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 glas...

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

<ol>
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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+printing+of+complex+biological+structures+by+freeform+reversible+embedding+of+suspended+hydrogels.">Three-dimensional printing of complex biological structures by freeform reversible embedding of suspended hydrogels.</a> Sci Adv. 1 (9), e1500758 (2015).</li><li>Wang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3d+bioprinting+of+neurovascular+tissue+modeling+with+collagen-based+low-viscosity+composites.">3d bioprinting of neurovascular tissue modeling with collagen-based low-viscosity composites.</a> Adv Healthcare Mater. 12 (25), e2300004 (2023).</li><li>Sreepadmanabh, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Jammed+microgel+growth+medium+prepared+by+flash-solidification+of+agarose+for+3d+cell+culture+and+3d+bioprinting.">Jammed microgel growth medium prepared by flash-solidification of agarose for 3d cell culture and 3d bioprinting.</a> Biomed Mater. 18 (4), 045011 (2023).</li><li>Zeng, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+the+residues+of+granular+support+bath+materials+on+printed+structures+in+embedded+extrusion+printing.">Comparative analysis of the residues of granular support bath materials on printed structures in embedded extrusion printing.</a> Biofabrication. 15 (3), 035013 (2023).</li><li>Terpstra, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioink+with+cartilage-derived+extracellular+matrix+microfibers+enables+spatial+control+of+vascular+capillary+formation+in+bioprinted+constructs.">Bioink with cartilage-derived extracellular matrix microfibers enables spatial control of vascular capillary formation in bioprinted constructs.</a> Biofabrication. 14 (3), 034104 (2022).</li><li>Compaan, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gellan+fluid+gel+as+a+versatile+support+bath+material+for+fluid+extrusion+bioprinting.">Gellan fluid gel as a versatile support bath material for fluid extrusion bioprinting.</a> ACS Appl Mater Inter. 11 (6), 5714-5726 (2019).</li><li>Compaan, A. M., Song, K., Chai, W., Huang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-linkable+microgel+composite+matrix+bath+for+embedded+bioprinting+of+perfusable+tissue+constructs+and+sculpting+of+solid+objects.">Cross-linkable microgel composite matrix bath for embedded bioprinting of perfusable tissue constructs and sculpting of solid objects.</a> ACS Appl Mater Inter. 12 (7), 7855-7868 (2020).</li><li>Zhang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+3D+printed+biomimetic+scaffolds+based+on+hydrogel+microparticles+for+cell+spheroid+growth.">Direct 3D printed biomimetic scaffolds based on hydrogel microparticles for cell spheroid growth.</a> Adv Funct Mater. 30 (13), 1910573 (2020).</li><li>Zhang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cation-crosslinked+&#954;-carrageenan+sub-microgel+medium+for+high-quality+embedded+bioprinting.">Cation-crosslinked &#954;-carrageenan sub-microgel medium for high-quality embedded bioprinting.</a> Biofabrication. 16, 025009 (2024).</li><li>Lee, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3d+bioprinting+of+collagen+to+rebuild+components+of+the+human+heart.">3d bioprinting of collagen to rebuild components of the human heart.</a> Science. 365 (6452), 482-487 (2019).</li><li>Yao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slightly+photo-crosslinked+chitosan/silk+fibroin+hydrogel+adhesives+with+hemostasis+and+anti-inflammation+for+pro-healing+cyclophosphamide-induced+hemorrhagic+cystitis.">Slightly photo-crosslinked chitosan/silk fibroin hydrogel adhesives with hemostasis and anti-inflammation for pro-healing cyclophosphamide-induced hemorrhagic cystitis.</a> Mater Today Bio. 25, 100947 (2024).</li><li>Senior, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+fluid+gels+formed+by+shear+processing+during+gelation+for+suspended+3d+bioprinting.">Agarose fluid gels formed by shear processing during gelation for suspended 3d bioprinting.</a> J Vis Exp. (195), e64458 (2023).</li><li>Roche, C. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=durability,+contractility+and+vascular+network+formation+in+3d+bioprinted+cardiac+endothelial+cells+using+alginate-gelatin+hydrogels.">durability, contractility and vascular network formation in 3d bioprinted cardiac endothelial cells using alginate-gelatin hydrogels.</a> Front Bioeng Biotech. 9, 63657 (2021).</li><li>Wang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+bioprinting+of+tough+hydrogel-based+vascular+conduits+for+functional+blood+vessels.">Microfluidic bioprinting of tough hydrogel-based vascular conduits for functional blood vessels.</a> Sci Adv. 8 (43), (2022).</li><li>Shao, L., Hou, R. X., Zhu, Y. B., Yao, Y. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3D bioprinter</td>
			<td>Custom-designed</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4&#8217;,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&#160;</td>
			<td>4%</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Solarbio Life Science</td>
			<td>MA0110</td>
			<td>100 &#180;</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.

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

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

3D bioprinting in a microgel-based suspension bath has provided a versatile platform for fabricating complex 3D structures using soft hydrogels, but it is still limited by the low print resolution and accuracy, hindering the practical applications of the printed products in tissue engineering. The present microgel baths often exhibit large sizes and poor dispersions, resulting in a big diameter and irregular morphology of the printed filaments. Therefore, it poses a significant challenge in preparing a microgel bath with a small particle size and uniform morphology.This protocol offers the fabrication of a homogenous and biocompatible cationic cross-linked kappa-carrageenan sub-microgel bath for high-fidelity bioprinting, through an easy-to-operate mechanical grinding strategy, Since kappa-carrageenan sub-microgels exhibit a uniform methodology with a small particle size of 565 nanometer, and a low state of 0.35%enabling precise tissue and organ construction with remarkable fidelity and high resolution.

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

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 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

Preparation of the &#954;-Carrageenan Sub-Microgel Suspension Bath for Embedded 3D Printing

Begin by adding 1.75 grams of Kappa Carrageenan powder to 500 milliliters of PBS in a 1000 milliliter glass bottle to prepare a Kappa Carrageenan suspension bath. Then introduce a 70 millimeter magnetic stir bar into the glass bottle. Tighten the glass bottle cap and then loosen it by half a turn.Heat the glass bottle in a 70 degree Celsius water bath. Then turn on the magnetic stir at 300 RPM. Place the bottle and stir until the polymer is completely dissolved.Using a 0.22 micrometer filter, filter the completely dissolved Kappa Carrageenan solution into the sterilized glass bottle. Store the Kappa Carrageenan solution at four degrees Celsius to induce a cation cross-linked gelation for 12 hours. Then using a 60 millimeter magnetic stir, mechanically grind the Kappa Carrageenan hydrogels until they successfully transform into a liquid state.

Printing Biomimetic Hydrogel Scaffolds Using a Custom-Designed Micro-Extrusion Bioprinter

To begin, switch on the micro-extrusion pump controller. Select the corresponding volume syringe and set the extrusion rate at 0.08 milliliters per minute. Then divide the hydrogel volume by the extrusion rate and coordinate it with the desired printing time to calculate the duration of extrusion.Next, fit the syringe with a needle featuring an inner diameter of 210 micrometers into the syringe slot of the bioprinter. Adjust the syringe controller to ensure that the plunger of the syringe is in close contact with the screw. Then place three milliliters of the prepared kappa-carrageenan suspension bath into a 35 millimeter cell culture dish.Based on the code settings, position the dish on the platform and confirm that the printing head is one millimeter above the bottom of the dish. After that, 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.Then expose the printed construct to 405 nanometers of blue light for one minute to initiate photo-crosslinking. Using a one milliliter pipette, remove the kappa-carrageenan gel, followed by the addition of three milliliters of PBS for washing and subsequent removal.

Embedded 3D Bioprinting of Esophageal Muscle-Layer Analogs for Tissue Engineering

To begin, take the cultured esophageal smooth muscle cells once they attain 80%co fluency. Aspirate the medium and wash the endothelial smooth muscle cells with five milliliters of PBS. Then add two milliliters of 0.2%Trypsin-EDTA to the flask and incubate for two minutes to digest the cells.Add two milliliters of DMEM to terminate the digestion process and transfer the cell suspension to a 15 milliliter centrifuge tube. Centrifuge the tube at 675G for three minutes. Carefully aspirate the supernatant, and resuspend the cell pellet in the composite gelatin methacrylate and silk fibroin methacryloyl bioinks.After bioprinting and washing the 3D esophageal muscle layer with PBS, carefully aspirate the PBS and replace it with three milliliters of DMEM, supplemented with 10%FBS, and 1%penicillin or streptomycin.

503 Views.  Ningbo University.  This study introduces a novel κ-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.

Advancing 3D Bioprinting with κ-Carrageenan Sub-Microgel Medium

Summary