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

Summary

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.

Abstract

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 κ-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 κ-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 κ-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 κ-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.

Introduction

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 function^1,2,3. 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 growth^5,6. 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 gelatin^7,8, agarose⁹, and gellan gum^10,11 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 printing^12,13,14. 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 κ-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 κ-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 κ-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.

Protocol

1. Preparation of the κ -carrageenan sub-microgel suspension bath

1. Prepare 500 mL of κ-carrageenan suspension bath (0.35% wt/vol) by adding 1.75 g of κ -carrageenan powder into 500 mL of phosphate-buffered saline (PBS, pH 7.4) solution within a 1,000 mL glass bottle.
2. 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.
3. Place the glass bottle in a 70 °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.
4. Take another glass bottle and place it in an autoclave for sterilization, running at 121 °C for 30 min.
5. After the high-pressure sterilizer cools to 80 °C, remove the bottle from the sterilizer and transfer it to the ultra-clean workbench for further handling.
6. Filter the completely dissolved κ-carrageenan solution into the sterilized glass bottle using a 0.22 µm filter.
   NOTE: Perform filtration swiftly while the solution is hot to prevent the κ-carrageenan from cooling and obstructing the filter.
7. Store the κ-carrageenan solution at 4 °C to induce a cation-crosslinked gelation for 12 h.
8. Mechanically grind the κ-carrageenan hydrogels using a 60 mm magnetic stirrer with a stirring speed of 1200 rpm at 25 °C until it successfully transforms into a liquid state, taking approximately 60 min.
   NOTE: Store the κ-carrageenan hydrogels between 25-37 °C to avoid gelation at low temperatures or dissolution at high temperatures.

2. Preparation of gelatin methacrylate/silk fibroin methacrylate composite bioinks

1. 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.
2. Slowly add the powders into two 50 mL centrifuge tubes. Add 10 mL of PBS solution separately into the 50 mL centrifuge tubes.
3. Add a 10 mm magnetic stirrer to each and constantly stir and heat in a water bath at 45 °C. Allow the GelMA and SFMA powders to completely dissolve, taking approximately 30 min.
4. Mix the GelMA and SFMA solutions in an equal volume under continuous stirring at 45 °C. Filter the composite bioinks using a 100 µm pore size filter.
5. 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.

3. Rheological characterization of the κ -carrageenan sub-microgel suspension bath

1. Start up and prepare rheology-related equipment.
   1. 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.
   2. Power on the rheometer and allow it to initialize. Turn on the circulation water switch and allow the temperature to reach 15 °C.
   3. Open the rheometer control software to establish the online connection.
2. Calibrate the rheometer and install the parallel plate geometry.
   1. 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.
   2. Select the Calibrate Manager in the control software and subsequently click the Inertia, Friction Calibration, and Rotational Mapping to calibrate the parallel plate geometry.
3. Zero the rheometer gap height by conducting a standard gap zeroing procedure to ensure precise measurement.
4. 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.
5. Drop 2 mL of 0.35% κ-carrageenan suspensions onto the rheometer's Peltier plate. 
6. Position the geometry gap to 510 μm and remove any excess overflow at the edge of the clamp device. Set the sample gap to 500 µm to allow the sample to extend slightly beyond the clamp edge.
7. Conduct a flow experiment by selecting the Flow Ramp from the experimental design options.
   1. 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 °C.
   2. Perform step 3.5 and the Flow Ramp experiment on the added sample.
8. Perform a cyclic Peak Hold test to assess the material's recovery performance.
   1. Choose Peak Hold sweep in the experimental design and set 5 consecutive experiments with a time of 120 s at 25 °C.
   2. 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.
   3. Add a new sample of 2 mL. Perform step 3.5 and the cyclic recovery test.
9. Perform viscoelasticity analysis to evaluate the potential gel-sol transition of the κ-carrageenan suspension bath.
   1. Choose the Amplitude Sweep test in experimental design. Set the oscillation strain from 0.1% to 100% at a temperature of 25 °C. Click Start to perform the Amplitude Sweep experiment on the added sample.
10. Perform alternative strain analysis to assess the material's elastic recovery performance.
    1. Choose Oscillation Time Sweep in the experimental design and set 5 consecutive experiments with a time of 60 s at 25 °C.
    2. Set the strain to 1% for the first, third, and fifth steps and to 100% for the second and fourth steps.
    3. 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.

4. Printing biomimetic hydrogel scaffolds using a custom-designed micro-extrusion bioprinter

1. 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/.).​
   1. 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.
   2. Input the estimated filament diameter (typically around 200-500 µm 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%.
   3. Export the final parameters as G-code onto an SD card.
2. Run the bioprinter and micro-extrusion pump.
   1. 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.
   2. Fit the syringe with a needle featuring an inner diameter of 210 µm into the syringe slot of the bioprinter.
   3. Adjust the syringe controller to ensure that the plunger of the syringe is in close contact with the screw.
   4. 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.
   5. 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.
3. Processing of the constructs
   1. Expose the printed construct to 405 nm blue light for 1 min to initiate photocrosslinking.
   2. Remove the κ-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.

5. Embedded 3D bioprinting of esophageal muscle-layer analogs

1. Culture rabbit esophageal smooth muscle cells (eSMCs).
   1. 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.
   2. Maintain the eSMCs at 37 °C in a humidified atmosphere of 5% CO₂ and 95% air until they reach 80% confluency.
2. Preparation of esophageal muscle layer bioink
   1. Aspirate the medium upon reaching confluency and wash the eSMCs with 5 mL of 1x PBS.
   2. 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.
   3. 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 µL of cell-suspension.
   4. 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.
3. Preparation of the 3D bioprinter and associated materials
   1. Autoclave the essential printing instruments, including syringe needles, forceps, a magnetic stirrer, and a 1000 mL silicate glass bottle, under standard sterilization conditions (121 °C for 30 min) to ensure an aseptic environment for the printing process.
   2. Place the bioprinter inside a biosafety cabinet and sterilize it with ultraviolet (UV) light for a duration of 30 min to maintain sterility.
   3. Prepare 50 mL of sterile κ-carrageenan and 5 mL of sterile GelMA/SFMA composite bioinks, as described in step 1 and step 2, respectively.
   4. Design the model, set the desired parameters, and prepare the bioprinter as outlined in step 4.1.
4. Initiation of the bioprinting process
   1. Perform the procedures described in steps 4.2 and 4.3 to bioprint the 3D esophageal muscle layer, followed by PBS washing and replacement.
   2. Perform cell printing with sterile techniques throughout the printing process to minimize the risk of cell contamination in the printed structure.
5. Post-printing cell culture medium replacement
   1. 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.
   2. Incubate the cell culture dish containing the printed constructs in an incubator set to 37 °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.

6. Evaluation of eSMCs viability within the printed constructs via Live/Dead assay staining

1. Remove the cell culture medium after 5 h of incubation and wash the cell-laden constructs with PBS.
2. Add 2 mL of the standard calcein-AM/propidium iodide solution (1:1000) to the printed cell-laden constructs and incubate.
3. Wash the constructs 3x with PBS after a 45 min incubation and then add fresh cell culture medium.
4. Observe live and dead cells and capture fluorescent images using confocal laser scanning microscopy (CLSM).

7. Observation of the eSMCs within the printed constructs via FITC-Phalloidin/DAPI staining

1. Stain the printed constructs using fluorescein isothiocyanate (FITC) labeled phalloidin (Green) for F-actin and 4',6-Diamidino-2-Phenylindole (DAPI, blue) for nuclear post 5 days of incubation.
2. Fixation solution preparation
   1. 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%.
   2. 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.
   3. Introduce 2.5 mL of 1x PBS to the culture dish, fully submerging the constructs to remove any residual staining reagents.
3. Cell membrane permeabilization
   1. Formulate a 0.5% Triton X-100 solution by adding 20 µL of Triton X-100 to 4 mL of 1x PBS in a 5 mL PE tube.
   2. Administer the Triton X-100 solution to the constructs for 30 min at room temperature to permeate cell membranes, then remove the solution.
   3. Introduce 2.5 mL of 1x PBS to the culture dish, fully submerging the constructs to remove any residual staining reagents.
4. Blocking non-specific binding
   1. 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.
   2. Introduce the BSA solution to the constructs for 30 min at room temperature to block non-specific sites, then discard the solution.
5. Actin staining with FITC-Phalloidin
   1. Prepare 0.2% FITC-phalloidin solution in a 5 mL PE tube with 4 mL of 1x PBS and 8 µL of staining reagent.
   2. Immerse the constructs in this solution for 60 min at room temperature in the dark, then discard the solution.
   3. Add 2.5 mL of 1x PBS to the culture dish, fully submerging the constructs to remove residual staining reagents.
6. Nuclear staining with DAPI
   1. Apply 4 mL of DAPI staining solution to the constructs, ensuring complete coverage.
   2. Incubate in the dark at room temperature for 20 min before removing the DAPI solution.
7. 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.
8. Observe using a confocal microscope: Inspect and image the stained constructs using confocal microscopy to evaluate cell growth, ensuring comprehensive documentation of the results.

8. Evaluation of eSMCs proliferation within the printed constructs using CCK-8 assay

1. Remove the culture medium from the constructs on days 7 and 14. Wash the constructs with PBS to remove residual medium and detached cells.
2. Add CCK-8 solution to each construct following the manufacturer's instructions. Ensure that the volume of the CCK-8 solution is adequate to cover each construct completely.
3. Incubate the constructs with the CCK-8 solution at 37 °C for 2 h. Following incubation, carefully transfer the supernatant to a new 96-well plate.
4. Measure the absorbance at 450 nm using a microplate reader.

Results

The granular κ-carrageenan gel bath was generated by mechanically breaking up the bulk hydrogels into a particulate gel slurry. The most recent study demonstrated that the κ-carrageenan particles exhibited an average diameter of approximately 642 ± 65 nm with uniform morphologies at 1000 rpm of mechanical blending¹⁵, significantly smaller than the dimensions of microgels previously reported in the literature^16,17...

Discussion

The preparation of κ-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 κ-carrageenan solution is prepared by dissolving the κ-carrageenan powder in deionized water at elevated temperatures, creating a homogeneous mixture. The concentration of th...

Materials

Name	Company	Catalog Number	Comments
3D bioprinter	Custom-designed
4’,6-Diamidino-2-Phenylindole	Solarbio Life Science	C0065	Ready-to-use
405 nm UV light	EFL	XY-WJ01
Cell Counter	Corning	Cyto smart 6749
Confocal laser scanning microscope	Leica	STELLARIS 5
DMEM high glucose	VivaCell	C3113-0500	High Glucose, with Sodium Pyruvate and L-Glutamine
Dynamic rotational rheometer	TA Instrument	Discovery HR-20
Esophageal smooth muscle cells	Supplied by the Department of Cell Biology and Regenerative Medicine, Health Science Center, Ningbo University		Primary cells from the rabbit esophagus
Fetal bovine serum	UE	F9070L
Fluorescein isothiocyanate labeled phalloidin	Solarbio Life Science	CA1610	300T
Gelatin methacrylate	EFL	EFL-GM-60	60% substitution
k-carrageenan	Aladdin	C121013-100g	Reagent grade
Lithium Phenyl (2,4,6-trimethylbenzoyl) phosphinate	Aladdin	L157759-1g	365~405 nm
Live-Dead kit	beyotime	C2015M
Microplate reader	Potenov	PT-3502B
Paraformaldehyde	Solarbio Life Science	P1110	4%
Penicillin/streptomycin	Solarbio Life Science	MA0110	100 ´
Phosphate buffered saline	VivaCell	C3580-0500	pH 7.2-7.4
Silk fibroin methacrylate	EFL	EFL-SilMA-001	39% substitution
Triton X-100	Solarbio Life Science	T8200
Trypsin-EDTA	VivaCell	C100C1	0.25%, without phenol red