent images using confocal laser scanning microscopy (CLSM).</li>
</ol>
7. Observation of the eSMCs within the printed constructs via FITC-Phalloidin/DAPI staining
<ol>
	<li>Stain the printed constructs using fluorescein isothiocyanate (FITC) labeled phalloidin (Green) for F-actin and 4&#39;,6-Diamidino-2-Phenylindole (DAPI, blue) for nuclear post 5 days of incubation.</li>
	<li>Fixation solution preparation
	<ol>
		<li>Combine 4 mL of 1x PBS with 0.16 mL of paraformaldehyde (PFA) in a 5 mL polyethylene (PE) tube, achieving a final concentration of 4%.</li>
		<li>Place the culture dish containing the bioprinted constructs on a clean surface. Remove the medium and gently cover the constructs with the PFA solution, ensuring their full immersion. Allow the fixation to proceed at room temperature for 20 min before discarding the solution. 
		NOTE: Handle liquids with care during the addition and removal processes to preserve the structural integrity of the bioprinted constructs.</li>
		<li>Introduce 2.5 mL of 1x PBS to the culture dish, fully submerging the constructs to remove any residual staining reagents.</li>
	</ol>
	</li>
	<li>Cell membrane permeabilization
	<ol>
		<li>Formulate a 0.5% Triton X-100 solution by adding 20 &#181;L of Triton X-100 to 4 mL of 1x PBS in a 5 mL PE tube.</li>
		<li>Administer the Triton X-100 solution to the constructs for 30 min at room temperature to permeate cell membranes, then remove the solution.</li>
		<li>Introduce 2.5 mL of 1x PBS to the culture dish, fully submerging the constructs to remove any residual staining reagents.</li>
	</ol>
	</li>
	<li>Blocking non-specific binding
	<ol>
		<li>Create a 3% bovine serum albumin (BSA) blocking solution in a 5 mL PE tube by adding 0.12 mL of BSA to 4 mL of 1x PBS.</li>
		<li>Introduce the BSA solution to the constructs for 30 min at room temperature to block non-specific sites, then discard the solution.</li>
	</ol>
	</li>
	<li>Actin staining with FITC-Phalloidin
	<ol>
		<li>Prepare 0.2% FITC-phalloidin solution in a 5 mL PE tube with 4 mL of 1x PBS and 8 &#181;L of staining reagent.</li>
		<li>Immerse the constructs in this solution for 60 min at room temperature in the dark, then discard the solution.</li>
		<li>Add 2.5 mL of 1x PBS to the culture dish, fully submerging the constructs to remove residual staining reagents.</li>
	</ol>
	</li>
	<li>Nuclear staining with DAPI
	<ol>
		<li>Apply 4 mL of DAPI staining solution to the constructs, ensuring complete coverage.</li>
		<li>Incubate in the dark at room temperature for 20 min before removing the DAPI solution.</li>
	</ol>
	</li>
	<li>Final washing and PBS submersion: Add 2.5 mL of 1x PBS to the culture dish, fully submerging the constructs to remove residual staining reagents. 
	NOTE: Prepare staining solutions in advance and handle them gently. At no point should the cells undergo desiccation within the culture vessel.</li>
	<li>Observe using a confocal microscope: Inspect and image the stained constructs using confocal microscopy to evaluate cell growth, ensuring comprehensive documentation of the results.</li>
</ol>
8. Evaluation of eSMCs proliferation within the printed constructs using CCK-8 assay
<ol>
	<li>Remove the culture medium from the constructs on days 7 and 14. Wash the constructs with PBS to remove residual medium and detached cells.</li>
	<li>Add CCK-8 solution to each construct following the manufacturer&#39;s instructions. Ensure that the volume of the CCK-8 solution is adequate to cover each construct completely.</li>
	<li>Incubate the constructs with the CCK-8 solution at 37 &#176;C for 2 h. Following incubation, carefully transfer the supernatant to a new 96-well plate.</li>
	<li>Measure the absorbance at 450 nm using a microplate reader.</li>
</ol>

Advancing 3D Bioprinting with &#954;-Carrageenan Sub-Microgel Medium

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The authors have no financial interest in the products described in this manuscript.

The preparation of &#954;-carrageenan sub-microgel suspension baths for use in bioprinting is a carefully orchestrated process that involves several critical steps to ensure the resulting medium exhibits the desired properties for supporting bioinks. Initially, a &#954;-carrageenan solution is prepared by dissolving the &#954;-carrageenan powder in deionized water at elevated temperatures, creating a homogeneous mixture. The concentration of th...

Tissue engineering scaffolds, including electro-spun fibers, porous sponges, and polymer hydrogels, play a pivotal role in the repair and reconstruction of damaged tissues and organs by providing a structural framework supporting cell growth, tissue regeneration, and the restoration of organ function1,2,3. However, traditional scaffolds encounter challenges in accurately replicating native tissue structures, leading to a mismatch between the engineered and natural tissues. This limitation hinders the efficient healing of defective tissues, emphasizing the urgent need for scaffold design advancements to achieve more accurate biomimicry. Three-dimensional (3D) bioprinting is an innovative manufacturing technique that precisely constructs complex biological tissue structures layer by layer using biomaterial inks and cells4. Among various biomaterials, polymer hydrogels emerge as ideal bioinks with their distinctive network that facilitates in situ encapsulation of cells and crucially supports their growth5,6. Nevertheless, many soft and highly hydrated hydrogels tend to induce blurring or rapid collapse of printed scaffold structures during the printing process when used as bioinks. To address this challenge, embedded 3D bioprinting technology employs a microgel bath as a support material, allowing precise soft bioink deposition. Upon gelation of the hydrogel bioinks, refined bionic scaffolds with intricate structures are obtained by removing the microgel bath. Materials like gelatin7,8, agarose9, and gellan gum10,11 have been employed to create microgel baths for embedded 3D bioprinting, significantly advancing the application of soft hydrogels in tissue engineering. However, the micron-level and non-uniform particle size of these particulate gels detrimentally impacts the resolution and fidelity of 3D printing12,13,14. There is an urgent need to fabricate a gel-like suspension float with small and uniformly dispersed particles, offering advantages in achieving high-fidelity bioprinting.
In this protocol, a novel sacrificial granulate &#954;-carrageenan suspension bath with a uniform sub-micron level is presented for embedded 3D printing. This innovative sub-microgel bath behavior of rapid jamming-unjamming transition facilitates the precise fabrication of biomimetic hydrogel scaffolds with high structural fidelity15. Utilizing this new suspension medium, a series of biomimetic tissue and organ constructs featuring multi-layer tissue structures are successfully printed, employing a composite bioink composed of gelatin methacrylate and silk fibro methacrylate. In this study, we chose the esophagus as the 3D bioprinting biomimetic object mainly because the esophagus not only has a multi-layered tissue structure but also its muscle layer exhibits an internal circular and external longitudinal complex layering structure. Ensuring proper alignment and organization of these layers is essential for functional tissue regeneration. Therefore, we highly desire to replicate the multilayered architecture of the esophagus. More importantly, we utilized &#954;-carrageenan sub-microgels as the suspension bath and GelMA/SFMA as the bioink to design and construct a biomimetic scaffold for tissue engineering. The printed esophagus can be easily released by repeated phosphate-buffered saline washing. Moreover, the &#954;-carrageenan sub-microgel bath is free of cytotoxic substances, ensuring high cytocompatibility15. The smooth muscle cells loaded within anisotropic scaffolds exhibit a notable spreading activity. This uniform sub-microgel suspension medium offers a new avenue for the fabrication of complex tissues and organs through embedded 3D bioprinting.

<table><tbody><tr><td>3D bioprinter</td><td>Custom-designed</td><td></td><td></td></tr><tr><td>4&amp;rsquo;,6-Diamidino-2-Phenylindole</td><td>Solarbio Life Science</td><td>C0065</td><td>Ready-to-use</td></tr><tr><td>405 nm UV light</td><td>EFL</td><td>XY-WJ01</td><td></td></tr><tr><td>Cell Counter</td><td>Corning</td><td>Cyto smart 6749</td><td></td></tr><tr><td>Confocal laser scanning microscope</td><td>Leica</td><td>STELLARIS 5</td><td></td></tr><tr><td>DMEM high glucose</td><td>VivaCell</td><td>C3113-0500</td><td>High Glucose, with Sodium Pyruvate and L-Glutamine</td></tr><tr><td>Dynamic rotational rheometer</td><td>TA Instrument</td><td>Discovery HR-20</td><td></td></tr><tr><td>Esophageal smooth muscle cells</td><td>Supplied by the Department of Cell Biology and Regenerative Medicine, Health Science Center, Ningbo University</td><td></td><td>Primary cells from the rabbit esophagus</td></tr><tr><td>Fetal bovine serum</td><td>UE</td><td>F9070L</td><td></td></tr><tr><td>Fluorescein isothiocyanate labeled phalloidin</td><td>Solarbio Life Science</td><td>CA1610</td><td>300T</td></tr><tr><td>Gelatin methacrylate</td><td>EFL</td><td>EFL-GM-60</td><td>60% substitution</td></tr><tr><td>k-carrageenan</td><td>Aladdin</td><td>C121013-100g</td><td>Reagent grade</td></tr><tr><td>Lithium Phenyl (2,4,6-trimethylbenzoyl) phosphinate</td><td>Aladdin</td><td>L157759-1g</td><td>365~405 nm</td></tr><tr><td>Live-Dead kit</td><td>beyotime</td><td>C2015M</td><td></td></tr><tr><td>Microplate reader</td><td>Potenov</td><td>PT-3502B</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Solarbio Life Science</td><td>P1110&amp;nbsp;</td><td>4%</td></tr><tr><td>Penicillin/streptomycin</td><td>Solarbio Life Science</td><td>MA0110</td><td>100 &amp;acute;</td></tr><tr><td>Phosphate buffered saline</td><td>VivaCell</td><td>C3580-0500</td><td>pH 7.2-7.4</td></tr><tr><td>Silk fibroin methacrylate</td><td>EFL</td><td>EFL-SilMA-001</td><td>39% substitution</td></tr><tr><td>Triton X-100</td><td>Solarbio Life Science</td><td>T8200</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>VivaCell</td><td>C100C1</td><td>0.25%, without phenol red</td></tr></tbody></table>

embedded bioprinting of tissue-like structures using &#954;-carrageenan sub-microgel medium

Embedded three-dimensional (3D) bioprinting utilizing a granular hydrogel supporting bath has emerged as a critical technique for creating biomimetic scaffolds. However, engineering a suitable gel suspension medium that balances precise bioink deposition with cell viability and function presents multiple challenges, particularly in achieving the desired viscoelastic properties. Here, a novel &#954;-carrageenan gel supporting bath is fabricated through an easy-to-operate mechanical grinding process, producing homogeneous sub-microscale particles. These sub-microgels exhibit typical Bingham flow behavior with small yield stress and rapid shear-thinning properties, which facilitate the smooth deposition of bioinks. Moreover, the reversible gel-sol transition and self-healing capabilities of the &#954;-carrageenan microgel network ensure the structural integrity of printed constructs, enabling the creation of complex, multi-layered tissue structures with defined architectural features. Post-printing, the &#954;-carrageenan sub-microgels can be easily removed by a simple phosphate-buffered saline wash. Further bioprinting with cell-laden bioinks demonstrates that cells within the biomimetic constructs have a high viability of 92% and quickly extend pseudopodia, as well as maintain robust proliferation, indicating the potential of this bioprinting strategy for tissue and organ fabrication. In summary, this novel &#954;-carrageenan sub-microgel medium emerges as a promising avenue for embedded bioprinting of exceptional quality, bearing profound implications for the in vitro development of engineered tissues and organs.

The granular &#954;-carrageenan gel bath was generated by mechanically breaking up the bulk hydrogels into a particulate gel slurry. The most recent study demonstrated that the &#954;-carrageenan particles exhibited an average diameter of approximately 642 &#177; 65 nm with uniform morphologies at 1000 rpm of mechanical blending15, significantly smaller than the dimensions of microgels previously reported in the literature16,17...

Read this Scientific Article Protocol about Embedded Bioprinting of Tissue-like Structures Using κ-Carrageenan Sub-microgel Medium at JoVE.com

Author Spotlight: Development of Homogeneous &#954;-Carrageenan Sub-Microgel Baths for High-Resolution 3D Bioprinting

This study introduces a novel &#954;-carrageenan sub-microgel suspension bath, displaying remarkable reversible jamming-unjamming transition properties. These attributes contribute to the construction of biomimetic tissues and organs in embedded 3D bioprinting. The successful printing of heart/esophageal-like tissues with high resolution and cell growth demonstrates high-quality bioprinting and tissue engineering applications.

Embedded Bioprinting of Tissue-like Structures Using &#954;-Carrageenan Sub-Microgel Medium

Embedded three-dimensional (3D) bioprinting utilizing a granular hydrogel supporting bath has emerged as a critical technique for creating biomimetic ...

embedded-bioprinting-tissue-like-structures-using-carrageenan-sub

Research

JoVE Journal

Engineering

1.1K Views.  Ningbo University. 3D-bioprinten in een suspensiebad op basis van microgel heeft een veelzijdig platform geboden voor het fabriceren van complexe 3D-structuren met behulp van zachte hydrogels, maar het wordt nog steeds beperkt door de lage printresolutie en nauwkeurigheid, waardoor de praktische toepassingen van de geprinte producten in tissue engineering worden belemmerd. De huidige microgelbaden vertonen vaak grote afmetingen en slechte dispersies, wat resulteert in een grote...

Video: Auteur in de schijnwerpers: ontwikkeling van homogene κ-carrageen sub-microgelbaden voor 3D-bioprinten met hoge resolutie 

Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact bioprinting1-4 (extrusion, dip pen and soft lithography), contactless bioprinting5-7 (laser forward transfer, ink-jet deposition) and laser based techniques such as two photon photopolymerization8. It can be used for many applications such as tissue engineering9-13, biosensor microfabrication14-16 and as a tool to answer basic biological questions such as influences of co-culturing of different cell types17. Unlike common photolithographic or soft-lithographic methods, extrusion bioprinting has the advantage that it does not require a separate mask or stamp. Using CAD software, the design of the structure can quickly be changed and adjusted according to the requirements of the operator. This makes bioprinting more flexible than lithography-based approaches.
Here we demonstrate the printing of a sacrificial mold to create a multi-material 3D structure using an array of pillars within a hydrogel as an example. These pillars could represent hollow structures for a vascular network or the tubes within a nerve guide conduit. The material chosen for the sacrificial mold was poloxamer 407, a thermoresponsive polymer with excellent printing properties which is liquid at 4 &deg;C and a solid above its gelation temperature ~20 &deg;C for 24.5% w/v solutions18. This property allows the poloxamer-based sacrificial mold to be eluted on demand and has advantages over the slow dissolution of a solid material especially for narrow geometries. Poloxamer was printed on microscope glass slides to create the sacrificial mold. Agarose was pipetted into the mold and cooled until gelation. After elution of the poloxamer in ice cold water, the voids in the agarose mold were filled with alginate methacrylate spiked with FITC labeled fibrinogen. The filled voids were then cross-linked with UV and the construct was imaged with an epi-fluorescence microscope.

A bioprinter was used to create patterned hydrogels based on a sacrificial mold. The poloxamer mold was backfilled with a second hydrogel and then eluted, leaving voids which were filled with a third hydrogel. This method uses fast elution and good printability of poloxamer to generate complex architectures from biopolymers.

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact ...

Immunology and Infection

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as organ replacements for implantation in patients, and also, when created on a smaller size scale as model "organoids" that can be used in in vitro systems for drug and toxicology screening.
Despite development of a wide variety of bioprinting devices, application of bioprinting technology can be limited by the availability of materials that both expedite bioprinting procedures and support cell viability and function by providing tissue-specific cues. Here we describe a versatile hyaluronic acid (HA) and gelatin-based hydrogel system comprised of a multi-crosslinker, 2-stage crosslinking protocol, which can provide tissue specific biochemical signals and mimic the mechanical properties of in vivo tissues. 
Biochemical factors are provided by incorporating tissue-derived extracellular matrix materials, which include potent growth factors. Tissue mechanical properties are controlled combinations of PEG-based crosslinkers with varying molecular weights, geometries (linear or multi-arm), and functional groups to yield extrudable bioinks and final construct shear stiffness values over a wide range (100 Pa to 20 kPa). Using these parameters, hydrogel bioinks were used to bioprint primary liver spheroids in a liver-specific bioink to create in vitro liver constructs with high cell viability and measurable functional albumin and urea output. This methodology provides a general framework that can be adapted for future customization of hydrogels for biofabrication of a wide range of tissue construct types.

We describe a set of protocols that together provide a tissue-mimicking hydrogel bioink with which functional and viable 3-D tissue constructs can be bioprinted for use in in vitro screening applications.

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as ...

Bioengineering

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell lines using conventional two-dimensional (2D) cell culture. These challenges can be overcome by using bioprinting techniques to build heterogeneous three-dimensional (3D) tumor models whereby different types of cells are embedded. Alginate and gelatin are two of the most common biomaterials employed in bioprinting due to their biocompatibility, biomimicry, and mechanical properties. By combining the two polymers, we achieved a bioprintable composite hydrogel with similarities to the microscopic architecture of a native tumor stroma. We studied the printability of the composite hydrogel via rheology and obtained the optimal printing window. Breast cancer cells and fibroblasts were embedded in the hydrogels and printed to form a 3D model mimicking the in vivo microenvironment. The bioprinted heterogeneous model achieves a high viability for long-term cell culture (&#62; 30 days) and promotes the self-assembly of breast cancer cells into multicellular tumor spheroids (MCTS). We observed the migration and interaction of the cancer-associated fibroblast cells (CAFs) with the MCTS in this model. By using bioprinted cell culture platforms as co-culture systems, it offers a unique tool to study the dependence of tumorigenesis on the stroma composition. This technique features a high-throughput, low cost, and high reproducibility, and it can also provide an alternative model to conventional cell monolayer cultures and animal tumor models to study cancer biology.

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

We developed a heterogeneous breast cancer model consisting of immortalized tumor and fibroblast cells embedded in a bioprintable alginate/gelatin bioink. The model recapitulates the in vivo tumor microenvironment and facilitates the formation of multicellular tumor spheroids, yielding insight into the mechanisms driving tumorigenesis.

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell ...

Core/shell Printing Scaffolds For Tissue Engineering Of Tubular Structures

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the interface with a liquid core. The latter is removed post-printing, leaving behind a hollow tube. Integrating an additive manufacturing technique (like the one described here with tailor-made [bio]inks, which structurally and biochemically mimic the native extracellular matrix [ECM]) is an important step towards advanced tissue engineering. However, precise fabrication of well-defined structures requires tailored fabrication strategies optimized for the material in use. Therefore, it is sensible to begin with a set-up that is customizable, simple-to-use, and compatible with a broad spectrum of materials and applications. This work presents an easy-to-manufacture core/shell nozzle with luer-compatibility to explore core/shell printing of woodpile structures, tested with a well-defined, alginate-based scaffold material formulation.

Presented here is a simple-to-use, core/shell, three-dimensional bioprinting set-up for one-step fabrication of hollow scaffolds, suitable for tissue engineering of vascular and other tubular structures.

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the ...

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed from mammal collagen. Thus, the arginine-glycine-aspartic acid (RGD) sequences and target motifs of matrix metalloproteinase (MMP) remain on the molecular chains, which help achieve cell attachment and degradation. Furthermore, formation properties of GelMA are versatile. The methacrylamide groups allow a material to become rapidly crosslinked under light irradiation in the presence of a photoinitiator. Therefore, it makes great sense to establish suitable methods for synthesizing three-dimensional (3D) structures with this promising material. However, its low viscosity restricts GelMA's printability. Presented here are methods to carry out 3D bioprinting of GelMA hydrogels, namely the fabrication of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. The resulting structures and biocompatibility of the materials as well as the printing methods are discussed. It is believed that this protocol may serve as a bridge between previously applied biomaterials and GelMA as well as contribute to the establishment of GelMA-based 3D architectures for biomedical applications.

Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...

3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also respond to neural injuries and act to protect the tissue from degenerative events. In vitro studies of astrocytes&#39; functionality are important to elucidate the mechanisms involved in such events and contribute to developing therapies to treat neurological disorders. This protocol describes a method to biofabricate a neural-like tissue structure rich in astrocytes by 3D bioprinting astrocytes-laden bioink. An extrusion-based 3D bioprinter was used in this work, and astrocytes were extracted from C57Bl/6 mice pups&#39; brain cortices. The bioink was prepared by mixing cortical astrocytes from up to passage 3 to a biomaterial solution composed of gelatin, gelatin-methacryloyl (GelMA), and fibrinogen, supplemented with laminin, which presented optimal bioprinting conditions. The 3D bioprinting conditions minimized cell stress, contributing to the high viability of the astrocytes during the process, in which 74.08% &#177; 1.33% of cells were viable right after bioprinting. After 1 week of incubation, the viability of astrocytes significantly increased to 83.54% &#177; 3.00%, indicating that the 3D construct represents a suitable microenvironment for cell growth. The biomaterial composition allowed cell attachment and stimulated astrocytic behavior, with cells expressing the specific astrocytes marker glial fibrillary acidic protein (GFAP) and possessing typical astrocytic morphology. This&#160;reproducible protocol provides a valuable method to biofabricate 3D neural-like tissue rich in astrocytes that resembles cells&#39; native microenvironment, useful to researchers that aim to understand astrocytes&#39; functionality and their relation to the mechanisms involved in neurological diseases.

Here we report a method of 3D bioprinting murine cortical astrocytes for biofabricating neural-like tissues to study the functionality of astrocytes in the central nervous system and the mechanisms involving glial cells in neurological diseases and treatments.

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also ...

Ceramic Omnidirectional Bioprinting in Cell-Laden Suspensions for the Generation of Bone Analogs

Structurally, bone tissue is an inorganic-organic composite containing metabolically active cells embedded within a hierarchical, highly mineralized matrix. This organization is challenging to replicate due to the heterogeneous environment of bone. Ceramic omnidirectional bioprinting in cell-suspensions (COBICS) is a microgel-based bioprinting technique that uniquely replicates the mineral and cellular structure of bone. COBICS prints complex, biologically relevant constructs without the need for sacrificial support materials or harsh postprocessing steps (e.g., radiation and high-temperature sintering), which are two of the biggest challenges in the additive manufacturing of bone mimetic constructs. This technique is enabled via the freeform extrusion of a novel calcium phosphate-based ink within a gelatin-based microgel suspension. The yield-stress properties of the suspension allow deposition and support the printed bone structure. UV crosslinking and nanoprecipitation then "lock" it in place. The ability to print nanostructured bone-mimetic ceramics within cell-laden biomaterials provides spatiotemporal control over macro- and micro-architecture and facilitates the real-time fabrication of complex bone constructs in clinical settings.

This protocol describes a 3D printing technique to fabricate bone-like structures by depositing a calcium phosphate ink in a gelatin-based granular support. Printed bone analogs are deposited in freeform, with flexibility for direct harvesting of the print or crosslinking within a living cell matrix for multiphasic constructs.

Structurally, bone tissue is an inorganic-organic composite containing metabolically active cells embedded within a hierarchical, highly mineralized ...

Microgel-Extracellular Matrix Composite Support for the Embedded 3D Printing of Human Neural Constructs

The embedded 3D printing of cells inside a granular support medium has emerged in the past decade as a powerful approach for the freeform biofabrication of soft tissue constructs. However, granular gel formulations have been restricted to a limited number of biomaterials that allow for the cost-effective generation of large amounts of hydrogel microparticles. Therefore, granular gel support media have generally lacked the cell-adhesive and cell-instructive functions found in the native extracellular matrix (ECM).
To address this, a methodology has been developed for the generation of self-healing annealable particle-extracellular matrix (SHAPE) composites. SHAPE composites consist of a granular phase (microgels) and a continuous phase (viscous ECM solution) that, together, allow for both programmable high-fidelity printing and an adjustable biofunctional extracellular environment. This work describes how the developed methodology can be utilized for the precise biofabrication of human neural constructs.
First, alginate microparticles, which serve as the granular component in the SHAPE composites, are fabricated and combined with a collagen-based continuous component. Then, human neural stem cells are printed inside the support material, followed by the annealing of the support. The printed constructs can be maintained for weeks to allow the differentiation of the printed cells into neurons. Simultaneously, the collagen continuous phase allows for axonal outgrowth and the interconnection of regions. Finally, this works provides information on how to perform live-cell fluorescence imaging and immunocytochemistry to characterize the 3D-printed human neural constructs.

This work describes a protocol for the freeform embedded 3D printing of neural stem cells inside self-healing annealable particle-extracellular matrix composites. The protocol enables the programmable patterning of interconnected human neural tissue constructs with high fidelity.

The embedded 3D printing of cells inside a granular support medium has emerged in the past decade as a powerful approach for the freeform biofabrication ...

Embedded Bioprinting of Tissue-like Structures Using κ-Carrageenan Sub-Microgel Medium

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