明胶美沙克里洛醇水凝胶生物油墨3D生物印刷方案
1State Key Laboratory of Fluid Power and Mechatronic Systems, School of Mechanical Engineering,Zhejiang University, 2Key Laboratory of 3D Printing Process and Equipment of Zhejiang Province, School of Mechanical Engineering,Zhejiang University, 3School of Mechanics and Safety Engineering,Zhengzhou University
Summary
这里介绍的是明胶甲酰胺的3D生物打印方法。
Abstract
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。
Introduction
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。
Protocol
1. 细胞培养 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。 用L-谷氨酰胺（DMEM/F-12）制备DMEM，辅以10S和1%青霉素/链霉素，用于培养骨髓间质干细胞（BMSC）线。 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…
Representative Results
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，?…
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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）。
Materials
| 0.22 μm filter membrane | Millipore | ||
| 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) | Yeasen Biological Technology Co., Ltd., Shanghai, China | ||
| 3D bioprinter | SuZhou Intelligent Manufacturing Research Institute, SuZhou, China | ||
| 405nm wavelength light | SuZhou Intelligent Manufacturing Research Institute, SuZhou, China | ||
| co-axial nozzle | SuZhou Intelligent Manufacturing Research Institute, SuZhou, China | ||
| confocal fluorescence microscope | OLYMPUS FV3000 | ||
| digital light processing (DLP) bioprinter | SuZhou Intelligent Manufacturing Research Institute, SuZhou, China | ||
| DLP printer | SuZhou Intelligent Manufacturing Research Institute, SuZhou, China | ||
| Dulbecco's Phosphate Buffered Saline (DPBS) | Tangpu Biological Technology Co., Ltd., Hangzhou, China | ||
| Dulbecco's Modified Eagle Medium (DMEM) | Tangpu Biological Technology Co., Ltd., Hangzhou, China | ||
| Dulbecco's Modified Eagle Medium with L-glutamine (DMEM/F-12) | Tangpu Biological Technology Co., Ltd., Hangzhou, China | ||
| EFL Software | SuZhou Intelligent Manufacturing Research Institute, SuZhou, China | ||
| fetal bovine serum (FBS) | Tangpu Biological Technology Co., Ltd., Hangzhou, China | ||
| gelatin | Sigma-Aldrich, Shanghai, China | ||
| gelatin methacryloyl (GelMA) | SuZhou Intelligent Manufacturing Research Institute, SuZhou, China | ||
| high voltage power | SuZhou Intelligent Manufacturing Research Institute, SuZhou, China | ||
| lithium phenyl-2, 4, 6-trimethylbenzoylphosphinate (LAP) | SuZhou Intelligent Manufacturing Research Institute, SuZhou, China | ||
| paraformaldehyde | Tangpu Biological Technology Co., Ltd., Hangzhou, China | ||
| penicillin/streptomycin | Tangpu Biological Technology Co., Ltd., Hangzhou, China | ||
| sodium alginate (Na-Alg) | Sigma-Aldrich, Shanghai, China | ||
| TRITC phalloidin | Yeasen Biological Technology Co., Ltd., Shanghai, China | ||
| Triton X-100 | Solarbio Co., Ltd., Shanghai, China |
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Cite This Article
Xie, M., Yu, K., Sun, Y., Shao, L., Nie, J., Gao, Q., Qiu, J., Fu, J., Chen, Z., He, Y. Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks. J. Vis. Exp. (154), e60545, doi:10.3791/60545 (2019).