This work was sponsored by the National Key Research and Development Program of China (2018YFA0703000), the National Nature Science Foundation of China (No.U1609207, 81827804), the Science Fund for Creative Research Groups of the National Natural Science Foundation of China (No. 51821093).

1. Cell culturing
<ol>
	<li>Prepare Dulbecco&#39;s modified Eagle medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin, used to culture human breast cancer cell (MDA-MB-231) lines and human umbilical vein endothelial cell (HUVEC) lines.</li>
	<li>Prepare DMEM with L-glutamine (DMEM/F-12), supplemented with 10% FBS and 1% penicillin/streptomycin, used to culture bone marrow mesenchymal stem cell (BMSC) lines.</li>
	<li>Set the culturing environment as 37 &#176;C and 5% CO2. Culture MDA-MB-231, HUVEC, and BMSC, and passage the cells in a 1:2 ratio when 90% confluence is reached.</li>
</ol>
2. Fabrication of GelMA microspheres
<ol>
	<li>Print the fixture as Figure 1A with polylactic acid (PLA) on a fused deposition modeling (FDM) printer. Place two metal ring electrodes in the fixture.</li>
	<li>Connect the two metal ring electrodes with ground and positive poles, respectively. Place the metal plate connected with the high voltage below the ring electrode and place a Petri dish with silicon oil on the metal plate as a droplet receiver.</li>
	<li>Dissolve freeze-dried GelMA (5% w/v) and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP, 0.5% w/v) in Dulbecco's phosphate-buffered saline (DPBS) as the bioink (10 mL). Filter the bioink through a 0.22 &#956;m filter for sterility and heat it in a 37 &#176;C water bath for 15 min.</li>
	<li>Detach MDA-MB-231 cells with 3 mL of 0.25% trypsin-0.02% EDTA solution for 3 min at 37 &#176;C. Centrifuge cells in a 15 mL centrifugal tube at 100 x g for 5 min to obtain a cell pellet.</li>
	<li>Remove the supernatant. Mix the cell pellet with 1 mL of prepared bioink by slowly pipetting it to prevent the production of bubbles.</li>
	<li>Place 1 mL of bioink (MDA-MB-231) into a 3 mL sterile syringe. Feed the bioink by the force of compressed air (~0.5 kPa). Put the syringe on the fixture. 
	NOTE: The printing environment should be strictly controlled at a temperature of 30 &#176;C and humidity of 50%.</li>
	<li>Switch on the high voltage power and set the voltage as 0&#8722;4 kV. Simultaneously, turn on the 405 nm wavelength light to crosslink the GelMA droplets in 5 mL of silicon oil.</li>
	<li>Pour the most part of the silicon oil away by decanting the Petri dish. Transfer the remained silicon oil and the GelMA microspheres into a 15 mL centrifugal tube using a spoon.</li>
	<li>Add 5 mL of DPBS and shake the mixture uniformly. Centrifuge the tube at 100 x g for 5 min and remove the supernatant fluid.</li>
	<li>Repeat step 2.9 3x.</li>
	<li>Take out the GelMA microspheres with a spoon and culture them in DMEM in a Petri dish at 37 &#176;C and 5% CO2 for 3 days.</li>
	<li>Discard the medium and wash the microspheres with DPBS. Fix with 2 mL of 4% paraformaldehyde (PFA) for 30 min at room temperature (RT).</li>
	<li>Discard the PFA and wash the microspheres with DPBS. Permeabilize with 2 mL of 0.5% nonionic surfactant (i.e., Triton X-100) for 5 min at RT.</li>
	<li>Discard the nonionic surfactant and wash the microspheres with DPBS. Stain them with 2 mL of tetramethylrhodamine (TRITC) phalloidin for 30 min in darkness at RT.</li>
	<li>Discard the TRITC and wash the microspheres with DPBS. Stain them with 2 mL of 4&#8211;,6-diamidino-2-phenylindole (DAPI) for 10 min in darkness at RT.</li>
	<li>Discard the DAPI and wash the microspheres with DPBS. Capture the morphology with a confocal fluorescence microscope.</li>
</ol>
3. Fabrication of GelMA fibers
<ol>
	<li>Prepare a coaxial nozzle as shown in Figure 2A. Fix an inner nozzle (25 G, OD = 510 &#956;m, ID = 250 &#956;m) and outer nozzle (18 G, OD = 1200 &#956;m, ID = 900 &#956;m) with soldering. Connect a glass tube (length = 50 mm, inside diameter = 1.2 mm) to the end of the coaxial nozzle.</li>
	<li>Dissolve sodium alginate (Na-Alg) powder that is sterilized under ultraviolet (UV) light for 30 min in deionized water at 2% (w/v).</li>
	<li>Prepare a sterile bioink solution following step 2.3. Heat the GelMA bioink and Na-Alg solution in a 37 &#176;C water bath for 15 min.</li>
	<li>Detach BMSCs cells with 3 mL of 0.25% trypsin-0.02% EDTA solution for 3 min at 37 &#176;C. Centrifuge cells in a 15 mL centrifugal tube at 100 x g for 5 min to obtain a cell pellet.</li>
	<li>Remove the supernatant fluid. Mix the cell pellet with 2 mL of prepared GelMA bioink by slowly pipetting it to prevent the production of bubbles.</li>
	<li>Place 2 mL of bioink (BMSCs) into a 10 mL syringe. Place 2 mL of Na-Alg solution into another syringe (10 mL). Feed them with two syringe pumps, respectively (here, bioink at 50 &#956;m/min and Na-Alg solution at 350 &#956;m/min). 
	NOTE: The printing environment should be strictly controlled at a temperature of 30 &#176;C and humidity of 50%.</li>
	<li>Turn on the 405 nm wavelength light to irradiate the transparent tube to crosslink the GelMA fibers. Use a Petri dish with DPBS to receive the fibers.</li>
	<li>Take out the GelMA fibers with a spoon from DPBS and culture them for 3 days in the prepared DMEM/F-12 in 37 &#176;C and 5% CO2.</li>
	<li>Follow steps 2.12&#8722;2.16 to prepare the GelMA fibers for morphological observation with a confocal fluorescence microscope.</li>
</ol>
4. Fabrication of complex 3D GelMA structures
NOTE: Figure 3A shows the fabrication sketch of the complex 3D GelMA structures.
<ol>
	<li>Wipe the DLP bioprinter (Figure 3E) with 75% alcohol and expose it to the UV irradiation for 30 min for sterility.</li>
	<li>Dissolve the freeze-dried GelMA (10% w/v) and LAP (0.5% w/v) in DPBS. Add magenta edible pigment into the solution (3% v/v) to improve the printing accuracy.</li>
	<li>Filter the solution through a 0.22 &#956;m filter for sterility and heat it in a 37 &#176;C water bath for 15 min.</li>
	<li>Build the 3D models with computer-aided design (CAD) software. Import the model documents to the upper software (EFL) of the applied DLP bioprinter.</li>
	<li>Add 10 mL of prepared bioink into the trough of the DLP bioprinter.</li>
	<li>Set the printing parameters in the upper software as follows: light intensity = 12 mW/cm2, irradiation duration = 30 s, and slice height = 100 &#956;m. Start printing.</li>
	<li>Remove the printed structure from the bioprinter and immerse it in DPBS in a Petri dish.</li>
	<li>Detach the MDA-MB-231s cells with 3 mL of 0.25% trypsin-0.02% EDTA solution for 3 min at 37 &#176;C. Centrifuge cells at 100 x g for 5 min in a 15 mL tube to obtain a cell pellet.</li>
	<li>Remove the supernatant fluid and mix the cell pellet with 2 mL of DMEM.</li>
	<li>Add 100 &#956;L of cell suspension on the printed structures. Culture them for 3 days in the prepared DMEM at 37 &#176;C and 5% CO2.</li>
	<li>Follow steps 2.12&#8722;2.16 to prepare the complex 3D structures for morphological observation with a confocal fluorescence microscope.</li>
</ol>
5. Fabrication of GelMA-based microfluidic chips
NOTE: Figure 4A shows the fabrication sketch of the GelMA-based microfluidic chip.
<ol>
	<li>Dissolve the freeze-dried GelMA 10% (w/v) and LAP (0.5% w/v) in DPBS. Filter the GelMA solution through a 0.22 &#956;m filter for sterility.</li>
	<li>Sterilize the gelatin powder under UV light for 30 min and add it to the GelMA-LAP solution prepared in step 5.1 to a final concentration of gelatin of 5% (w/v). Heat the mixture in a 37 &#176;C water bath for 15 min.</li>
	<li>Design a group of molds (Figure 4B,C) with CAD software and manufacture them with photopolymer resin on a DLP printer.</li>
	<li>Fill the molds fully with the prepared bioink.</li>
	<li>Put the molds into a 4 &#176;C refrigerator to crosslink the gelatin for 30 min.</li>
	<li>Remove the molds and demold with a blade the partially (physically) crosslinked hydrogel sheets from the molds.</li>
	<li>Combine the two demolded hydrogel sheets and bond them with the help of GelMA by irradiating at 405 nm for 1 min.</li>
	<li>Detach the HUVECs cells with 3 mL of 0.25% trypsin-0.02% EDTA solution for 3 min at 37 &#176;C. Centrifuge cells in a 15 mL centrifugal tube to obtain a cell pellet at 100 x g for 5 min.</li>
	<li>Remove the supernatant fluid and mix the cell pellet with 2 mL of DMEM.</li>
	<li>Fill the microchannel fully by injecting the cell suspension with a nozzle and syringe.</li>
	<li>Flip the chip upside down every 15 min during the next 3 h to achieve uniform and complete cell seeding. Culture the chips in the Petri dish for 3 days in the prepared DMEM at 37 &#176;C and 5% CO2.</li>
	<li>Follow steps 2.12&#8722;2.16 to prepare the microfluidic chips for morphological observation with a confocal fluorescence microscope.</li>
</ol>

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The authors have nothing to disclose.

This article describes several strategies to fabricate GelMA 3D structures, namely GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. GelMA has promising biocompatibility and formation capability and is widely used in the field of biofabrication. Microsphere structures are suitable for controlled drug release, tissue culturing, and injection into organisms for further therapy21,22,23<...

Hydrogels are thought to be a suitable material in the field of biofabrication1,2,3,4. Among them, gelatin methacryloyl (GelMA) has become one of the most versatile biomaterials, initially proposed in 2000 by Van Den Bulcke et al.5. GelMA is synthesized by the direct reaction of gelatin with methacrylic anhydride (MA). The gelatin, which is hydrolyzed by the mammal collagen, is composed of target motifs of matrix metalloproteinase (MMP). Thus, in vitro three-dimensional (3D) tissue models established by GelMA can ideally mimic the interactions between cells and extracellular matrix (ECM) in vivo. Furthermore, arginine-glycine-aspartic acid (RGD) sequences, which are absent in some other hydrogels such as alginates, remain on the molecular chains of GelMA. This makes it possible to realize the attachment of encapsulated cells inside the hydrogel networks6. Additionally, the formation capability of GelMA is promising. The methacrylamide groups on the GelMA molecular chains react with the photoinitiator under mild reaction conditions and form covalent bonds upon exposure to light irradiation. Therefore, the printed structures can be rapidly crosslinked to maintain the designed shapes in a simple way.
Based on these properties, a series of fields utilize GelMA to carry out various applications, such as tissue engineering, basic cytology analysis, drug screening, and biosensing. Accordingly, various fabrication strategies have been also demonstrated7,8,9,10,11,12,13,14. However, it is still challenging to carry out 3D bioprinting based on GelMA, which is due to its fundamental properties. GelMA is a temperature-sensitive material. During the printing process, the temperature of the printing atmosphere has to be strictly controlled in order to maintain the physical state of the bioink. Besides, the viscosity of GelMA is generally lower than other common hydrogels (i.e., alginate, chitosan, hyaluronic acid, etc.). However, other obstacles are faced when building 3D architectures with this material15.
This article summarizes several approaches for the 3D bioprinting of GelMA proposed by our lab and describes the printed samples (i.e., the synthesis of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips). Each method has specialized functions and can be adopted in different situations with different requirements. GelMA microspheres are generated by an electroassisted module, which forms extra external electric force to shrink the droplet size. In terms of GelMA fibers, they are extruded by a coaxial bioprinting nozzle with the help of viscous sodium alginate. In addition, the establishment of complex 3D structures is achieved with a digital light processing (DLP) bioprinter. Finally, a twice crosslinking strategy is proposed to build GelMA-based microfluidic chips, combining GelMA hydrogel and traditional microfluidic chips. It is believed that this protocol is a significant summary of the GelMA bioprinting strategies used in our lab and may inspire other researchers in relative fields.

<table><tbody><tr><td>0.22 &amp;mu;m filter membrane</td><td>Millipore</td><td></td><td></td></tr><tr><td>2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI)</td><td>Yeasen Biological Technology Co., Ltd., Shanghai, China</td><td></td><td></td></tr><tr><td>3D bioprinter</td><td>SuZhou Intelligent Manufacturing Research Institute, SuZhou, China</td><td></td><td></td></tr><tr><td>405nm wavelength light</td><td>SuZhou Intelligent Manufacturing Research Institute, SuZhou, China</td><td></td><td></td></tr><tr><td>co-axial nozzle</td><td>SuZhou Intelligent Manufacturing Research Institute, SuZhou, China</td><td></td><td></td></tr><tr><td>confocal fluorescence microscope</td><td>OLYMPUS FV3000</td><td></td><td></td></tr><tr><td>digital light processing (DLP) bioprinter</td><td>SuZhou Intelligent Manufacturing Research Institute, SuZhou, China</td><td></td><td></td></tr><tr><td>DLP printer</td><td>SuZhou Intelligent Manufacturing Research Institute, SuZhou, China</td><td></td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline (DPBS)</td><td>Tangpu Biological Technology Co., Ltd., Hangzhou, China</td><td></td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Tangpu Biological Technology Co., Ltd., Hangzhou, China</td><td></td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium with L-glutamine (DMEM/F-12)</td><td>Tangpu Biological Technology Co., Ltd., Hangzhou, China</td><td></td><td></td></tr><tr><td>EFL Software</td><td>SuZhou Intelligent Manufacturing Research Institute, SuZhou, China</td><td></td><td></td></tr><tr><td>fetal bovine serum (FBS)</td><td>Tangpu Biological Technology Co., Ltd., Hangzhou, China</td><td></td><td></td></tr><tr><td>gelatin</td><td>Sigma-Aldrich, Shanghai, China</td><td></td><td></td></tr><tr><td>gelatin methacryloyl (GelMA)</td><td>SuZhou Intelligent Manufacturing Research Institute, SuZhou, China</td><td></td><td></td></tr><tr><td>high voltage power</td><td>SuZhou Intelligent Manufacturing Research Institute, SuZhou, China</td><td></td><td></td></tr><tr><td>lithium phenyl-2, 4, 6-trimethylbenzoylphosphinate (LAP)</td><td>SuZhou Intelligent Manufacturing Research Institute, SuZhou, China</td><td></td><td></td></tr><tr><td>paraformaldehyde</td><td>Tangpu Biological Technology Co., Ltd., Hangzhou, China</td><td></td><td></td></tr><tr><td>penicillin/streptomycin</td><td>Tangpu Biological Technology Co., Ltd., Hangzhou, China</td><td></td><td></td></tr><tr><td>sodium alginate (Na-Alg)</td><td>Sigma-Aldrich, Shanghai, China</td><td></td><td></td></tr><tr><td>TRITC phalloidin</td><td>Yeasen Biological Technology Co., Ltd., Shanghai, China</td><td></td><td></td></tr><tr><td>Triton X-100</td><td>Solarbio Co., Ltd., Shanghai, China</td><td></td><td></td></tr></tbody></table>

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.

During the fabrication of GelMA microspheres, the GelMA droplets were separated by the external electric field force. When the droplets fell into the receiving silicon oil, they remained standard spheroid shape without tails. This is because the GelMA droplets were in an aqueous phase, while the silicon oil was in an oil phase. The surface tension that formed between the two phases caused the GelMA droplets to maintain a standard spheroid shape. In terms of the cell-laden microspheres, cells experienced the high voltage ...

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Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

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 ...

protocols-3d-bioprinting-gelatin-methacryloyl-hydrogel-based

Research

JoVE Journal

Bioengineering

18.4K Views.  Zhejiang University. Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

Video: Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

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

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

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

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

Immunology and Infection

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

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

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

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

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 ...

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.

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 ...

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 ...

Preparation and Characterization of Graphene-Based 3D Biohybrid Hydrogel Bioink for Peripheral Neuroengineering

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global problem that occurs in 1.5%-5% of emergency patients and may lead to significant job losses. Today, tissue engineering-based approaches, consisting of scaffolds, appropriate cell lines, and biosignals, have become more applicable with the development of three-dimensional (3D) bioprinting technologies. The combination of various hydrogel biomaterials with stem cells, exosomes, or bio-signaling molecules is frequently studied to overcome the existing problems in peripheral nerve regeneration. Accordingly, the production of injectable systems, such as hydrogels, or implantable conduit structures formed by various bioprinting methods has gained importance in peripheral neuro-engineering. Under normal conditions, stem cells are the regenerative cells of the body, and their number and functions do not decrease with time to protect their populations; these are not specialized cells but can differentiate upon appropriate stimulation in response to injury. The stem cell system is under the influence of its microenvironment, called the stem cell niche. In peripheral nerve injuries, especially in neurotmesis, this microenvironment cannot be fully rescued even after surgically binding severed nerve endings together. The composite biomaterials and combined cellular therapies approach increases the functionality and applicability of materials in terms of various properties such as biodegradability, biocompatibility, and processability. Accordingly, this study aims to demonstrate the preparation and use of graphene-based biohybrid hydrogel patterning and to examine the differentiation efficiency of stem cells into nerve cells, which can be an effective solution in nerve regeneration.

In this manuscript, we demonstrate the preparation of a biohybrid hydrogel bioink containing graphene for use in peripheral tissue engineering. Using this 3D biohybrid material, the neural differentiation protocol of stem cells is performed. This can be an important step in bringing similar biomaterials to the clinic.

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global ...

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

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

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

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

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 ...

Phototunable Bioprinted Hydrogels to Probe Cellular Responses to ECM Stiffening

3D Bioprinting Phototunable Hydrogels to Study Fibroblast Activation

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture platforms and dynamically triggering changes, such as increasing microenvironmental stiffness, enables researchers to model changes in the extracellular matrix (ECM) that occur during fibrotic disease progression. Herein, a method is presented for 3D bioprinting a phototunable hydrogel biomaterial capable of two sequential polymerization reactions within a gelatin support bath. The technique of Freeform Reversible Embedding of Suspended Hydrogels (FRESH) bioprinting was adapted by adjusting the pH of the support bath to facilitate a Michael addition reaction. First, the bioink containing poly(ethylene glycol)-alpha methacrylate (PEG&#945;MA) was reacted off-stoichiometry with a cell-degradable crosslinker to form soft hydrogels. These soft hydrogels were later exposed to photoinitator and light to induce the homopolymerization of unreacted groups and stiffen the hydrogel. This protocol covers hydrogel synthesis, 3D bioprinting, photostiffening, and endpoint characterizations to assess fibroblast activation within 3D structures. The method presented here enables researchers to 3D bioprint a variety of materials that undergo pH-catalyzed polymerization reactions and could be implemented to engineer various models of tissue homeostasis, disease, and repair.

Author Spotlight: Understanding Chronic Lung Diseases Using 3D Printed Phototunable Hydrogels 

This article describes how to 3D bioprint phototunable hydrogels to study extracellular matrix stiffening and fibroblast activation.

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture ...

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Summary

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Chapters in this video

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

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

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

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

Preparation and Characterization of Graphene-Based 3D Biohybrid Hydrogel Bioink for Peripheral Neuroengineering

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

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

3D Bioprinting Phototunable Hydrogels to Study Fibroblast Activation