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>

<ol><li>Ahmed, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hydrogel%3A+Preparation%2C+characterization%2C+and+applications%3A+A+review%5BTitle%5D)+AND+%22Journal+of+Advanced+Research%22%5BJournal%5D)">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/pubmed?term=((Scaffolds+based+on+degradable+alginate+hydrogels+and+poly%28lactide-co-glycolide%29+microspheres+for+stem+cell+culture%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">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/pubmed?term=((A+review+of+trends+and+limitations+in+hydrogel-rapid+prototyping+for+tissue+engineering%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">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/pubmed?term=((A+review+on+biocompatibility+nature+of+hydrogels+with+3D+printing+techniques%2C+tissue+engineering+application+and+its+future+prospective%5BTitle%5D)+AND+%22Bio-Design+and+Manufacturing%22%5BJournal%5D)">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/pubmed?term=((Structural+and+Rheological+Properties+of+Methacrylamide+Modified+Gelatin+Hydrogels%5BTitle%5D)+AND+%22Biomacromolecules%22%5BJournal%5D)">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/pubmed?term=((Synthesis+and+Properties+of+Gelatin+Methacryloyl+%28GelMA%29+Hydrogels+and+Their+Recent+Applications+in+Load-Bearing+Tissue%5BTitle%5D)+AND+%22Polymers%22%5BJournal%5D)">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/pubmed?term=((3D+printing+of+complex+GelMA-based+scaffolds+with+nanoclay%5BTitle%5D)+AND+%22Biofabrication%22%5BJournal%5D)">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/pubmed?term=((Ultrastrong+and+flexible+hybrid+hydrogels+based+on+solution+self-assembly+of+chitin+nanofibers+in+gelatin+methacryloyl+%28GelMA%29%5BTitle%5D)+AND+%22Journal+of+Materials+Chemistry+B%22%5BJournal%5D)">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/pubmed?term=((3D+bioprinting+of+GelMA+scaffolds+triggers+mineral+deposition+by+primary+human+osteoblasts%5BTitle%5D)+AND+%22Biofabrication%22%5BJournal%5D)">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/pubmed?term=((Vessel-on-a-chip+with+Hydrogel-based+Microfluidics%5BTitle%5D)+AND+%22Small%22%5BJournal%5D)">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/pubmed?term=((Bioprinting+of+Cell-Laden+Microfiber%3A+Can+It+Become+a+Standard+Product%5BTitle%5D)+AND+%22Advanced+Healthcare+Materials%22%5BJournal%5D)">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/pubmed?term=((Fiber-Based+Mini+Tissue+with+Morphology-Controllable+GelMA+Microfibers%5BTitle%5D)+AND+%22Small%22%5BJournal%5D)">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/pubmed?term=((Electro-Assisted+Bioprinting+of+Low-Concentration+GelMA+Microdroplets%5BTitle%5D)+AND+%22Small%22%5BJournal%5D)">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/pubmed?term=((Synthesis%2C+properties%2C+and+biomedical+applications+of+gelatin+methacryloyl+%28GelMA%29+hydrogels%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">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/pubmed?term=((Gelatin-Methacrylamide+Hydrogels+as+Potential+Biomaterials+for+Fabrication+of+Tissue-Engineered+Cartilage+Constructs%5BTitle%5D)+AND+%22Macromolecular+Bioscience%22%5BJournal%5D)">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/pubmed?term=((On-chip+Microfluidic+Multimodal+Swimmer+toward+3D+Navigation%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">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/pubmed?term=((An+integrated+microfluidic+chip+for+immunomagnetic+detection+and+isolation+of+rare+prostate+cancer+cells+from+blood%5BTitle%5D)+AND+%22Biomedical+Microdevices%22%5BJournal%5D)">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/pubmed?term=((Characterization+and+evaluation+of+3D+printed+microfluidic+chip+for+cell+processing%5BTitle%5D)+AND+%22Microfluidics+and+Nanofluidics%22%5BJournal%5D)">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/pubmed?term=((A+biomimetic+microfluidic+chip+to+study+the+circulation+and+mechanical+retention+of+red+blood+cells+in+the+spleen%5BTitle%5D)+AND+%22American+Journal+of+Hematology%22%5BJournal%5D)">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/pubmed?term=((Materials+for+Microfluidic+Chip+Fabrication%5BTitle%5D)+AND+%22Accounts+of+Chemical+Research%22%5BJournal%5D)">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/pubmed?term=((Covalently+antibacterial+alginate-chitosan+hydrogel+dressing+integrated+gelatin+microspheres+containing+tetracycline+hydrochloride+for+wound+healing%5BTitle%5D)+AND+%22Materials+Science+and+Engineering%3A+C%22%5BJournal%5D)">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/pubmed?term=((Covalent+and+injectable+chitosan-chondroitin+sulfate+hydrogels+embedded+with+chitosan+microspheres+for+drug+delivery+and+tissue+engineering%5BTitle%5D)+AND+%22Materials+Science+and+Engineering%3A+C%22%5BJournal%5D)">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/pubmed?term=((Preparation+of+black-pearl+reduced+graphene+oxide-sodium+alginate+hydrogel+microspheres+for+adsorbing+organic+pollutants%5BTitle%5D)+AND+%22Journal+of+Colloid+and+Interface+Science%22%5BJournal%5D)">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/pubmed?term=((Synthesis+and+characterization+of+thermosensitive+gelatin+hydrogel+microspheres+in+a+microfluidic+system%5BTitle%5D)+AND+%22Macromolecular+Research%22%5BJournal%5D)">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/pubmed?term=((Injectable+Hydrogel-Microsphere+Construct+with+Sequential+Degradation+for+Locally+Synergistic+Chemotherapy%5BTitle%5D)+AND+%22ACS+Applied+Materials%2C+Interfaces%22%5BJournal%5D)">Injectable Hydrogel-Microsphere Construct with Sequential Degradation for Locally Synergistic Chemotherapy.</a> ACS Applied Materials, Interfaces. 9 (4), 3487-3496 (2017).</li>
<li>Fernández de la Mora, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Fluid+Dynamics+of+Taylor+Cones%5BTitle%5D)+AND+%22Annual+Review+of+Fluid+Mechanics%22%5BJournal%5D)">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/pubmed?term=((Smooth+muscle-like+tissue+constructs+with+circumferentially+oriented+cells+formed+by+the+cell+fiber+technology%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">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/pubmed?term=((Microfluidic+generation+of+hollow+Ca-alginate+microfibers%5BTitle%5D)+AND+%22Lab+on+a+Chip%22%5BJournal%5D)">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/pubmed?term=((Continuous+fabrication+of+multi-stimuli+responsive+graphene+oxide+composite+hydrogel+fibres+by+microfluidics%5BTitle%5D)+AND+%22RSC+Advances%22%5BJournal%5D)">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/pubmed?term=((Micropassage-embedding+composite+hydrogel+fibers+enable+quantitative+evaluation+of+cancer+cell+invasion+under+3D+coculture+conditions%5BTitle%5D)+AND+%22Lab+on+a+Chip%22%5BJournal%5D)">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/pubmed?term=((Microfluidic+synthesis+of+chemically+and+physically+anisotropic+hydrogel+microfibers+for+guided+cell+growth+and+networking%5BTitle%5D)+AND+%22Soft+Matter%22%5BJournal%5D)">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/pubmed?term=((Tissue+engineered+bio-blood-vessels+constructed+using+a+tissue-specific+bioink+and+3D+coaxial+cell+printing+technique%3A+a+novel+therapy+for+ischemic+disease%5BTitle%5D)+AND+%22Advanced+Functional+Materials%22%5BJournal%5D)">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>

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 therapy21,22,23,24,25. Because the viscosity of GelMA solution is low, its formation is challenging. Thus, during the fabrication of the GelMA microspheres, the electrohydrodynamic (EHD) principle was chosen to solve this problem. The voltage applied was relatively low, and the microdroplets were generated one-by-one. To fabricate microspheres of a smaller size, the applied voltage can be increased, and the fluid would be in another state with the Taylor cone26.
Because of the Coulomb explosion phenomenon, the dropping droplets were further separated by their excessive electric density, resulting in smaller GelMA microspheres. Furthermore, monocomponent GelMA fibers were fabricated with the help of a coaxial nozzle and sodium alginate solution. A coaxial nozzle was applied here. As mentioned above, because of the low viscosity of GelMA, sodium alginate provided resistance to help maintain the shape of fiber. Hydrogel fiber structures are suitable for mimicking the fiber-shaped tissues in vivo (i.e., muscles, vessels, etc.27,28,29,30,31,32). For GelMA fibers with more complicated components, the applied bioprinting nozzle can be further modified. For example, a three-layer coaxial nozzle can be assembled to generate multilayer GelMA fibers.
In the establishment of complex GelMA 3D structures, it was found that the DLP bioprinter breaks through the printing obstacle caused by the low viscosity of GelMA. With the help of CAD software, GelMA 3D structures were fabricated on demand. Finally, a new GelMA fabrication method, the twice cross-linking strategy, was demonstrated and applied to the combination of GelMA and a traditional microfluidic chip. The hydrogels have higher biocompatibility, and researchers can encapsulate cells inside the chip body. The proposed GelMA-based microfluidic chip can be further improved by encapsulating cells in the chips to serve as suitable models in vitro for drug screening, cellular interaction studies, etc. We believe that the methods for fabrication of GelMA described here will increase the rate of development in this field and can be applied in further biomedical research.

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 electric field force in this process. From the morphology of the stained MDA-MB-231s (Figure 1B–E), it was found that the encapsulated MDA-MB-231s maintained its spreading capability, verifying the biocompatibility of this electroassisted fabrication method.
In terms of the GelMA fibers, GelMA and sodium alginate solution flowed in the inner and outer nozzles of the coaxial nozzle, respectively. As the sodium alginate had higher viscosity than GelMA, GelMA was restricted in the sodium alginate solution and maintained a line shape. The irradiation by light (405 nm wavelength) caused the inner GelMA to become crosslinked, forming the GelMA fibers (Figure 2B). Besides, BMSCs were encapsulated in the GelMA fibers (Figure 2C,D). As shown, the encapsulated BMSCs maintained spreading capability in the GelMA hydrogel networks after the fabrication process (Figure 2E).
A DLP bioprinter was chosen to fabricate GelMA structures with more complex shapes. As shown in Figure 3B–D, the structures of "nose", "ear", and "multichamber" were established. On the surface of the crosslinked GelMA structures, the seeded HUVECs attached to the GelMA materials and spread (Figure 3F). This demonstrated the possibility that the establishment of GelMA complex 3D structures with the help of a DLP bioprinter holds great potential in applications in the field of tissue engineering.
Unlike the traditional microfluidic chip that is based on materials without biodegradation properties16,17,18,20 (i.e, resin, glass, polydimethylsiloxane [PDMS], and polymethyl methacrylate [PMMA]), a GelMA-based microfluidic chip was fabricated here using a twice cross-linking strategy. Two components in the bioink were crosslinked successively. Chips with various microchannels were built by designing different molds on demand (Figure 4B,C). Besides, it was verified that HUVECs were seeded in the channels and attached to the channel wall, forming the macroscopic vessel shape (Figure 4D,E).
<img alt="Fabrication diagram of GelMA microspheres using light, microscopy of MDA-MB-231s, Phalloidin/DAPI 3D view." class="xfigimg" src="/files/ftp_upload/60545/60545fig01.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: GelMA microspheres. (A) Fabrication sketch of the GelMA microspheres. (B) Optical microscope image of the GelMA microspheres. (C) Optical microscope image of the MDA-MB-231s in GelMA. (D) 2D view of the F-actin and nucleus of the encapsulated MDA-MB-231s. (E) 3D view of the F-actin and nucleus of the encapsulated MDA-MB-231s. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60545/60545fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Fabrication of GelMA fibers: diagram, confocal microscopy, BMSCs-laden fiber, Phalloidin/DAPI staining." class="xfigimg" src="/files/ftp_upload/60545/60545fig02.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: GelMA fibers. (A) Fabrication sketch of the GelMA fibers. (B) Optical microscope image of the GelMA fibers (with blue ink). (C) Confocal fluorescence microscope image of the GelMA fibers (with green fluorescence particles). (D) Optical microscope image of the BMSCs in GelMA fibers. (E) The F-actin and nucleus of the encapsulated BMSCs. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60545/60545fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="GelMA fabrication diagram with DLP printer, complex structures, UV projection, Phalloidin/DAPI staining." class="xfigimg" src="/files/ftp_upload/60545/60545fig03.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: GelMA complex 3D structures. (A) Fabrication sketch of the complex GelMA 3D structures. (B) Optical microscope image of the GelMA "nose". (C) Optical microscope image of the GelMA "ear". (D) Optical microscope image of the GelMA "multichamber". (E) The applied DLP bioprinter. (F) The F-actin and nucleus of the seeded MDA-MB-231s. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60545/60545fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Fabrication of GelMA microfluidic chip with 405 nm crosslinking, HUVECs-seeding, Phalloidin/DAPI imaging." class="xfigimg" src="/files/ftp_upload/60545/60545fig04.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4: GelMA-based microfluidic chip. (A) Fabrication sketch of the GelMA-based microfluidic chip. (B,C) Optical microscope images of the GelMA-based microfluidic chip. (D) Optical microscope image of the seeded HUVECs on the channel wall. (E) The F-actin and nucleus of the seeded HUVECs on the channel wall. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60545/60545fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Bioengineering

18.6K Views.  Zhejiang University. GelMA hydrogel gebaseerde bionica worden erg populair in 3D bioprinting. De lage viscositeit beperkt echter de afdrukbaarheid. Hier delen we strategieën voor het printen van GelMA in ons lab met andere onderzoekers.In deze video maken we volledig gebruik van de speciale eigenschappen van dit biomateriaal en stellen we verschillende printmethoden voor voor verschillende 3D-structuren die kunnen bijdragen aan verdere biomedische toepassingen. Om bijvoorbeeld orgaanschade op te nemen, ku...

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

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 Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area, which are commonly used as carriers for large-scale cell cultures. Microcarrier culture technology has become one of the main techniques in cytological research and is commonly used in the field of large-scale cell expansion. Microcarriers have also been shown to play an increasingly important role in in vitro tissue engineering construction and clinical drug screening. Current methods for preparing microcarriers include microfluidic chips and inkjet printing, which often rely on complex flow channel design, an incompatible two-phase interface, and a fixed nozzle shape. These methods face the challenges of complex nozzle processing, inconvenient nozzle changes, and excessive extrusion forces when applied to multiple bioink. In this study, a 3D printing technique, called alternating viscous-inertial force jetting, was applied to enable the construction of hydrogel microcarriers with a diameter of 100-300 &#181;m. Cells were subsequently seeded on microcarriers to form tissue engineering modules. Compared to existing methods, this method offers a free nozzle tip diameter, flexible nozzle switching, free control of printing parameters, and mild printing conditions for a wide range of bioactive materials.

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

Presented here is a mild 3D printing technique driven by alternating viscous-inertial forces to enable the construction of hydrogel microcarriers. Homemade nozzles offer flexibility, allowing easy replacement for different materials and diameters. Cell binding microcarriers with a diameter of 50-500 &#181;m can be obtained and collected for further culturing.

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area, which are commonly used as carriers for large-scale 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 ...

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 ...

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 ...

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

Samenvatting

Meer video's verkennen