来自微凝胶棒的互连大孔3D支架
Summary
具有互补反应基团的微凝胶棒通过微流体生产，具有在水溶液中相互连接的能力。与基于球形的系统相比，各向测量微凝胶可卡住并相互连接成具有更大孔的稳定结构。用GRGDS-PC修饰的微凝胶形成可用于细胞培养的大孔3D构建体。
Abstract
来自微流体的双组分功能化微凝胶系统允许在水溶液中快速互连成3D大孔结构，而无需进一步的添加剂。连续的光引发片上凝胶可以改变微凝胶纵横比，这决定了所获得的构建体的构建块特性。甲基丙烯酸缩水甘油酯（GMA）或甲基丙烯酸2-氨基乙酯（AMA）单体被共聚合到基于聚乙二醇（PEG）星形聚合物的微凝胶网络中，以实现环氧或胺官能团。将聚焦油流引入微流体出口结构，以确保功能化微凝胶棒的连续收集。根据最近的出版物，基于微凝胶棒的构建体会产生数百微米的更大孔隙，同时与基于球形的模型相比，导致整体更高的支架稳定性。通过这种方式，可以生产具有更多自由体积的大批量结构，同时减少所需的材料量。相互连接的大孔支架可以拾取和运输，而不会损坏或分解。不参与互连的胺和环氧基团保持活性，可以独立用于后改性。该协议描述了一种优化的方法，用于制造微凝胶棒以形成可用于后续细胞实验的大孔互连支架。
Introduction
为了研究 3D 构建体中复杂的协作细胞行为，支架平台需要在可重复性方面表现出一致的性能，具有适合细胞迁移的几何形状，同时在参数改变方面具有一定的灵活性，以研究它们对活组织的影响1。近年来，由Segura等人首次描述的大孔退火颗粒（MAP）概念发展成为3D脚手架生产的高效多功能平台2。微凝胶的定制组成是最终3D支架的构建块，预定义了诸如结构的刚度，凝胶网络的选择性化学反应性以及支架的最终孔径2，3，4，5，6等特性。细胞粘附肽作为支架-细胞相互作用的线索被掺入微凝胶的聚合物网络中以允许细胞附着，并且可以改变以研究它们对培养细胞的特异性影响。3D支架通过共价或超分子键使退火的可注射微凝胶相互连接而稳定，从而为细胞培养2，3，5，7，8提供稳健且明确的构建体。
微流体已成为制备确定的颗粒水凝胶的最准确和适应性最强的方法之一9。在连续过程中生产大量所需构建块的可能性，同时保持其化学、机械和物理单分散性，大大有助于该工艺的适用性。此外，生产的微凝胶的大小和形状可以通过各种方法进行操作，例如批量乳液，微流体，光刻，电动喷涂或机械碎裂，这些方法决定了构建块的几何形状，从而决定了最终支架的3D结构1，10。
最近，已经报道了由功能化微凝胶棒组成的大孔3D支架的概念，该支架在水溶液中快速互连而无需进一步的添加剂11。与在本研究中使用球形微凝胶相比，微凝胶棒的各向异性导致更高的孔隙率和孔分布以及更大的孔径11。通过这种方式，更少的材料会产生具有各种不同孔隙几何形状的较大孔隙，同时保持 3D 脚手架的稳定性。该系统由两种类型的微凝胶棒组成，具有互补的伯胺和环氧官能团，当它们相互接触时，它们在相互连接的反应中消耗。不参与互连过程的官能团保持活性，可用于细胞粘附肽或其他生物活性因子的选择性后修饰。成纤维细胞在3D支架内培养时附着，扩散和增殖，首先在微凝胶表面上生长，并在5天后填充大部分大孔。人成纤维细胞和人脐静脉内皮细胞（HUVECs）的初步共培养研究表明，在相互连接的3D支架11内形成血管样结构的结果很有希望。
Protocol
Representative Results
Discussion
该方案中的关键步骤之一是用作伯胺官能化的共聚单体的2-氨基乙基甲基丙烯酸酯（AMA）的质量。AMA应该是细粒的，最好是无色的粉末，装在气密的棕色玻璃容器中。应避免使用绿色和块状材料，因为它会显着损害凝胶反应并对结果的可重复性产生负面影响。如果凝胶化不良和微凝胶棒不稳定，可以考虑更换供应商。
如果胺和环氧微凝胶棒的混合导致多个相互连接的团簇而不…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们感谢我们以前工作的共同作者，这种方法是基于Céline Bastard，Luis P. B. Guerzoni，Yonca Kittel，Rostislav Vinokur，Nikolai Born和Tamás Haraszti。我们非常感谢德国研究协会（DFG）在B5和C3 SFB 985“功能性微凝胶和微凝胶系统”项目中的资助。我们感谢莱布尼茨参议院竞争委员会（SAW）在Professorinnenprogramm（SAW-2017-PB62：BioMat）下提供的资金。我们衷心感谢欧盟委员会（EUSMI，731019）的资助。这项工作部分是在化学聚合物技术中心（CPT）进行的，该中心得到了欧盟和北莱茵-威斯特法伦州联邦州的支持（授予EFRE 30 00 883 02）。
Materials
| ABIL EM 90 | Evonik | 144243-53-8 | non-ionic surfactant |
| 2-Aminoethyl methacrylate hydrochloride | TCI Chemicals | A3413 | >98.0%(T)(HPLC) |
| 8-Arm PEG-acrylate 20 kDa | Biochempeg Scientific Inc. | A88009-20K | ≥ 95 % |
| AutoCAD 2019 | Autodesk | computer-aided design (CAD) software; modeling of microfluidic designs | |
| CHROMAFIL MV A-20/25 syringe filter | XH49.1 | pore size 0.20 µm; Cellulose Mixed Esters (MV) | |
| Cover glass | Marienfeld-Superior | type No. 1 | |
| EMS Swiss line core sampling tool 0.75 mm | Electron Microscopy Sciences | 0.77 mm inner diameter, 1.07 mm outer diameter | |
| Ethanol absolut | VWR Chemicals | ||
| FL3-U3-13Y3M 150 FPS series high-speed camera | FLIR Systems | ||
| Fluoresceinamine isomer I | Sigma-Aldrich | 201626 | |
| Fluorescein isothiocyanate | Thermo Fisher Scientific | 46424 | |
| 25G x 5/8’’ 0,50 x 16 mm needles | BD Microlance 3 | ||
| Glycidyl methacrylate | Sigma-Aldrich | 779342 | ≥97.0% (GC) |
| GRGDS-PC | CPC Scientific | FIBN-015A | |
| Hamilton 1000 Series Gastight syringes | Thermo Fisher Scientific | 10772361/10500052 | PFTE Luer-Lock |
| Hexane | Sigma-Aldrich | 1,04,367 | |
| Lithium phenyl-2,4,6-trimethylbenzoylphosphinate | Sigma-Aldrich | 900889 | ≥95 % |
| Motic AE2000 trinocular microscope | Ted Pella, Inc. | 22443-12 | |
| Novec 7100 | Sigma-Aldrich | SHH0002 | |
| Oil Red O | Sigma-Aldrich | O9755 | |
| Paraffin | VWR Chemicals | 24679320 | |
| Pavone Nanoindenter Platform | Optics11Life | ||
| Phosphate buffered saline | Thermo Fisher Scientific | AM9624 | |
| Polyethylene Tubing 0.38×1.09mm medical grade | dropletex | ID 0.38 mm OD 1.09 mm | |
| 2-Propanol | Sigma-Aldrich | 190764 | ACS reagent, ≥99.5% |
| Protein LoBind Tubes | Eppendorf | 30108132 | |
| Pump 11 Pico Plus Elite Programmable Syringe Pump | Harvard Apparatus | ||
| RPMI 1640 medium | Gibco | 11530586 | |
| SYLGARD 184 silicone elastomer kit | Dow SYLGARD | 634165S | |
| Trichloro-(1H,1H,2H,2H-perfluoroctyl)-silane | Sigma-Aldrich | 448931 | |
| UVC LED sterilizing box | UVLED Optical Technology Co., Ltd. | 9S SZH8-S2 |
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