Name: protocols of 3d bioprinting of gelatin methacryloyl hydrogel based bioinks
Uploaded: 2019-12-21T13:00:00
Description: Watch this Scientific Journal Video about Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks at JoVE.com

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</s...

<ol>
	<li>Ahmed, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogel:+Preparation,+characterization,+and+applications:+A+review.">Hydrogel: Preparation, characterization, and applications: A review.</a> Journal of Advanced Research. 6 (2), 105-121 (2015).</li><li>Ashton, R. S., Banerjee, A., Punyani, S., Schaffer, D. V., Kane, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaffolds+based+on+degradable+alginate+hydrogels+and+poly(lactide-co-glycolide)+microspheres+for+stem+cell+culture.">Scaffolds based on degradable alginate hydrogels and poly(lactide-co-glycolide) microspheres for stem cell culture.</a> Biomaterials. 28 (36), 5518-5525 (2007).</li><li>Billiet, T., Vandenhaute, M., Schelfhout, J., Van Vlierberghe, S., Dubruel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+trends+and+limitations+in+hydrogel-rapid+prototyping+for+tissue+engineering.">A review of trends and limitations in hydrogel-rapid prototyping for tissue engineering.</a> Biomaterials. 33 (26), 6020-6041 (2012).</li><li>Saroia, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+on+biocompatibility+nature+of+hydrogels+with+3D+printing+techniques,+tissue+engineering+application+and+its+future+prospective.">A review on biocompatibility nature of hydrogels with 3D printing techniques, tissue engineering application and its future prospective.</a> Bio-Design and Manufacturing. 1 (4), 265-279 (2018).</li><li>Van Den Bulcke, A. I., 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=Structural+and+Rheological+Properties+of+Methacrylamide+Modified+Gelatin+Hydrogels.">Structural and Rheological Properties of Methacrylamide Modified Gelatin Hydrogels.</a> Biomacromolecules. 1 (1), 31-38 (2000).</li><li>Sun, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+Properties+of+Gelatin+Methacryloyl+(GelMA)+Hydrogels+and+Their+Recent+Applications+in+Load-Bearing+Tissue.">Synthesis and Properties of Gelatin Methacryloyl (GelMA) Hydrogels and Their Recent Applications in Load-Bearing Tissue.</a> Polymers. 10 (11), 1290 (2018).</li><li>Gao, Q., 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+printing+of+complex+GelMA-based+scaffolds+with+nanoclay.">3D printing of complex GelMA-based scaffolds with nanoclay.</a> Biofabrication. 11 (3), 035006 (2019).</li><li>Hassanzadeh, P., 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=Ultrastrong+and+flexible+hybrid+hydrogels+based+on+solution+self-assembly+of+chitin+nanofibers+in+gelatin+methacryloyl+(GelMA).">Ultrastrong and flexible hybrid hydrogels based on solution self-assembly of chitin nanofibers in gelatin methacryloyl (GelMA).</a> Journal of Materials Chemistry B. 4 (15), 2539-2543 (2016).</li><li>McBeth, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+bioprinting+of+GelMA+scaffolds+triggers+mineral+deposition+by+primary+human+osteoblasts.">3D bioprinting of GelMA scaffolds triggers mineral deposition by primary human osteoblasts.</a> Biofabrication. 9 (1), 015009 (2017).</li><li>Nie, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vessel-on-a-chip+with+Hydrogel-based+Microfluidics.">Vessel-on-a-chip with Hydrogel-based Microfluidics.</a> Small. 14 (45), 1802368 (2018).</li><li>Shao, 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=Bioprinting+of+Cell-Laden+Microfiber:+Can+It+Become+a+Standard+Product.">Bioprinting of Cell-Laden Microfiber: Can It Become a Standard Product.</a> Advanced Healthcare Materials. 8 (9), 1900014 (2019).</li><li>Shao, 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=Fiber-Based+Mini+Tissue+with+Morphology-Controllable+GelMA+Microfibers.">Fiber-Based Mini Tissue with Morphology-Controllable GelMA Microfibers.</a> Small. 14 (44), 1802187 (2018).</li><li>Xie, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electro-Assisted+Bioprinting+of+Low-Concentration+GelMA+Microdroplets.">Electro-Assisted Bioprinting of Low-Concentration GelMA Microdroplets.</a> Small. 15 (4), 1804216 (2019).</li><li>Yue, 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=Synthesis,+properties,+and+biomedical+applications+of+gelatin+methacryloyl+(GelMA)+hydrogels.">Synthesis, properties, and biomedical applications of gelatin methacryloyl (GelMA) hydrogels.</a> Biomaterials. 73, 254-271 (2015).</li><li>Schuurman, W., 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=Gelatin-Methacrylamide+Hydrogels+as+Potential+Biomaterials+for+Fabrication+of+Tissue-Engineered+Cartilage+Constructs.">Gelatin-Methacrylamide Hydrogels as Potential Biomaterials for Fabrication of Tissue-Engineered Cartilage Constructs.</a> Macromolecular Bioscience. 13 (5), 551-561 (2013).</li><li>Barbot, A., Decanini, D., Hwang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On-chip+Microfluidic+Multimodal+Swimmer+toward+3D+Navigation.">On-chip Microfluidic Multimodal Swimmer toward 3D Navigation.</a> Scientific Reports. 6, 19041 (2016).</li><li>Esmaeilsabzali, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+microfluidic+chip+for+immunomagnetic+detection+and+isolation+of+rare+prostate+cancer+cells+from+blood.">An integrated microfluidic chip for immunomagnetic detection and isolation of rare prostate cancer cells from blood.</a> Biomedical Microdevices. 18 (1), 22 (2016).</li><li>Lee, J. M., Zhang, M., Yeong, W. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+evaluation+of+3D+printed+microfluidic+chip+for+cell+processing.">Characterization and evaluation of 3D printed microfluidic chip for cell processing.</a> Microfluidics and Nanofluidics. 20 (1), 5 (2016).</li><li>Picot, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+biomimetic+microfluidic+chip+to+study+the+circulation+and+mechanical+retention+of+red+blood+cells+in+the+spleen.">A biomimetic microfluidic chip to study the circulation and mechanical retention of red blood cells in the spleen.</a> American Journal of Hematology. 90 (4), 339-345 (2015).</li><li>Ren, K., Zhou, J., Wu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Materials+for+Microfluidic+Chip+Fabrication.">Materials for Microfluidic Chip Fabrication.</a> Accounts of Chemical Research. 46 (11), 2396-2406 (2013).</li><li>Chen, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Covalently+antibacterial+alginate-chitosan+hydrogel+dressing+integrated+gelatin+microspheres+containing+tetracycline+hydrochloride+for+wound+healing.">Covalently antibacterial alginate-chitosan hydrogel dressing integrated gelatin microspheres containing tetracycline hydrochloride for wound healing.</a> Materials Science and Engineering: C. 70, 287-295 (2017).</li><li>Fan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Covalent+and+injectable+chitosan-chondroitin+sulfate+hydrogels+embedded+with+chitosan+microspheres+for+drug+delivery+and+tissue+engineering.">Covalent and injectable chitosan-chondroitin sulfate hydrogels embedded with chitosan microspheres for drug delivery and tissue engineering.</a> Materials Science and Engineering: C. 71, 67-74 (2017).</li><li>Feng, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+black-pearl+reduced+graphene+oxide-sodium+alginate+hydrogel+microspheres+for+adsorbing+organic+pollutants.">Preparation of black-pearl reduced graphene oxide-sodium alginate hydrogel microspheres for adsorbing organic pollutants.</a> Journal of Colloid and Interface Science. 508, 387-395 (2017).</li><li>Park, K. S., Kim, C., Nam, J. O., Kang, S. M., Lee, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+characterization+of+thermosensitive+gelatin+hydrogel+microspheres+in+a+microfluidic+system.">Synthesis and characterization of thermosensitive gelatin hydrogel microspheres in a microfluidic system.</a> Macromolecular Research. 24 (6), 529-536 (2016).</li><li>Zheng, Y., 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=Injectable+Hydrogel-Microsphere+Construct+with+Sequential+Degradation+for+Locally+Synergistic+Chemotherapy.">Injectable Hydrogel-Microsphere Construct with Sequential Degradation for Locally Synergistic Chemotherapy.</a> ACS Applied Materials, Interfaces. 9 (4), 3487-3496 (2017).</li><li>Fern&#225;ndez de la Mora, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Fluid+Dynamics+of+Taylor+Cones.">The Fluid Dynamics of Taylor Cones.</a> Annual Review of Fluid Mechanics. 39 (1), 217-243 (2006).</li><li>Hsiao, A. Y., 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=Smooth+muscle-like+tissue+constructs+with+circumferentially+oriented+cells+formed+by+the+cell+fiber+technology.">Smooth muscle-like tissue constructs with circumferentially oriented cells formed by the cell fiber technology.</a> PLoS ONE. 10, 0119010 (2015).</li><li>Meng, Z. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+generation+of+hollow+Ca-alginate+microfibers.">Microfluidic generation of hollow Ca-alginate microfibers.</a> Lab on a Chip. 16 (14), 2673-2681 (2016).</li><li>Peng, L., Liu, Y., Gong, J., Zhang, K., Ma, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+fabrication+of+multi-stimuli+responsive+graphene+oxide+composite+hydrogel+fibres+by+microfluidics.">Continuous fabrication of multi-stimuli responsive graphene oxide composite hydrogel fibres by microfluidics.</a> RSC Advances. 7 (31), 19243-19249 (2017).</li><li>Sugimoto, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropassage-embedding+composite+hydrogel+fibers+enable+quantitative+evaluation+of+cancer+cell+invasion+under+3D+coculture+conditions.">Micropassage-embedding composite hydrogel fibers enable quantitative evaluation of cancer cell invasion under 3D coculture conditions.</a> Lab on a Chip. 18 (9), 1378-1387 (2018).</li><li>Yamada, M., Sugaya, S., Naganuma, Y., Seki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+synthesis+of+chemically+and+physically+anisotropic+hydrogel+microfibers+for+guided+cell+growth+and+networking.">Microfluidic synthesis of chemically and physically anisotropic hydrogel microfibers for guided cell growth and networking.</a> Soft Matter. 8 (11), 3122-3130 (2012).</li><li>Gao, G., 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=Tissue+engineered+bio-blood-vessels+constructed+using+a+tissue-specific+bioink+and+3D+coaxial+cell+printing+technique:+a+novel+therapy+for+ischemic+disease.">Tissue engineered bio-blood-vessels constructed using a tissue-specific bioink and 3D coaxial cell printing technique: a novel therapy for ischemic disease.</a> Advanced Functional Materials. 27 (33), 1700798 (2017).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#956;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&#39;s Phosphate Buffered Saline (DPBS)</td>
			<td>Tangpu Biological Technology Co., Ltd., Hangzhou, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Tangpu Biological Technology Co., Ltd., Hangzhou, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;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 ...

Watch this Scientific Journal Video about Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks at JoVE.com

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 ...

GelMA hydrogel based bionics become very popular in 3D bioprinting. However the low viscosity restricts its printability. Here we are sharing strategies for printing GelMA in our lab with other researchers.In this video we fully utilize the special properties of this biomaterial and propose several printing methods for different 3D structures which could contribute to further biomedical applications. To take organ damage for example implications are potential to be used into the corresponding therapy by injecting embedding or tracing the printed GelMA structures. It would be not easy to totally dissolve the freeze-dried GelMA in a short time.To speed up the dissolving process a vortex mixer could be used. Every biomaterial will use special properties intimate of GelMA. I suggested a new chemer should properly take a wander of what is conditioning critical when printing.The operation details determine the success of printing procedures. It is why we choose a visual demonstration to share with other researchers. To fabricate microspheres start by connecting the two metal ring electrodes with ground and positive poles.Place the metal plate connected with the high voltage below the ring electrode and a Petri dish with silicon oil on the metal plate as a droplet receiver. Prepare bio-ink by dissolving freeze-dried GelMA and LAP and DPBS filter it through 0.22 micrometer filter for sterility then heat it in a 37 degree Celsius water bath for 15 minutes. Detach MDA-MB-231 cells with three milliliters of 0.25 percent trypsin and 0.02 percent EDTA solution for three minutes at 37 degrees Celsius.Transfer the cells to a 15 milliliter tube and centrifuge them at 100 times G for five minutes. Remove the supernatant and mix the cells with one milliliter of prepared bio-ink by slowly pipetting up and down making sure to avoid air bubbles. Aspirate one milliliter of the bio-ink and cell mixture into a three milliliter sterile syringe.Feed the bio-ink by the force of compressed air and put the syringe on the fixture. Switch on the high voltage power and set the voltage as zero to four kilovolts. Simultaneously turn on the 405 nanometer wavelength light to cross-link the GelMA droplets in five milliliters of silicon oil.Decant the Petri dish to get rid of most of the silicon oil and use a spoon to transfer the remaining oil and microspheres into a 15 milliliter centrifuge tube. Add five milliliters of DPBS and shake the mixture centrifuge the tube at 100 times G and remove the supernatant. Transfer the microspheres into a Petri dish with DMEM and culture them at 37 degrees Celsius and five percent carbon dioxide for three days.After three days discard the medium and wash the microspheres with DPBS. Fix them with two milliliters of four percent PFA for 30 minutes at room temperature then discard the PFA and repeat the wash. Permeabilize the cells with two milliliters of 0.5 percent non-ionic surfactant for five minutes at room temperature discard the surfactant and wash the microspheres with DPBS.Next stain the cells with TRITC-phalloidin for 30 minutes then DAPI for 10 minutes in the dark discarding the dyes and washing the spheres with DPBS after each staining. Image the microspheres with a confocal fluorescence microscope. Dissolve sterilized sodium alginate powder in deionized water at a two percent weight to volume ratio.Prepare sterile bio-ink solution as previously described then heat the bio-ink and sodium alginate in a 37 degrees Celsius water bath for 15 minutes. Trypsinize and centrifuge bMSCs according to the previously described procedure for MDA-MB 231 cells then remove the supernatant fluid and resuspend the cell pellet with 2 milliliters of the GelMA bio-ink. Aspirate two milliliters of the bio-ink cell mixture into a 10 milliliter syringe and two milliliters of the sodium alginate mixture into another syringe.Feed the solutions with two syringe pumps bio-ink at 50 micrometers per minute and sodium alginate at 350 micrometers per minute. Turn on the 405 nanometer wavelength light to irradiate the transparent tube and cross-link the GelMA fibers. Use a Petri dish to receive the fibers and collect them with a spoon.Transfer the fibers into a dish with DMEM/F12 and culture them for three days at 37 degrees Celsius and five percent carbon dioxide. After culturing the cells follow the previously described directions for observing the fibers under a confocal fluorescence microscope. Dissolve freeze-dried GelMA and LAP and DPBS and add magenta edible pigment into the solution to improve the printing accuracy.Filter the solution through a 0.22 micrometer filter for sterility and heat it in a 37 degree Celsius water bath for 15 minutes. Build the 3D models with CAD software and import the model documents to the upper software of the applied DLP bioprinter. Add ten milliliters of the prepared bio-ink to the trough of the bioprinter.Set the printing parameters according to the manuscript directions then start printing. When complete remove the printed structure from the bioprinter and immerse it in DPBS in a Petri dish. Detach and pellet MDA-MB 231 cells as previously described.Resuspend them with two milliliters of DMEM and add the suspension to the printed structure. Culture the cells at 37 degrees Celsius and five percent carbon dioxide for three days then prepare the structures for observation with a confocal fluorescence microscope as previously described. Prepare a 10 percent weight to volume GelMA and 0.5 percent weight to volume LAP solution then filter it through a 0.22 micrometer filter.Sterilize gelatin powder under UV light for 30 minutes and add it to the GelMA LAP solution at a five percent weight to volume ratio. Heat the mixture in a 37 degrees Celsius water bath for 15 minutes. Fill the molds with the prepared bio-ink and place them in a four degrees Celsius refrigerator for 30 minutes.Use a blade to remove the partially cross-linked hydrogel sheets from the molds. Combine 2D molded hydrogel sheets and bond them with GelMA by irradiating at 405 nanometers for one minute. Detach and pellet HUVECs mix the cells with two milliliters of DMEM and inject the suspension into the microchannel with the nozzle and syringe.For the next three hours flip the chip upside down every 15 minutes to achieve uniform and complete cell seating. Culture the chips in a Petri dish for three days in DMEM at 37 degrees Celsius and five percent carbon dioxide then prepare them for morphological observation. When the GelMA droplets fell into the receiving silicone oil they kept a standard spheroid shape without tails.The encapsulated cells experiencing the high voltage electric field force maintained their spreading capability which verified the biocompatibility of the electro-assisted fabrication method. A DLP bio printer was chosen to fabricate GelMA structures with more complex shapes such as nose ear and multi chamber. Seeded HUVECs attached to the GelMA materials and spread on the surface of the cross-linked GelMA structures demonstrating that this application holds potential for tissue engineering.A twice cross-linking strategy was used to fabricate GelMA based microfluidic chips. Chips with various microchannels were built by designing different molds on demand and HUVECs were seeded in the channels and attached to the channel wall forming the macroscopic vessel shape. High voltage source is applied when fabricating microspheres.Researchers must examine circuit before and don't touch exposed millibars protecting operators or makes the cells from a damage by short-circuit. The ultrasonic water-base could be another choice to accelerate the deserving speed. Definitely this contribution summarized the 3D bioprinting methods of GelMA in our lab.Researchers could refer to the video and improve or expand printing strategies to serve further biomedical requirements.

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Research

JoVE Journal

Bioengineering

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 ...

Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and fibroblasts are first isolated, counted and mixed at desired cell ratios. They are co-cultured in individual wells in ultra-low attachment 96-well plates. Within 3 days, beating spheroids form. These spheroids are then picked up by a nozzle using vacuum suction and assembled on a needle array using a 3D bioprinter. The spheroids are then allowed to fuse on the needle array. Three days after 3D bioprinting, the spheroids are removed as an intact patch, which is already spontaneously beating. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

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 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 ...

Formulating and Characterizing an Exosome-based Dopamine Carrier System

Exosomes between 40 and 200 nm in size constitute the smallest subgroup of extracellular vesicles. These bioactive vesicles secreted by cells play an active role in intercellular cargo and communication. Exosomes are mostly found in body fluids such as plasma, cerebrospinal fluid, urine, saliva, amniotic fluid, colostrum, breast milk, joint fluid, semen, and pleural acid. Considering the size of exosomes, it is thought that they may play an important role in central nervous system diseases because they can pass through the blood-brain barrier (BBB). Hence, this study aimed to develop an exosome-based nanocarrier system by encapsulating dopamine into exosomes isolated from Wharton's jelly mesenchymal stem cells (WJ-MSCs). Exosomes that passed the characterization process were incubated with dopamine. The dopamine-loaded exosomes were recharacterized at the end of incubation. Dopamine-loaded exosomes were investigated in drug release and cytotoxicity assays. The results showed that dopamine could be successfully encapsulated within the exosomes and that the dopamine-loaded exosomes did not affect fibroblast viability.

Here, we aimed to obtain a formulation by dopamine loading of the isolated exosomes of stem cells from Wharton's jelly mesenchymal stem cells. Exosome isolation and characterization, drug loading into the resulting exosomes, and the cytotoxic activity of the developed formulation are described in this protocol.

Exosomes between 40 and 200 nm in size constitute the smallest subgroup of extracellular vesicles. These bioactive vesicles secreted by cells play an ...

Synthesis of Strong Adhesive Hydrogel, Gelatin O-Nitrosobenzaldehyde

Adhesive materials have become popular biomaterials in the field of biomedical and tissue engineering. In our previous work, we presented a new material - gelatin o-nitrosobenzaldehyde (gelatin-NB) - which is mainly used for tissue regeneration and has been validated in animal models of corneal injury and inflammatory bowel disease. This is a novel hydrogel formed by modifying biological gelatin with o-nitrosobenzaldehyde (NB). Gelatin-NB was synthesized by activating the carboxyl group of NB-COOH and reacting with gelatin through 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The obtained compound was purified to generate the final product, which can be stably stored for at least 18 months. NB has a strong adhesion to -NH2 on the tissue, which can form many C = N bonds, thus increasing the adhesion of gelatin-NB to the tissue interface. The preparation process comprises steps for the synthesis of the NB-COOH group, modification of the group, synthesis of gelatin-NB, and purification of the compound. The goal is to describe the specific synthesis process of gelatin-NB in detail and to demonstrate the application of gelatin-NB to damage repair. Moreover, the protocol is presented to further strengthen and expand the nature of the material produced by the scientific community for more applicable scenarios.

The protocol presented here shows the synthesis of a strong adhesive hydrogel gelatin o-nitrosobenzaldehyde (gelatin-NB). Gelatin-NB has rapid and efficient tissue adhesion ability, which can form a strong physical barrier to protect wound surfaces, so it is expected to be applied to the field of injury repair biotechnology.

Adhesive materials have become popular biomaterials in the field of biomedical and tissue engineering. In our previous work, we presented a new material - ...

Gelatin Methacryloyl Granular Hydrogel Scaffolds: High-throughput Microgel Fabrication, Lyophilization, Chemical Assembly, and 3D Bioprinting

The emergence of granular hydrogel scaffolds (GHS), fabricated via assembling hydrogel microparticles (HMPs), has enabled microporous scaffold formation in situ. Unlike conventional bulk hydrogels, interconnected microscale pores in GHS facilitate degradation-independent cell infiltration as well as oxygen, nutrient, and cellular byproduct transfer. Methacryloyl-modified gelatin (GelMA), a (photo)chemically crosslinkable, protein-based biopolymer containing cell adhesive and biodegradable moieties, has widely been used as a cell-responsive/instructive biomaterial. Converting bulk GelMA to GHS&#160;may open a plethora of opportunities for tissue engineering and regeneration. In this article, we demonstrate the procedures of high-throughput GelMA microgel fabrication, conversion to resuspendable dry microgels (micro-aerogels), GHS formation via the chemical assembly of microgels, and granular bioink fabrication for extrusion bioprinting. We show how a sequential physicochemical treatment via cooling and photocrosslinking enables the formation of mechanically robust GHS.&#160;When light is inaccessible (e.g., during deep tissue injection), individually crosslinked GelMA HMPs may be bioorthogonally assembled via enzymatic crosslinking using transglutaminases. Finally, three-dimensional (3D) bioprinting of microporous GHS&#160;at low HMP packing density is demonstrated via the interfacial self-assembly of heterogeneously charged nanoparticles.

This article describes protocols for high-throughput gelatin methacryloyl microgel fabrication using microfluidic devices, converting microgels to resuspendable powder (micro-aerogels), the chemical assembly of microgels to form granular hydrogel scaffolds, and developing granular hydrogel bioinks with preserved microporosity for 3D bioprinting.

The emergence of granular hydrogel scaffolds (GHS), fabricated via assembling hydrogel microparticles (HMPs), has enabled microporous scaffold formation ...

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 ...