Name: protocols of 3d bioprinting of gelatin methacryloyl hydrogel based bioinks
Uploaded: 2019-12-21T13:00:00
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这项工作由中国国家重点研究发展计划（2018YFA07030000）、国家自然科学基金（No.U1609207，81827804）、国家自然科学创新研究团体科学基金赞助。中国基金会（第51821093号）。

1. 细胞培养
<ol><li>制备Dulbeco的改性鹰培养基（DMEM），辅以10%胎儿牛血清（FBS）和1%青霉素/链霉素，用于培养人类乳腺癌细胞（MDA-MB-231）线和人类脐带静脉内皮细胞（HUVEC）线。</li>
	<li>用L-谷氨酰胺（DMEM/F-12）制备DMEM，辅以10%FBS和1%青霉素/链霉素，用于培养骨髓间质干细胞（BMSC）线。</li>
	<li>将培养环境设置为 37°C 和 5% CO2。培养MDA-MB-231、HUVEC和BMSC，当达到90%汇合时，以1：2的比例通过...

<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>

本文介绍了几种制造GelMA 3D结构的策略，即凝胶微球、凝胶母纤维、凝胶MA复杂结构和基于GelMA的微流体芯片。GelMA具有广阔的生物相容性和形成能力，在生物制造领域有着广泛的应用。微球结构适用于受控药物释放、组织培养和注射到生物体中，以进行进一步治疗21、22、23、24、25。<sup class="xr...

水凝胶被认为是生物制造领域1、2、3、4的合适材料。其中，明胶甲酰胺（GelMA）已成为用途最广的生物材料之一，最初由范登布尔克等人于2000年提出。GelMA由明胶与冰毒氢化物（MA）的直接反应合成。明胶由哺乳动物胶原蛋白水解，由基质金属蛋白酶（MMP）的目标图案组成。因此，GelMA建立的体外三维（3D）组织模型可以理想地模拟细胞与体内细胞外基质（ECM）之间的相互作用。此外，精氨酸-甘氨酸-阿斯巴酸（RGD）序列，在一些其他水凝胶（如藻酸盐）中不存在，留在GelMA的分子链上。这使得它有可能实现在水凝胶网络6内封装的细胞的附着。此外，GelMA的形成能力是有希望的。GelMA分子链上的甲酰胺组在轻度反应条件下与光射器发生反应，在暴露于光照射时形成共价键。因此，打印结构可以快速交联，以简单的方式保持设计的形状。
基于这些特性，一系列领域利用GelMA进行各种应用，如组织工程、基础细胞学分析、药物筛选和生物传感。因此，各种制造策略也已显示7，8，9，10，11，12，13，14。然而，基于GelMA进行3D生物打印仍然具有挑战性，这是由于其基本特性。GelMA 是一种温度敏感材料。在印刷过程中，必须严格控制印刷环境的温度，以保持生物墨水的物理状态。此外，GelMA的粘度一般低于其他常见的水凝胶（即藻酸盐、甲酸、透明质酸等）。然而，在用这种材料建造3D架构时，还面临着其他障碍。
本文总结了我们实验室提出的GelMA3D生物打印的几种方法，并描述了印刷样品（即凝胶微球、GelMA纤维、GelMA复杂结构和基于GelMA的微流体芯片的合成）。每种方法都有专门的功能，可以在不同情况下采用，有不同的要求。GelMA 微球由电辅助模块生成，形成额外的外部电力以缩小液滴大小。就GelMA纤维而言，它们由同轴生物印刷喷嘴挤出，借助粘性藻酸钠。此外，通过数字光处理 （DLP） 生物打印机实现复杂 3D 结构的建立。最后，提出了两次交联策略，将凝胶水凝胶与传统微流体芯片相结合，构建基于GelMA的微流体芯片。据认为，该协议是我们实验室使用的GelMA生物打印策略的重要总结，并可能激励相关领域的其他研究人员。

<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

明胶甲酰胺（GelMA）已成为生物印刷领域流行的生物材料。这种材料的衍生物是明胶，它是从哺乳动物胶原蛋白水解。因此，基质金属蛋白酶（MMP）的精氨酸-甘氨酸-阿斯巴酸（RGD）序列和目标图案留在分子链上，有助于实现细胞附着和降解。此外，GelMA 的地层特性是通用的。甲酰胺组允许材料在光射光下在光开射器的存在下迅速交联。因此，建立合适的方法，用这种有前途的材料合成三维（3D）结构是十分有意义的。然而，其低粘度限制了GelMA的可打印性。这里介绍了对GelMA水凝胶进行3D生物打印的方法，即制造凝胶微球、凝胶纤维、GelMA复杂结构和基于GelMA的微流体芯片。讨论了材料的结构和生物相容性以及印刷方法。据信，该协议可作为以前应用的生物材料和GelMA之间的桥梁，并有助于建立基于GelMA的生物医学应用3D架构。

在制造GelMA微球过程中，凝胶电滴被外部电场力分离。当液滴落入接收硅油时，它们保持标准球形形状，没有尾巴。这是因为GelMA液滴处于水相，而硅油处于油相。两个阶段之间形成的表面张力导致GelMA液滴保持标准球形形状。就电池载载的微球而言，细胞在这个过程中经历了高压电场力。从染色MDA-MB-231的形态（图1B+E）中发现，封装的MDA-MB-231保持其扩散能力，?...

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

这里介绍的是明胶甲酰胺的3D生物打印方法。

明胶美沙克里洛醇水凝胶生物油墨3D生物印刷方案

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...

基于凝胶水凝胶的仿生学在3D生物印刷中非常流行。然而，低粘度限制了其可打印性。在这里，我们与其他研究人员分享在实验室中打印 GelMA 的策略。在这段视频中，我们充分利用了这种生物材料的特殊特性，并提出了几种用于不同3D结构的打印方法，这些方法有助于进一步的生物医药应用。以器官损伤为例，通过注射嵌入或追踪印刷的GelMA结构，有可能用于相应的治疗。在短时间内完全溶解冻干的凝胶是不容易的。为了加快溶解过程，可以使用涡流混合器。每个生物材料将使用凝胶MA的特殊属性。我建议一个新的化学器应该正确地采取什么是条件关键在打印时徘徊。操作详细信息确定打印过程是否成功。这就是为什么我们选择一个视觉演示与其他研究人员分享。制造微球首先将两个金属环电极与接地极和正极连接。将金属板与环电极下方的高压连接，将带硅油的培养盘作为液滴接收器放在金属板上。通过溶解冷冻干燥的GelMA和 LAP和DPBS过滤它通过0.22微米过滤器进行无菌，然后在37摄氏度的水浴中加热15分钟，准备生物墨水。分离MDA-MB-231细胞与三毫升0.25%的三普辛和0.02%的EDTA溶液在37摄氏度下三分钟。将细胞转移到15毫升管中，并在100次G下离心5分钟。去除上流水，通过缓慢上下移液，将细胞与一毫升准备好的生物墨水混合，确保避免气泡。将一毫升的生物墨水和细胞混合物吸进三毫升无菌注射器中。用压缩空气的力量喂给生物墨水，然后将注射器放在夹具上。打开高压电源，将电压设置为零至四千伏。同时打开 405 纳米波长的光，将 GelMA 液滴在五毫升硅油中交叉连接。去掉培养皿以去除大部分硅油，用勺子将剩余的油和微球转移到15毫升的离心管中。加入五毫升的 DPBS，以 100 倍 G 摇动混合物离心管，然后取出上盖。将微球转移到带DMEM的培养皿中，并在37摄氏度和5%的二氧化碳下培养它们，为期三天。三天后丢弃介质，用 DPBS 清洗微球。在室温下用两毫升 4% PFA 的毫升将其修复 30 分钟，然后丢弃 PFA 并重复洗涤。在室温下用两毫升0.5%的非离子表面活性剂对细胞进行渗透，5分钟，丢弃表面活性剂，用DPBS清洗微球。接下来用TRITC-phalloidin将细胞染色30分钟，然后DAPI在黑暗中染色10分钟，每次染色后丢弃染料，用DPBS清洗球体。使用共和荧光显微镜成像微球。以2%的重量和体积比在脱离子水中溶解灭菌的藻酸钠粉末。准备无菌生物墨水溶液，如前所述，然后在37摄氏度的水浴中加热生物墨水和藻酸钠15分钟。根据前面描述的MDA-MB 231细胞程序，对bMSC进行试穿和离心，然后去除上流液，然后用2毫升的GelMA生物墨水重新泵化细胞颗粒。将两毫升的生物墨水细胞混合物吸进10毫升注射器和两毫升藻酸钠混合物中，放入另一个注射器中。以每分钟 50 微米的生物墨水和每分钟 350 微米的生物墨水为解决方案提供解决方案。打开 405 纳米波长的光，照射透明管，并交叉连接 GelMA 光纤。使用培养皿接收纤维，然后用勺子收集。将纤维转移到带DMEM/F12的培养皿中，并在37摄氏度和5%的二氧化碳下培养三天。培养后，细胞遵循前面描述的在共和荧光显微镜下观察纤维的方向。溶解冷冻干燥的凝胶和 LAP 和 DPBS，并在溶液中加入洋红色食用颜料，以提高打印精度。通过0.22微米过滤器过滤溶液，使无菌，并在37摄氏度的水浴中加热15分钟。使用 CAD 软件构建 3D 模型，将模型文档导入应用 DLP 生物打印机的上部软件。在生物打印机的槽中加入十毫升的准备好的生物墨水。根据手稿方向设置打印参数，然后开始打印。完成后，从生物打印机上取下打印结构，并将其浸入培养皿中的 DPBS 中。分离和颗粒MDA-MB 231细胞，如前所述。用两毫升 DMEM 重新悬浮，并将悬浮液添加到打印结构中。培养37摄氏度的细胞和5%的二氧化碳三天，然后准备结构观察与共理荧光显微镜，如前面所述。准备 10% 的重量到体积 GelMA 和 0.5% 的重量到体积 LAP 解决方案，然后通过 0.22 微米过滤器进行过滤。在紫外光下消毒明胶粉 30 分钟，以 5% 的重量与体积比将其添加到 GelMA LAP 溶液中。在37摄氏度的水浴中加热混合物15分钟。用准备好的生物墨水填充模具，并把它们放在4摄氏度的冰箱中30分钟。使用刀片从模具中去除部分交对水凝胶片。将 2D 模制水凝胶片混合，在 405 纳米照射下照射一分钟，将其与 GelMA 粘合。分离和颗粒 HUVEC 将细胞与两毫升 DMEM 混合，然后用喷嘴和注射器将悬浮液注入微通道。接下来的三个小时，每 15 分钟翻转一次芯片，以实现均匀和完整的电池座椅。在37摄氏度和5%的二氧化碳下培养皿中的碎屑，然后准备进行形态学观察。当GelMA液滴落入接收硅油中时，它们保持了标准的球形形状，没有尾巴。经历高压电场力的封装电池保持了其扩散能力，验证了电辅助制造方法的生物相容性。选择DLP生物打印机制造具有更复杂的形状的GelMA结构，如鼻耳和多腔室。种子HUVEC附着在GelMA材料上，并扩散在交链接的GelMA结构的表面，表明此应用具有组织工程的潜力。两次交叉链接策略用于制造基于凝胶MA的微流体芯片。各种微通道的芯片是按需设计不同模具的，HUVEC在通道中播种，并连接到通道壁上，形成宏体形状。制造微球时应用高压源。研究人员必须检查电路之前，不要触摸暴露的毫巴保护运营商或使细胞免受短路损坏。超声波水基可能是另一种选择，以加快值得的速度。这一贡献肯定总结了我们实验室中GelMA的3D生物打印方法。研究人员可以参考视频，改进或扩展打印策略，以满足进一步的生物医学需求。

Research

JoVE Journal

Bioengineering

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

生物印刷细胞化构造使用组织特异性水凝胶生物油墨

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

科研

生物工程

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

使用3D生物印刷创建心脏组织展示球体的机械整合

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

Bioink Precellularization 无源混合装置打印软骨及皮肤组织类似物的应用

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

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

Bioprintable 藻酸盐/明胶水凝胶 3D体外模型诱导细胞球形形成

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

用于工程神经样组织的小鼠皮质星形胶质细胞的3D生物打印

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

用于外周神经工程的石墨烯基3D生物杂化水凝胶生物墨水的制备与表征

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

基于外泌体的多巴胺载体系统的配方和表征

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

强粘性水凝胶明胶O-亚硝基苯甲醛的合成

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

明胶甲基丙烯酰基颗粒水凝胶支架：高通量微凝胶制造、冻干、化学组装和 3D 生物打印

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

Understanding Chronic Lung Diseases Using 3D Printed Phototunable Hydrogels (Video) | JoVE

3D生物打印光可调水凝胶研究成纤维细胞活化

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