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
Summary
在这里，我们报道了一种3D生物打印小鼠皮质星形胶质细胞的方法，用于生物制造神经样组织，以研究星形胶质细胞在中枢神经系统中的功能以及涉及神经胶质细胞在神经系统疾病和治疗中的机制。
Abstract
星形胶质细胞是神经胶质细胞，在中枢神经系统（CNS）中起着至关重要的作用，包括神经元的支持和功能。这些细胞也对神经损伤有反应，并保护组织免受退行性事件的影响。星形胶质细胞功能的 体外 研究对于阐明此类事件所涉及的机制并有助于开发治疗神经系统疾病的疗法非常重要。该协议描述了一种通过3D生物打印含有星形胶质细胞的生物墨水生物制造富含星形胶质细胞的神经样组织结构的方法。在这项工作中使用了基于挤出的3D生物打印机，并从C57Bl / 6小鼠幼崽的脑皮质层中提取星形胶质细胞。通过将皮质星形胶质细胞从第3代混合到由明胶，明胶 – 甲基丙烯酰（GelMA）和纤维蛋白原组成的生物材料溶液中，并补充层粘连蛋白来制备生物墨水，其提供了最佳的生物打印条件。3D生物打印条件最大限度地减少了细胞应激，有助于星形胶质细胞在此过程中的高活力，其中74.08%±1.33%的细胞在生物打印后立即存活。孵育1周后，星形胶质细胞的活力显着增加到83.54%±3.00%，表明3D构建体代表了适合细胞生长的微环境。生物材料组成允许细胞附着并刺激星形细胞行为，细胞表达特定的星形胶质细胞标志物神经胶质纤维酸性蛋白（GFAP）并具有典型的星形细胞形态。这种可重复的方案提供了一种有价值的方法来生物制造富含星形胶质细胞的3D神经样组织，类似于细胞的天然微环境，对于旨在了解星形胶质细胞的功能及其与神经系统疾病中涉及的机制的关系的研究人员很有用。
Introduction
星形胶质细胞是中枢神经系统（CNS）中最丰富的细胞类型，在大脑稳态中起着关键作用。除了持久的神经元支持外，星形胶质细胞还负责调节神经递质摄取，维持血脑屏障完整性，并调节神经元突触发生1，2。星形胶质细胞在中枢神经系统炎症中也具有重要作用，在导致星体反应性或反应性星形胶质细胞增多症3，4的过程中对大脑损伤作出反应，形成神经胶质疤痕，阻止健康组织暴露于退行性因子5。该事件导致星形胶质细胞的基因表达，形态和功能的变化6，7。因此，涉及星形胶质细胞功能的研究有助于开发治疗神经系统疾病的疗法。
体外 模型对于研究与神经损伤相关的机制至关重要，尽管已经建立了成功的皮质星形胶质细胞的分离和二维（2D）培养8，但该模型未能提供模拟天然细胞行为的现实环境并重现大脑的复杂性9.在2D条件下，较差的机械和生化支持，低细胞 – 细胞和细胞 – 基质相互作用以及细胞扁平化导致基底 – 顶端极性缺失，影响细胞信号传导动力学和实验结果，导致细胞形态和基因表达改变，从而损害对治疗的反应10。因此，开发提供更逼真的神经环境的替代方案至关重要，旨在将结果转化为临床。
三维（3D）细胞培养代表了一种更先进的模型，该模型概括了器官和组织（包括CNS11）的保真度增加的特征。关于神经胶质培养，3D模型有助于维持星形胶质细胞形态，细胞基底 – 顶端极性和细胞信号传导12，13。3D生物打印技术成为一种强大的工具，通过使用细胞和生物材料重建天然组织的结构和性质，以受控的方式生物制造3D活组织。该技术的使用导致了结果预测的实质性改进，并有助于再生医学应用于CNS14，15，16。
这里描述的方案详细介绍了皮质星形胶质细胞的分离和培养。该协议还详细介绍了一种可重复的方法，用于生物打印嵌入明胶/明胶甲基丙烯酰（GelMA）/纤维蛋白原中的星形胶质细胞，并补充层粘连蛋白。在这项工作中，使用基于挤出的生物打印机以1×106个细胞/ mL的密度打印含有皮质星形胶质细胞的生物材料组合物。通过控制打印速度将生物打印剪切应力降至最低，并且星形胶质细胞在该过程后显示出高活力。生物打印构建体培养1周，星形胶质细胞能够在水凝胶内扩散，附着和存活，维持星形细胞形态并表达特异性标志物胶质纤维酸性蛋白（GFAP）4 。
该程序与活塞驱动的基于挤出的生物打印机兼容，可用于生物打印来自不同来源的星形胶质细胞。这里提出的3D生物打印模型适用于广泛的神经工程应用，例如研究健康组织中星形胶质细胞功能所涉及的机制以及了解神经病理学的进展和治疗发展。
Protocol
所有涉及动物的程序都遵循国际动物在研究中使用的指导方针（http://www.iclas.org），并得到圣保罗联邦大学研究伦理委员会（CEUA 2019 / 9292090519）的批准。 1. 小鼠脑部解剖 将 10 mL 冷汉克斯缓冲盐溶液 （HBSS） 转移到 100 mm 培养皿中，将 1 mL 转移到 1.5 mL 微管中。每只动物准备一个微管。注意：培养皿和微管都需要保持在冰上。 使用DMEM F12 + 10%胎牛血清（FBS）?…
Representative Results
这项工作旨在使用3D生物打印技术开发一种神经样组织，以逐层沉积含有原代星形胶质细胞的明胶/ GelMA / 纤维蛋白原生物墨水。从小鼠幼崽的大脑皮层中提取和分离星形胶质细胞（图1），添加到生物材料组合物中，允许生物制造活的3D构建体。 计算机辅助设计（CAD）是使用G代码（补充文件）作为方形（0.6 x 0.6毫米）的互连框架开发的，孔?…
Discussion
3D生物打印技术已经成为一种生物制造替代方案，可以设计出在结构和生理上类似于天然组织22的精制结构，包括大脑23。神经样组织的生物制造允许在体外进行天然微环境建模，是了解与影响CNS11的许多疾病的发展和治疗相关的细胞和分子机制的重要工具。由于神经胶质细胞在神经功能中的重要作用，皮质星形胶质细胞已被用于许多研?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了圣保罗研究基金会（FAPESP）的支持，资助号为2018/23039-3和2018/12605-8;国家科学技术发展委员会（CNPq），批准号465656/2014-5和309679/2018-4;和高等教育人员改进协调（CAPES），财务代码001。
Materials
| 3D Bioprinter | 3D Biotechnology Solutions | Extrusion-based bioprinter | |
| Blunt-tip forceps | Integra Miltex | 6–30 | Forceps for brain dissection previously sterilized |
| Bovine serum albumin | Sigma-Aldrich | 9048-46-8 | Protease free, fatty acid free, essentially globulin free |
| CaCl2 | Sigma-Aldrich | 10043-52-4 | |
| Cell culture flask | Fisher Scientific | 156340 | Culture flask T25 |
| Cell strainer | Corning Incorporated | 352340 | Cell strainer 40 µm |
| Confocal microscope | Leica | Confocal TCS SP8 microscopy coupled with an Olympus FluoView 300 confocal system | |
| Conical tubes | Thermo Scientific | 339651, 339652 | Sterile tubes of 15 mL and 50 mL |
| DAPI | Abcam | ab224589 | DAPI staining solution |
| DMEM/F12 | Gibco; Life Technologies Corporation | 12500062 | DMEM/F-12 50/50, 1X (Dulbecco's Mod. Of Eagle's Medium/Ham's F12 50/50 Mix) with L-glutamine |
| Dyalisis tubing | Sigma-Aldrich | D9527 | Molecular weight cut-off = 14 kDa |
| Ethanol | Fisher Scientific | 64-15-5 | Reagent grade |
| Fetal Bovine Serum | Gibco; Life Technologies Corporation | 12657011 | Research Grade |
| Fibrinogen | Sigma-Aldrich | 9001-32-5 | Fibrinogen cristalline powder from bovine plasma |
| Gelatin | Sigma-Aldrich | 9000-70-8 | Gelatin powder from porcine skin |
| Glycine | Sigma-Aldrich | 56-40-6 | Glycine powder |
| Hanks Buffered Salt Solution (HBSS) | Gibco; Life Technologies Corporation | 14175095 | No calcium, no magnesium, no phenol red |
| L-Glutamine | Sigma-Aldrich | 56-85-9 | L-Glutamine crystalline powder |
| Laminin | Sigma-Aldrich | 114956-81-9 | Laminin 1-2 mg/mL L in 50 mM Tris-HCl |
| Live dead kit cell imaging kit | Thermo Scientific | R37601 | Green fluorescence in live cells (ex/em 488 nm/515 nm). Red fluorescence in dead cells (ex/em 570 nm/602 nm) |
| Methacrylic anhydride | Sigma-Aldrich | 760-93-0 | For GelMA preparation |
| Microtubes | Corning Incorporated | MCT-150-C | Microtubes of 1,5 mL |
| NaCl | Sigma-Aldrich | 7647-14-5 | |
| Needle 22G | Fisher Scientific | NC1362045 | Sterile blunt needle |
| Operating scissor | Integra Miltex | 05–02 | Sharp scissor for brain dissection previously sterilized |
| Paraformaldehyde | Sigma-Aldrich | 30525-89-4 | Paraformaldehyde powder |
| Penicillin/Streptomycin | Gibco; Life Technologies Corporation | 15070063 | Pen Strep (5,000 Units/ mL Penicillin; 5,000 ug/mL Streptomycin) |
| Petri dish | Corning Incorporated | 430591, 430588 | Sterile petri dishes of 35 and 100 mm |
| Phalloidin | Abcam | ab176753 | iFluor 488 reagent |
| Photoinitiator | Sigma-Aldrich | 106797-53-9 | 2-Hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone |
| Phosphate buffer saline (PBS) | Gibco; Life Technologies Corporation | 10010023 | PBS 1 x, culture grade, no calcium, no magnesium |
| Poly-L-lysine | Sigma-Aldrich | 25988-63-0 | Poly-L-lysine hydrobromide mol wt 30,000-70,000 |
| Primary antobody | Abcam | ab4674 | Chicken polyclonal to GFAP |
| Secondary antibody | Abcam | ab150176 | Alexa fluor 594 anti-chicken |
| Spatula | Miltex | V973-70 | Number 24 cement spatula previously sterilized |
| Stereomicroscope | Fisherbrand | 3000038 | Microscope for brain dissection |
| Syringe 5 mL | BD | 1222C84 | Sterile syringe |
| Syringe filter 2 µm | Fisher Scientific | 09-740-105 | Polypropylene filter for sterilization |
| Thrombin | Sigma-Aldrich | 9002–04-4 | Thrombin cristalline powder from bovine plasma |
| Triton X-100 | Sigma-Aldrich | 9002-93-1 | Laboratory grade |
| Trypsin-EDTA | Gibco; Life Technologies Corporation | 15400054 | Trypsin no phenol red 1 x diluted in PBS |
| Versene solution | Gibco; Life Technologies Corporation | 15040066 | Versene Solution (0.48 mM) formulated as 0.2 g EDTA(Na4) per liter of PBS |
| Well plate | Thermo Scientific | 144530 | Sterile 24-well plate |
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Cite This Article
de Melo, B. A. G., Cruz, E. M., Ribeiro, T. N., Mundim, M. V., Porcionatto, M. A. 3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue. J. Vis. Exp. (173), e62691, doi:10.3791/62691 (2021).