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.
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.
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 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.
1. Preparation of the κ -carrageenan sub-microgel suspension bath
2. Preparation of gelatin methacrylate/silk fibroin methacrylate composite bioinks
3. Rheological characterization of the κ -carrageenan sub-microgel suspension bath
4. Printing biomimetic hydrogel scaffolds using a custom-designed micro-extrusion bioprinter
5. Embedded 3D bioprinting of esophageal muscle-layer analogs
6. Evaluation of eSMCs viability within the printed constructs via Live/Dead assay staining
7. Observation of the eSMCs within the printed constructs via FITC-Phalloidin/DAPI staining
8. Evaluation of eSMCs proliferation within the printed constructs using CCK-8 assay
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 literature16,17,18. Longer breaking times and larger speeds result in smaller microparticle sizes, with a blending time of 60 min at 1200 rpm yielding sub-microparticles with an average diameter of 565 ± 86 nm in this study (Supplementary Figure 1). Such small particle size is anticipated to enhance the resolution of printing processes. These particles assemble into a granular gel via the formation of hydrogen bonding and cation interactions (Figure 1A). When subjected to stress, the κ-carrageenan sub-microgel medium exhibits characteristic behaviors such as yielding, shear-thinning, and a reversible jamming-unjamming transition, all of which are essential for successful embedded 3D bioprinting. Figure 1B shows representative yield behavior and shear thinning of granular κ-carrageenan gels upon a flow ramp test. With increasing shear rate from 0.001 to 10 s-1, the shear stresses for 0.35% (wt/vol) granular κ-carrageenan gels remain relatively unchanged at shear rates below 0.3 s-1, indicating a low yield stress of 3.5 Pa. Subsequently, it is observed that the shear stresses linearly increase with the shear rates, suggesting flow behavior resembling that of a Bingham fluid. Furthermore, strain-sweeping rheological measurements of the κ-carrageenan sub-microgels reveal a distinctive transition from gel to sol. These gels yield at strains exceeding 10% and undergo a gel-sol transition at a 35% strain (Figure 1C). The small yield strain of the sub-microparticle gels is ascribed to the physical network established through hydrogen bonds and cationic interactions. Such gel-sol transition behavior is reversible. As shown in Figure 1D, at a strain of 100%, the storage modulus of the κ-carrageenan sub-microgels plummets from 38 Pa to 6 Pa, lower than the loss modulus, indicating a sol state. Conversely, upon a sudden reduction in strain to 1%, the storage modulus swiftly reverts to approximately 38 Pa. It surpasses the loss modulus within 5 s, suggesting that the κ-carrageenan sub-microgels are capable of rapidly re-establishing the physical cross-links among the particles.
The granular κ-carrageenan gels, characterized by their small and homogeneous morphologies, exhibit shear-thinning properties, rapid self-recovery, and yield stress behavior, which is advantageous for their use in embedded 3D bioprinting applications. To assess the efficacy of the κ–carrageenan sub-microgel suspension as a support bath for embedded printing, a widely used bioink comprised of 10% (wt/vol) gelatin methacrylate and 6% (wt/vol) silk fibroin methacrylate composites (GelMA/SFMA) is employed for the printing trials within the κ-carrageenan medium19,20. The GelMA was labeled with rhodamine for enhanced visualization21. As illustrated in Figure 2A, the granular κ-carrageenan gel bath is highly transparent, facilitating real-time monitoring of the printing process. The shear rates generated by the nozzle range from 0.66 to 6 s-1 when the nozzle speeds vary within the range of 10-90 mm/s. The κ-carrageenan sub-microgel bath still exhibits self-healing behavior within 20 s upon continuous cyclic shearing at alternating rates of 6 s-1 and 0.01 s-1 (Supplementary Figure 2). A grid structure measuring 15 x 15 x 2 mm3, with a layer thickness of 150 µm, was sustained within the κ-carrageenan sub-microgel bath. Notably, the grid demonstrated remarkable integrity and resolution, with individual filaments measuring approximately 200 µm in width and inter-filament spacing of about 350 µm, as verified by confocal laser scanning microscopy (CLSM) images (Figure 2B,C). Moreover, the 3D grid structure featured a porous network with interconnected pores. These are vital attributes that influence printability and ultimately affect cellular behaviors, such as cell survival, proliferation, and differentiation potential.
To further confirm the suitability of the κ-carrageenan sub-microgel medium for generating complex tissue and organ structures, a human heart model and a tri-leaflet heart valve were fabricated using the GelMA/SFMA ink at a high infill density of 80%. The printed heart model accurately replicates the 3D anatomical shape, including major veins and arteries (Figure 2D), and exhibits mechanical stability post-water removal (Figure 2E). The tri-leaflet heart valve shows distinct valve leaflets and supporting structures (Figure 2F). Additionally, the filaments within the constructs are easily peeled off (Figure 2G). Furthermore, a hierarchical cyclic model like the esophagus is accurately recreated. As shown in Figure 2H and 2I, the printed esophagus exhibits clear stratification, with distinct demarcations between the individual layers to replicate the corresponding esophageal tissue layers, suggesting that the κ-carrageenan sub-microgel medium supports the structural complexity required for tissue engineering. While the current model falls short of replicating the full functionality of authentic human tissues and organs, the κ-carrageenan sub-microgel bath potentially offers a viable and promising strategy for the in vitro fabrication of intricate tissues and organs, with potential applications in research, therapy, and diagnosis.
Grid scaffolds with longitudinal and transverse anisotropy mimic the muscular layer of the esophagus (Figure 3A,B), consisting of a distinct inner circular muscle layer and an outer longitudinal muscle layer. To replicate this anisotropic architecture in vitro, rabbit esophageal smooth muscle cells (eSMCs) were carefully embedded within the GelMA/SFMA bioinks. By utilizing the κ-carrageenan sub-microgel medium, a biomimetic scaffold with eSMCs that closely resembled the dual-layer muscular structure of the esophagus was successfully achieved. The viability of cells encapsulated within hydrogels was assessed with the Live/Dead staining after 5 h of culture. It revealed a predominant presence of live cells (stained green) and a minimal proportion of dead cells (stained red) within the hydrogel networks, indicating high cell viability (Figure 3C). Quantitative analysis showed a remarkably high survival rate of up to 92% (Figure 3D). These results indicate the biocompatibility of the hydrogel material, as well as the efficacy of the printing and crosslinking methodologies in preserving cell integrity. To further examine the cell morphology within the GelMA/SFMA constructs, phalloidin (green)/DAPI (blue) images were captured after 3 days of cell culture. As shown in Figure 3E, a significant number of eSMCs within the biomimetic constructs have projected an abundance of pseudopodia and exhibited an alignment along the microfibers, as shown after 3 days of culture. The 3D reconstruction further confirms the precise replication of the esophageal muscle layers (Supplementary Figure 3). Additionally, eSMC proliferation was evaluated using CCK-8 assay on days 1, 7, and 14. The absorbance degree values at 450 nm markedly increased from day 1 to 7 and 14, reflecting a robust proliferative behavior (Supplementary Figure 4). These results indicate the significant potential of the κ-carrageenan sub-microgel medium in facilitating high-quality biomimetic bioprinting.
Figure 1: Self-assembled κ-carrageenan sub-microgel medium and its rheology analysis. (A) Schematic illustration of κ-carrageenan sub-microgel float formed through hydrogen bonding and cation interactions for embedded 3D bioprinting. (B) Flow ramp on 0.35% (wt/vol) jammed κ-carrageenan sub-microgels at 25 °C. (C) Measurements of gel-sol transition in κ-carrageenan sub-microgels through an amplitude sweep ranging from 0.1% to 100% at 25 °C. (D) Analysis of self-healing in 0.35% (wt/vol) κ-carrageenan sub-microgels upon continuous cyclic shearing at alternating 1% and 100% strains at 25 °C. Please click here to view a larger version of this figure.
Figure 2: High-fidelity embedded 3D bioprinting of rhodamine-labeled GelMA/SFMA inks using the κ-carrageenan sub-microgel suspension bath. (A) Real-time observation of embedded 3D printing within κ-carrageenan sub-microgel suspension bath. (B, C) Confocal laser scanning microscope images illustrating the (B) 3D reconstruction and (C) inner structure of the printed grids. (D, E) Photographs of 3D printed heart-like model in a water solution (D) and in its natural state (E). (F) Pictures of a tri-leaflet heart valve and (G) the filaments being peeled off from the constructs. (H) Top and (I) front-view photographs capturing an esophagus model printed with the GelMA/SFMA ink, showcasing hierarchical four-layer structures. Please click here to view a larger version of this figure.
Figure 3: Embedded cell-laden bioprinting using the κ-carrageenan sub-microgel suspension bath. (A, B) Schematic diagrams of (A) the inner circular and outer longitudinal muscular structure of the esophagus and (B) an anisotropic esophageal muscular layer biomimetic scaffold produced by embedded 3D bioprinting. (C) Live/Dead staining images and (D) quantitative viability of eSMCs grown in GelMA/SFMA scaffold at 5 h. (E) Projection images of eSMCs grown in GelMA/SFMA scaffolds on day 3. From left to right: the nucleus, F-actin, and the merge view. Please click here to view a larger version of this figure.
Supplementary Figure 1: (A) Scanning electron microscopy image showing the homogeneous morphology of κ–carrageenan sub-microgels. (B) The diameter distribution of κ-carrageenan microparticles was measured by a laser nanometer particle size analyzer. Please click here to download this File.
Supplementary Figure 2: Self-recovery analysis on κ–carrageenan sub-microgels of 0.35% (wt/vol) upon continually cyclic shearing at alternate 0.01 s-1 and 6 s-1 rates at 25 °C. Please click here to download this File.
Supplementary Figure 3: 3D reconstruction image of the printed grids. Please click here to download this File.
Supplementary Figure 4: The proliferation activity of eSMCs encapsulated into the GelMA/SFMA hydrogel after 1, 7, and 14 days was assessed using the CCK-8 assay (n = 3). Statistical significance was analyzed by one-way ANOVA followed by a Tukey post hoc analysis between three groups, **p<0.01, ****p < 0.0001. Data are shown as the mean ± standard deviation. Please click here to download this File.
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 the solution is key and must be optimized for subsequent gelation. 0.3-0.6% (wt/vol) κ–carrageenan demonstrated excellent performance in embedded 3D bioprinting15. Upon cooling, this solution transitions into a bulk gel.
The bulk gel is then subjected to a mechanical grinding strategy to produce sub-microgel particles. This involves high-shear mechanical forces that break down the gel into uniformly sized particles. The grinding process parameters, including shear intensity, time, and temperature, are precisely controlled to achieve the desired particle size distribution. Following grinding, the sub-microgel particles are stored at 25-37 °C to prevent aggregation or dissolution, which is essential for maintaining a uniform suspension that supports intricate bioprinting tasks. It is recommended that the gel be prepared and used immediately.
The production of κ–carrageenan sub-microgel suspension baths is easy to operate, ensuring alignment with industrial applications and the growing demand for large-scale tissue engineering and regenerative medicine endeavors. Moreover, κ–carrageenan sub-microgel suspension baths employ cell-compatible PBS or culture medium as the solvent, which ensures excellent biocompatibility and makes it an ideal medium for supporting cells within bioprinted constructs. The shelf life of the granular gel is around 1 month under aseptic preparation conditions.
Characterized by its Bingham plastic flow behavior (Figure 1B), the κ–carrageenan sub-microgel bath demonstrates excellent shear-thinning properties coupled with rapid self-healing abilities (Figure 1C,D), which are pivotal in enhancing the precision of printed constructs. The application of this sub-microgel bath has proven efficacious in the creation of tissues featuring multi-tiered architectures (Figure 2), ensuring the production of structures with high fidelity and resolution. It is noteworthy that the capacity for rapid pseudopodia extension in cells embedded within cell-laden bioinks (Figure 3), underscoring the bath's compatibility with cellular processes. Consequently, this κ–carrageenan sub-microgel bath holds transformative promise for the field of in vitro tissue and organ fabrication. It stands to make a profound impact on tissue engineering and regenerative medicine by enabling the development of more complex, viable, and functionally integrative tissue constructs.
Despite the considerable potential of κ–carrageenan sub-microgel bath for applications in bioprinting and tissue engineering, several challenges and limitations still exist. A primary concern is that its composition should closely resemble the cell culture medium, which is crucial for maintaining cell viability during the extended printing of large organs. κ–carrageenan sub-microgels are predominantly produced using PBS as the solvent, which necessitates the incorporation of various cell culture media to dissolve the material. In addition to PBS, κ-carrageenan is also gelatinized using a calcium chloride solution. However, calcium chloride solution may cause toxicity to the cells or create an osmotic pressure imbalance. Therefore, the use of a cell culture medium would be ideal for κ-carrageenan solvents and cross-linking agents. Moreover, achieving high-throughput sterile production of microgels presents a significant challenge. Sterilization of κ-carrageenan solutions necessitates filtration through a 0.22 µm filter at elevated temperatures. However, the yield of sterile κ-carrageenan is often hindered by various factors, including the quality of the PBS solution and the cooling of the κ-carrageenan during the filtration process. Each filtration batch typically results in only 20-50 mL of sterile κ-carrageenan, which poses a significant barrier to the high-throughput production of κ-carrageenan microgels. Consequently, future research will focus on optimizing the biofabrication process, evaluating long-term cell viability and function, and exploring the integration of printed tissues into living systems.
The authors have nothing to disclose.
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.
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 |