生成基于3-D胶原蛋白的水凝胶,分析神经系统发育过程中的Axonal生长和行为
1Molecular and Cellular Neurobiotechnology, Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology (BIST),Parc Científic de Barcelona, 2Department of Cell Biology, Physiology, and Immunology,Universitat de Barcelona, 3Center for Networked Biomedical Research on Neurodegenerative Diseases (CIBERNED), 4Institute of Neuroscience,University of Barcelona
Summary
在这里,我们提供了一个方法,用于分析在3D矩阵中生长斧子的行为,模仿它们的自然发育。
Abstract
该协议使用天然I型胶原蛋白生成三维(3-D)水凝胶,用于监测和分析斧状生长。该方案的核心是在大鼠尾巴肌腱衍生的I型胶原蛋白中形成的3D水凝胶内培养小块胚胎或产后啮齿动物大脑,具有特定的孔隙度。组织片在水凝胶内培养,并面对特定的脑碎片或转基因细胞聚集物,以产生和分泌适合在多孔基质内形成梯度的分子。该协议的步骤简单且可重现,但包含开发过程中要仔细考虑的关键步骤。此外,在免疫细胞化学方法固定后,可直接使用相对比显微镜或单光/多光子荧光显微镜监测和分析生长的斧子的行为。
Introduction
神经性斧头,以斧头生长锥结束,通过胚胎的细胞外基质(ECM)在特定的路径上迁移长距离,以达到适当的目标。生长锥体是斧子的远端部分,它专门用于感知细胞1,2的物理和分子环境。从分子的角度来看,生长锥体至少受四种不同的分子机制的引导:接触吸引、化学吸引、接触排斥和由不同斧突引导线索触发的切口3,4,5,6.接触介导过程可在微图案基板上的二维(2D)培养物中进行部分监测(例如,带条纹7、8或点9含有分子)。然而,斧子可以通过从环境中的引导柱细胞中感应到几个有吸引力的和令人厌恶的分子,以非扩散的方式导航到它们的目标。在这里,我们描述了一种简单的三维培养方法,以检查分泌分子是否对开发斧子产生化学吸引力或化学吸引力。
最早的研究旨在确定在三维(3-D)矩阵中使用外植培养物的斧子引导线索的影响,以生成模拟体内条件11、12的梯度。这种方法,连同体内实验,允许识别四个主要的家族的指导线索:内特林,斯利茨,塞马波林,和Ephrins4,5,6。这些分子线索和其他因素13被生长的斧突整合,触发粘附复合物的动态,并通过细胞骨架14、15、16传递机械力。为了在三维培养物中产生分子梯度,用于异形导航,先驱研究人员使用等离子凝固基质17,也用于有机切片制剂18。然而,在1958年,一个新的协议,以产生3D胶原水凝胶报告用于研究与Maximow的设备19,一个培养平台,用于几个研究适合微观观察20。另一项先驱研究报告说,胶原蛋白凝胶作为一种工具,将人类成纤维细胞嵌入到伤口愈合过程中的肌纤维细胞中。同时,Lumsden和Davies应用牛皮毛蛋白的胶原蛋白,分析神经生长因子(NGF)对感觉神经纤维22的引导作用。随着不同公司和实验室开发新的培养平台(例如多孔板),胶原蛋白培养物被适应这些新设备6、23、24、25 ,26.同时,从恩格尔布雷斯-霍尔姆-肖姆-肖姆肿瘤细胞系中提取的ECM物质的提取物也用于商业用途,以扩大这些研究27。
最近,已经开发出几种协议,利用三维水凝胶(例如胶原蛋白、纤维蛋白等),在斧子指导中产生具有假定作用的分子梯度。28.或者,候选分子可以在多孔基质(例如NGF29)中以不同浓度固定,或通过在分泌分子的3-D水凝胶细胞聚集体的小区域内培养产生,从而产生径向梯度4,23,24,25,26 。最后一种可能性将在本协议中解释。
这里介绍的程序是一种简单、快速和高度可重复的方法,基于对胚胎小鼠大脑三维水凝胶培养物的斧头生长的分析。与其他方法相比,该协议非常适合未经培训的研究人员,可在短期培训(1-2 周)后完全开发。在此协议中,我们首先从成年大鼠尾部分离胶原蛋白,以进一步生成三维基质,其中转基因细胞聚集物在胚胎神经元组织前培养。这些细胞聚集体形成候选分子的径向化学梯度,从而引起对生长的斧子的反应。最后,使用相对比显微镜或免疫细胞化学方法,可以轻松评估分子对生长的斧子的影响。
Protocol
所有动物实验均根据巴塞罗那大学动物实验伦理委员会(CEEA)的指导方针和规程进行,该研究中的啮齿动物使用协议由巴塞罗那大学欧洲动物实验中心审查和批准。巴塞罗那大学(CEEA批准#276/16和141/15)。 1. 大鼠尾部胶原蛋白的纯化 收集成年斯普拉格-道利大鼠尾巴(8-9周大)后牺牲动物遵循伦理准则,并在95%乙醇冲洗。将 2-4 尾鱼放在冰上 (4 °C),并在过程中将其覆盖在冰上。</li…
Representative Results
在这里,我们提出了一个广泛可访问的方法,研究胚胎小鼠神经系统的3D水凝胶胶原蛋白培养物的斧头生长。为此,我们从成年大鼠尾部分离出胶原蛋白,以生成三维基质,其中我们培养的转基因细胞聚集体,表达Netrin-1或Sema3E,与胚胎神经元组织(例如,海马的CA区域)对抗。这些细胞聚集体在胶原蛋白基质内形成候选分子的径向分布梯度。最后,为了评估神经元对不同分子的反应,我们使用免疫细胞化学方法(例…
Discussion
发育的斧子的生长主要是侵入性的,包括ECM降解和重塑。利用这里介绍的程序,研究人员可以获得由自然型I胶原蛋白形成的均质三维基质,其中斧子(或细胞)可以响应转基因细胞在体内分泌的化学梯度。对吸引或抑制性线索(蛋白质、脂质等)梯度的不同斧状反应可以很容易地与特定控制(模拟转染细胞)进行比较。作为优点,我们必须提到,肌腱很容易分离,事实上,它们可能是动物实验的残余物。此外,肌腱?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
作者感谢汤姆·约汉南的编辑建议和M.塞古拉-费柳的技术援助。这项工作由CERCA方案和加泰罗尼亚大学、大学和企业部大学与研究委员会资助(SGR2017-648)。这项工作由西班牙研究、创新和大学部(MEICO)通过BFU2015-67777-R和RTI2018-099773-B-100、西班牙普里昂网络(西班牙Prionet AGL2017-90665-REDT)和卡洛斯三世研究所资助。PRY-2018-2)。
Materials
| Material | |||
| 3,3′-Diaminobenzidine tetrahydrochloride 10 mg tablets (DAB) | Sigma | D5905 | |
| Adult Sprague-Dawley rats (8 to 9 weeks old) | Criffa-Credo, Lyon, France | ||
| Avidin-biotin-peroxidase complex (ABC) | Vector Labs | PK-4000 | |
| B27 serum-free supplement 50x | Invitrogen | 17504-044 | |
| Bicinchoninic acid (BCA) protein assay kit | Pierce | 23225 | |
| cDNA plasmid vectors | |||
| COS1 cell lines | ATTC | CRL-1570 | |
| D-(+)-Glucose | Sigma | 16325 | |
| D-(+)-glucose (45% solution in water) for complete Neurobasal medium | Sigma | G8769 | |
| D-MEM (Dulbecco's Modified Eagle Medium 1x ) for COS1 culture medium | Invitrogen | 41966-029 | |
| Dulbecco’s phosphate buffered saline 10x (without Ca2+ and Mg2+) (D-PBS) for cultures | Invitrogen | 14200 | |
| Ethanol | merck | 108543 | |
| Ethylenediaminetetraacetic acid dihydrate disodium salt (EDTA) | Sigma | E5134 | |
| Fluorescence mounting media (e.g., Fluoromount-G or similar) | Electron Microscopy Sciences (EMS) | 17984-25 | |
| Gelatin powder | Sigma | G1890 | |
| Glacial acetic acid (Panreac, cat. no. 211008) | Panreac | 211008 | |
| Hank’s balanced salt solution | Invitrogen | 24020083 | |
| Heat-inactivated foetal bovine serum | Invitrogen | 10108-165 | |
| Heat-inactivated horse serum | Invitrogen | 26050-088 | |
| Hydrogen peroxide (H2O2, 32 to 33% in water) | Sigma | 316989 | |
| L-glutamine 200 mM solution (100x) for complete Neurobasal and COS1 medium | Invitrogen | 25030-024 | |
| Lipofectamine 2000 Reagent | Invitrogen | 11668-019 | |
| Mice pregnant female (embryonic day 12.5 to 16.5; E12.5-16.5) | Criffa-Credo, Lyon, France | ||
| Modified Minimum Essential Medium Eagle (MEM) | Invitrogen | 11012-044 | |
| Monoclonal antibody against class III β-tubulin (clone TUJ-1) | Biolegend | 801201 | |
| N-2 supplement 100x | Invitrogen | 17502-048 | |
| Neurobasal medium | Invitrogen | 21103049 | |
| Paraformaldehyde | Merck | 1,040,051,000 | |
| Penicillin/streptomycin solution 100x | Invitrogen | 15140-22 | |
| Phosphate buffered saline 10x (PBS) for immunocytochemistry | Invitrogen | AM9624 | |
| Secondary antibody: biotinylated horse anti-mouse | Vector Labs | BA-2000 | |
| Serum-free medium (Opti-MEM) | Invitrogen | 11058-021 | |
| Sodium azide | Panreac | 162712 | |
| Sodium bicarbonate solution 7.5% | Invitrogen | 25080-094 | |
| Sterile culture grade H2O | Sigma | W3500 | |
| TritonTM X-100 | Sigma | X100 | |
| Trizma base | Sigma | T1503 | |
| Trypsin-EDTA (Trypsin (0.05% (wt/vol) with EDTA (1x) | Invitrogen | 25300-054 | |
| Equipment | |||
| 1 large and 1 small curved scissors for dissection | Fine tools Instruments or similar | ||
| 1.5-ml conical centrifuge tubes | Eppendorf or similar | ||
| 15-ml conical centrifuge tubes | Corning or similar | ||
| 2 haemostats | Fine tools Instruments or similar | ||
| 2 small straight dissecting scissors | Fine tools Instruments or similar | ||
| 200-ml centrifuge tubes for centrifugation | Nalgene or similar | ||
| 200-ml sterile glass conical flasks | |||
| 2-litre glass beaker | |||
| 4- and 6-well culture plates | Nunc | 176740 and 140675 | |
| Automatic pipette pumps and disposable 10 ml and 25 ml filter-containing sterile plastic pipettes. | Gilson, Brand, Eppendorf or similar | ||
| Automatic pipettes, sterile filter tips and current sterile tips | Gilson, Eppendorf or similar | ||
| Bench top microcentrifuge with angle fixed rotor | Eppendorf, Beckman Coulter or similar | ||
| Bench top refrigerated centrifuge with swing-bucket rotor (with 1.5, 15 and 50 ml tube adaptors) | Eppendorf, Beckman Coulter or similar | ||
| Cell culture incubator at 37 ºC, 5% CO2 and 95% air | |||
| Dialysis tubing cellulose membrane | Sigma | D9402 | |
| Dialysis tubing closures | Sigma | Z37101-7 | |
| Disposable glass pipettes | |||
| Dissecting microscope with dark field optics | Olympus SZ51 or similar | ||
| High-speed refrigerated Beckman Coulter centrifuge or similar with angle fixed rotor | |||
| Laminar flow hood | |||
| Large 100-mm, 60-mm and small 35-mm Ø cell culture dishes | Nunc | 150679 , 150288 and 150318, respectively | |
| Magnetic stirrer and magnetic spin bars | IKA or similar | ||
| McIlwain tissue chopper | Mickle Laboratory Engineering | ||
| One pair of fine straight forceps and one pair of curved forceps | Fine tools Instruments or similar | ||
| Razor blades for the tissue chopper | |||
| Scalpels (number 15 and 11) | |||
| Two pairs of fine spatulas for transferring collagen and tissue pieces | Fine tools Instruments or similar | ||
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Citazione di questo articolo
Gil, V., Del Río, J. A. Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development. J. Vis. Exp. (148), e59481, doi:10.3791/59481 (2019).