The authors acknowledge grants from the National Natural Science Foundation of China (81771971, 81970442, 81703470 and 81570422), National Key R&#38;D Program of China (2018YFC1005002), Science and Technology Commission of Shanghai Municipality (17JC1400200), Shanghai Municipal Science and Technology Major Project (Grant No. 2017SHZDZX01), and Shanghai Municipal Education Commission (Innovation Program 2017-01-07-00-07-E00027).

1. Preparation of cell-laden SF-GelMA
<ol>
	<li>Sterilize all the materials by using 0.22 &#956;m syringe filter units. Perform all the steps in a biological safety cabinet.</li>
	<li>Warm 1x PBS to 50 &#176;C, and dissolve gelatin in the heated 1x PBS with stirring. The final concentration of gelatin in PBS should be 10% (w/v).</li>
	<li>Add methacrylic anhydride into the gelatin solution (weight ratio of methacrylic anhydride to gelatin of 0.6 to 1) slowly with stirring, and mix the complex for at least 1 h (50 &#176;C). Typically, prepare 200 mL of 10% gelatin solution with 12 g of methacrylic anhydride; the volume depends on the needs of the study.</li>
	<li>Transfer the mixed solution containing gelatin and methacrylic anhydride to a 50 mL sterile tube.</li>
	<li>Centrifuge the mixed solution at 3,500 x g. It usually takes 3-5 min to obtain two layers. Collect the upper layer (GelMA) and discard the bottom layer (unreacted methacrylic anhydride).</li>
	<li>Dilute the upper layer solution obtained in step 1.5 with two volumes of deionized water (40&#8722;50 &#176;C).</li>
	<li>Dialyze the solution obtained in step 1.6 with a 12-14 kDa molecular weight cutoff dialysis membrane against deionized water for 5&#8722;7 days (40&#8722;50 &#176;C). Change the water twice every day.</li>
	<li>Collect and freeze the GelMA solution at &#8722;80 &#176;C overnight.</li>
	<li>Lyophilize the GelMA solution for 3&#8722;5 days in a freeze dryer with the temperature set to -45 &#176;C and the pressure set to 0.2 mbar.</li>
	<li>Dissolve the lyophilized GelMA (degree of substitution of approximately 75%) in 1x PBS containing 10% FBS (fetal bovine serum, v/v), 25 mM HEPES (N-2-hydroxyethylpiperazine-N-ethane-sulfonic acid), and photoinitiator (0.5%, w/v) to obtain the GelMA bioink preparation. 
	NOTE: The degree of substitution of GelMA can be calculated by a ninhydrin assay3.</li>
	<li>Mix the GelMA solution (10%, w/v) with different volumes of initial SF solution (5%, w/v) and different volumes of 1x PBS to obtain SF-GelMA bioinks with different concentrations of SF. The proportion per 1 mL of GelMA and SF solution in the different bioinks is presented in Table 1. 
	NOTE: All bioinks must contain a final concentration of 5% (w/v) GelMA, but the concentration of SF varies: 0.5, 0.75, 1.0, 1.25, and 1.5% (w/v) in the different SF-GelMA bioinks. Use SF-M-Layered-GelMA to term the GelMA bioink modified with SF in a layer-by-layer manner. Use SF-X-GelMA to term those GelMA modified with SF in a homogeneous manner (e.g., GelMA with modification of 1% SF was termed SF-1-GelMA).</li>
	<li>Sonicate all bioinks for 10-20 min.</li>
	<li>Grow NIH3T3 cells using DMEM (Dulbecco&#39;s modified Eagle medium) containing 10% FBS and 1% penicillin-streptomycin in an incubator (37 &#176;C with 5% CO2). Passage the cells at a ratio of 1:3 when the density reaches 80%.</li>
	<li>Centrifuge the suspension of NIH3T3 cells at 1500 rpm for 5 min. Remove the supernatant with suction and resuspend the cell pellet with fresh bioink solution in a 15 mL sterile tube. 
	NOTE: The concentration of the encapsulated cells in the bioink should be 1 x 106 cells/mL, and the concentration needs to be calculated with a cytometer.</li>
	<li>Use 2 mL of different SF-GelMA bioinks to suspend the cell pellets to obtain various cell-laden bioinks.</li>
</ol>
2. Loading, reheating and bioprinting of the SF-M-Layered-GelMA
<ol>
	<li>Load 0.4 mL of cell-laden SF-0.5-GelMA into the bottom layer of the syringe. 
	NOTE: Use a 2 mL syringe as the bioink reservoir in this study. Load different SF-GelMA bioinks into the syringe in a layer-by-layer manner.</li>
	<li>Place the syringe in ice water bath (0 &#176;C) for 5 min to cause the bottom-layer bioink to transform into a gel state.</li>
	<li>Load 0.4 mL of cell-laden SF-0.75-GelMA bioink above the bottom layer.</li>
	<li>Place the syringe with the two layers of bioinks in an ice water bath (0 &#176;C) for 5 min to cause the two layers of bioinks to transform into a gel state.</li>
	<li>Cycle the loading and cooling steps another 3 times with the remaining 3 kinds of bioinks (SF-1-GelMA, SF-1.25-GelMA, SF-1.5-GelMA) to obtain a multilayered bioink system with different concentrations of SF in the different layers. The volume used for the loading of all bioinks should be 0.4 mL.</li>
	<li>Reheat the multilayered bioink by placing the syringe in an incubator (37 &#176;C) for 30 min prior to bioprinting.</li>
	<li>Use a 2 mL syringe as the bioink reservoir with a 27 G printing nozzle. Set the speed of flow at 50 &#956;L/min, the moving speed of the nozzle at 2 mm/s, and the height of the nozzle at 1 mm. Perform the bioprinting procedure at room temperature (approximately 20 &#176;C).
	<ol>
		<li>Print the tissue construct in an extrusion manner using a custom-made bioprinter under the parameters adapted from our previous studies11,12.</li>
	</ol>
	</li>
	<li>Use ultraviolet light (365 nm, 800 mW) for 40 s to crosslink the bioprinted tissue construct.</li>
	<li>Culture the crosslinked tissue construct in DMEM (Dulbecco&#39;s modified Eagle medium) containing 10% FBS and 1% penicillin-streptomycin in an incubator (37 &#176;C with 5% CO2). The medium was changed every 8 h during the first 2 days and every 2-3 days thereafter.</li>
</ol>

<ol>
	<li>Khademhosseini, A., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+decade+of+progress+in+tissue+engineering.">A decade of progress in tissue engineering.</a> Nature Protocol. 11 (10), 1775-1781 (2016).</li><li>Heinrich, M. 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:+from+benches+to+translational+applications.">3D bioprinting: from benches to translational applications.</a> Small. , 1805510 (2019).</li><li>Daniela, 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=Functionalization,+preparation+and+use+of+cell-laden+gelatin+methacryloyl-based+hydrogels+as+modular+tissue+culture+platforms.">Functionalization, preparation and use of cell-laden gelatin methacryloyl-based hydrogels as modular tissue culture platforms.</a> Nature Protocols. 11 (4), 727-746 (2016).</li><li>Pedde, R. 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=Emerging+biofabrication+strategies+for+engineering+complex+tissue+constructs.">Emerging biofabrication strategies for engineering complex tissue constructs.</a> Advanced Materials. 29 (19), (2017).</li><li>Holzl, K., Lin, S., Tytgat, L., Van Vlierberghe, S., Gu, L., Ovsianikov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioink+properties+before,+during+and+after+3D+bioprinting.">Bioink properties before, during and after 3D bioprinting.</a> Biofabrication. 8 (3), 032002 (2016).</li><li>Guillotin, B., Guillemot, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+patterning+technologies+for+organotypic+tissue+fabrication.">Cell patterning technologies for organotypic tissue fabrication.</a> Trends in Biotechnology. 29 (4), 183-190 (2011).</li><li>Murphy, S. V., Atala, A. <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+tissues+and+organs.">3D bioprinting of tissues and organs.</a> Nature Biotechnology. 32, 773-785 (2014).</li><li>Dubbin, K., Hori, Y., Lewis, K. K., Heilshorn, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-stage+crosslinking+of+a+gel-phase+bioink+improves+cell+viability+and+homogeneity+for+3D+bioprinting.">Dual-stage crosslinking of a gel-phase bioink improves cell viability and homogeneity for 3D bioprinting.</a> Advanced Healthcare Materials. 5 (19), 2488-2492 (2016).</li><li>Rutz, A. L., Hyland, K. E., Jakus, A. E., Burghardt, W. R., Shah, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multimaterial+bioink+method+for+3D+printing+tunable,+cell-compatible+hydrogels.">A multimaterial bioink method for 3D printing tunable, cell-compatible hydrogels.</a> Advanced Materials. 27 (9), 1607-1614 (2015).</li><li>Chahal, D., Ahmadi, A., Cheung, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+piezoelectric+cell+printing+accuracy+and+reliability+through+neutral+buoyancy+of+suspensions.">Improving piezoelectric cell printing accuracy and reliability through neutral buoyancy of suspensions.</a> Biotechnology and Bioengineering. 109 (11), 2932-2940 (2012).</li><li>Chen, N., 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=Hydrogel+bioink+with+multilayered+interfaces+improves+dispersibility+of+encapsulated+cells+in+extrusion+bioprinting.">Hydrogel bioink with multilayered interfaces improves dispersibility of encapsulated cells in extrusion bioprinting.</a> ACS Applied Materials &amp; Interfaces. 11, 30585-30595 (2019).</li><li>Zhu, K., 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=A+general+strategy+for+extrusion+bioprinting+of+bio-macromolecular+bioinks+through+alginate-templated+dual-stage+crosslinking.">A general strategy for extrusion bioprinting of bio-macromolecular bioinks through alginate-templated dual-stage crosslinking.</a> Macromolecular Bioscience. 18 (9), 1800127 (2018).</li><li>Nauman, J. V., Campbell, P. G., Lanni, F., Anderson, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+of+insulin-like+growth+factor-I+and+ribonuclease+through+fibrin+gels.">Diffusion of insulin-like growth factor-I and ribonuclease through fibrin gels.</a> Biophysical Journal. 92 (12), 4444-4450 (2007).</li></ol>

The authors have nothing to disclose.

The stability of the multilayered system is a key point to perform this protocol successfully. We theoretically calculated the diffusion of SF molecules in the GelMA solution based on Nauman&#8217;s study13. It was found that the diffusion of proteins in solution was related to their molecular weight. The average molecular weight (MW) of bovine serum albumin (BSA) is 66.5 kDa, and its diffusion coefficient is 64-72 &#956;m2/s. The average MW of fibrinogen is 339.7 kDa, and its diffusion...

Three-dimensional (3D) bioprinting has been a promising method to manufacture complex architectural and functional replicas of native tissues in biofabrication and regenerative medicine1,2,3. The common strategies of bioprinting, including inkjet, extrusion, and stereolithography printing, have pros and cons from different perspectives4. Among these techniques, the extrusion procedure is most commonly used due to its cost-effectiveness. Bioink plays a key role in the process stability of extrusion bioprinting. The ideal cell-laden bioink should not only be biocompatible but also be suitable for mechanical properties5. Bioinks with low viscosity are typically presented as a liquid-like state. These bioinks can be easily and quickly deposited and avoid cell membrane damage induced by high shear stress during extrusion. However, in complex cases requiring long-term printing periods, low viscosity often gives rise to the inevitable sedimentation of the encapsulated cells in the bioink reservoir, which is usually driven by gravity and leads to an inhomogeneous cell dispersion in the bioink6,7. Consequently, a bioink with inhomogeneous cell dispersity hampers the in vitro bioprinting of a functional tissue construct.
Several recent studies focusing on bioinks have reported the promotion of homogenous dispersity of encapsulated cells. A modified alginate bioink based on dual-stage crosslinking was used for extrusion bioprinting8. An alginate polymer was modified with peptides and proteins in this study. Cells presented a more homogeneous distribution in this modified alginate than in the commonly used alginate due to the attachment sites provided by the peptides and the proteins. Alternatively, blended bioinks have been utilized to solve the sedimentation of cells in bioink. A blended bioink containing polyethylene glycol (PEG) and gelatin or gelatin methacryloyl (GelMA) with improved mechanical robustness was used in another study9. The encapsulated cells presented a homogeneous distribution mainly because the viscosity of the blended bioink was improved. In general, there are several factors influencing the dispersity of the encapsulated cells in the bioink, such as the viscosity of the bioink, the gravity of the cells, the density of the cells, and the duration of the working period. Among these factors, the gravity of cells plays a critical role in promoting sedimentation. The buoyancy and friction provided by the viscous bioink have been investigated as the main forces against gravity to date10.
Herein, we developed a novel strategy to promote homogeneous dispersity of the encapsulated cells in bioink by manipulating multiple liquid interfaces in the bioink reservoir. These liquid interfaces created by the multilayered modification of bioink can not only provide interfacial retention, which retards the sedimentation of cells, but also maintain a suitable biocompatibility and rheological behavior of the bioink. In practice, we modified aqueous GelMA solution (5%, w/v) with silk fibroin (SF) in a multilayered manner to longitudinally produce four interfaces, providing interfacial tensions in the blended bioink. As a result, the gravity loading on the cells was offset by the man-made interfacial tension, and a nearly homogeneous dispersion of the encapsulated cells in the bioink was obtained due to less sedimentation across the adjacent layers of cells. No similar protocol to slow down the sedimentation of encapsulated cells by manipulating interfacial retention in liquid bioinks has been reported to date. We present our protocol here to demonstrate a new way to solve cell sedimentation in bioprinting.

<table><tbody><tr><td>2-Hydroxy-4&amp;prime;-(2-hydroxyethoxy)-2-methylpropiophenone (PI2959)</td><td>TCI</td><td>M64BK-QD</td><td></td></tr><tr><td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td><td>Gibco</td><td>15630080</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium (DMEM)</td><td>Gibco</td><td>10569044</td><td></td></tr><tr><td>fetal bovine serum (FBS)</td><td>Gibco</td><td>10091</td><td></td></tr><tr><td>Gelatin</td><td>Sigma-Aldrich</td><td>V900863MSDS</td><td></td></tr><tr><td>Methacrylic anhydride (MA)</td><td>Sigma-Aldrich</td><td>276685MSDS</td><td></td></tr><tr><td>Penicillin&amp;ndash;streptomycin antibiotics</td><td>Gibco</td><td>15140163</td><td></td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>Gibco</td><td>10010049</td><td></td></tr><tr><td>Silk fibroin</td><td>Advanced BioMatrix</td><td>5154</td><td></td></tr></tbody></table>

using multilayered hydrogel bioink in three-dimensional bioprinting for homogeneous cell distribution

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by shear stress and improve the survival of the encapsulated cells. However, rapid gravity-driven cell sedimentation in the reservoir could lead to an inhomogeneous cell distribution in bioprinted structures and therefore hinder the application of liquid-like bioinks. Here, we developed a novel multilayered modified strategy for liquid-like bioinks (e.g., gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells. Multiple liquid interfaces were manipulated in the multilayered bioink to provide interfacial retention. Consequently, the cell sedimentation action going across adjacent layers in the multilayered system was retarded in the bioink reservoir. It was found that the interfacial retention was much higher than the sedimental pull of cells, demonstrating a critical role of the interfacial retention in preventing cell sedimentation and promoting a more homogeneous dispersion of cells in the multilayered bioink.

A schematic of the preparation of cell-laden bioinks is shown in Figure 1. After preparation of the different bioinks, loading, reheating and bioprinting were performed (Figure 2). To evaluate the distribution of the encapsulated cells in the bioink reservoir, a bioprinting procedure was performed using three different cell-laden bioinks in three 96-well plates (Figure 3A). Two control groups (pristine GelMA and SF-1-GelMA bioinks) ...

Watch this Scientific Journal Video about Using Multilayered Hydrogel Bioink in Three-Dimensional Bioprinting for Homogeneous Cell Distribution at JoVE.com

Using Multilayered Hydrogel Bioink in Three-Dimensional Bioprinting for Homogeneous Cell Distribution

Here, we developed a novel multilayered modified strategy for liquid-like bioinks (gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells.

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by ...

using-multilayered-hydrogel-bioink-three-dimensional-bioprinting-for

Research

JoVE Journal

Bioengineering

6.4K Views.  Fudan University. Here, we developed a novel multilayered modified strategy for liquid-like bioinks (gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells.

Video: Using Multilayered Hydrogel Bioink in Three-Dimensional Bioprinting for Homogeneous Cell Distribution

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

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of three-dimensional bioprinting in tissue engineering has generated new methods for the printing of cells and matrix to fabricate biomimetic tissue constructs. The solid freeform fabrication (SFF) method developed for three-dimensional bioprinting uses an additive manufacturing approach by depositing droplets of cells and hydrogels in a layer-by-layer fashion. Bioprinting fabrication is dependent on the specific placement of biological materials into three-dimensional architectures, and the printed constructs should closely mimic the complex organization of cells and extracellular matrices in native tissue. This paper highlights the use of the Palmetto Printer, a Cartesian bioprinter, as well as the process of producing spatially organized, viable constructs while simultaneously allowing control of environmental factors. This methodology utilizes computer-aided design and computer-aided manufacturing to produce these specific and complex geometries. Finally, this approach allows for the reproducible production of fabricated constructs optimized by controllable printing parameters.

A Cartesian bioprinter was designed and fabricated to allow multi-material deposition in precise, reproducible geometries, while also allowing control of environmental factors. Utilizing the three-dimensional bioprinter, complex and viable constructs may be printed and easily reproduced.

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of ...

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 ...

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

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

Engineering

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 ...

3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with controlled pore structure and mechanical properties. Cellulose nanocrystals (CNCs, 69.62 wt%) based hydrogel ink with matrix (sodium alginate and gelatin) was developed and 3D printed into scaffolds with uniform and gradient pore structure (110-1,100 &#181;m). The scaffolds showed compression modulus in the range of 0.20-0.45 MPa when tested in simulated in vivo conditions (in distilled water at 37 &#176;C). The pore sizes and the compression modulus of the 3D scaffolds matched with the requirements needed for cartilage regeneration applications. This work demonstrates that the consistency of the ink can be controlled by the concentration of the precursors and porosity can be controlled by the 3D printing process and both of these factors in return defines the mechanical properties of the 3D printed porous hydrogel scaffold. This process method can therefore be used to fabricate structurally and compositionally customized scaffolds according to the specific needs of patients.&#160;

The three critical steps of this protocol are i) developing the right composition and consistency of the cellulose hydrogel ink, ii) 3D printing of scaffolds into various pore structures with good shape fidelity and dimensions and iii) demonstration of the mechanical properties in simulated body conditions for cartilage regeneration.

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with ...

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 ...

Fabrication of a Crystalline Nanocellulose Embedded Agarose Biomaterial Ink for Bone Marrow-Derived Mast Cell Culture

Three-dimensional (3D) bioprinting utilizes hydrogel-based composites (or biomaterial inks) that are deposited in a pattern, forming a substrate onto which cells are deposited. Because many biomaterial inks can be potentially cytotoxic to primary cells, it is necessary to determine the biocompatibility of these hydrogel composites prior to their utilization in costly 3D tissue engineering processes. Some 3D culture methods, including bioprinting, require that cells be embedded into a 3D matrix, making it difficult to extract and analyze the cells for changes in viability and biomarker expression without eliciting mechanical damage. This protocol describes as proof of concept, a method to assess the biocompatibility of a crystalline nanocellulose (CNC) embedded agarose composite, fabricated into a 24-well culture system, with mouse bone marrow-derived mast cells (BMMCs) using flow cytometric assays for cell viability and biomarker expression.
After 18 h of exposure to the CNC/agarose/D-mannitol matrix, BMMC viability was unaltered as measured by propidium iodide (PI) permeability. However, BMMCs cultured on the CNC/agarose/D-mannitol substrate appeared to slightly increase their expression of the high-affinity IgE receptor (Fc&#949;RI) and the stem cell factor receptor (Kit; CD117), although this does not appear to be dependent on the amount of CNC in the bioink composite. The viability of BMMCs was also assessed following a time course exposure to hydrogel scaffolds that were fabricated from a commercial biomaterial ink composed of fibrillar nanocellulose (FNC) and sodium alginate using a 3D extrusion bioprinter. Over a period of 6-48 h, the FNC/alginate substrates did not adversely affect the viability of the BMMCs as determined by flow cytometry and microtiter assays (XTT and lactate dehydrogenase). This protocol describes an efficient method to rapidly screen the biochemical compatibility of candidate biomaterial inks for their utility as 3D scaffolds for post-print seeding with mast cells.

This protocol highlights a method to rapidly assess the biocompatibility of a crystalline nanocellulose (CNC)/agarose composite hydrogel biomaterial ink with mouse bone marrow-derived mast cells in terms of cell viability and phenotypic expression of the cell surface receptors, Kit (CD117) and high-affinity IgE receptor (Fc&#949;RI).

Three-dimensional (3D) bioprinting utilizes hydrogel-based composites (or biomaterial inks) that are deposited in a pattern, forming a substrate onto ...

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 ...

Agarose Fluid Gels Formed by Shear Processing During Gelation for Suspended 3D Bioprinting

The use of granular matrices to support parts during the bioprinting process was first reported by Bhattacharjee et al. in 2015, and since then, several approaches have been developed for the preparation and use of supporting gel beds in 3D bioprinting. This paper describes a process to manufacture microgel suspensions using agarose (known as fluid gels), wherein particle formation is governed by the application of shear during gelation. Such processing produces carefully defined microstructures, with subsequent material properties that impart distinct advantages as embedding print media, both chemically and mechanically. These include behaving as viscoelastic solid-like materials at zero shear, limiting long-range diffusion, and demonstrating the characteristic shear-thinning behavior of flocculated systems. 
On the removal of shear stress, however, fluid gels have the capacity to rapidly recover their elastic properties. This lack of hysteresis is directly linked to the defined microstructures previously alluded to; because of the processing, reactive, non-gelled polymer chains at the particle interface facilitate interparticle interactions-similar to a Velcro effect. This rapid recovery of elastic properties enables bioprinting high-resolution parts from low-viscosity biomaterials, as rapid reformation of the support bed traps the bioink in situ, maintaining its shape. Furthermore, an advantage of agarose fluid gels is the asymmetric gelling/melting transitions (gelation temperature of ~30 &#176;C and melting temperature of ~90 &#176;C). This thermal hysteresis of agarose makes it possible to print and culture the bioprinted part in situ without the supporting fluid gel melting. This protocol shows how to manufacture agarose fluid gels and demonstrates their use to support the production of a range of complex hydrogel parts within suspended-layer additive manufacture (SLAM).

Shear processing during hydrogel formation results in the production of microgel suspensions that shear-thin but rapidly restructure following the removal of shear forces. Such materials have been used as a supporting matrix for bioprinting complex, cell-laden structures. Here, methods used to manufacture the supporting bed and compatible bioinks are described.

The use of granular matrices to support parts during the bioprinting process was first reported by Bhattacharjee et al. in 2015, and since then, several ...

Using Multilayered Hydrogel Bioink in Three-Dimensional Bioprinting for Homogeneous Cell Distribution

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Chapters in this video

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

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

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

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

3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Fabrication of a Crystalline Nanocellulose Embedded Agarose Biomaterial Ink for Bone Marrow-Derived Mast Cell Culture

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

Agarose Fluid Gels Formed by Shear Processing During Gelation for Suspended 3D Bioprinting