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Bioengenharia

改良的3D 水凝胶培养的原发胶质细胞的体外Neuroinflammation 模型的建立

Published: December 08, 2017
doi:
10.3791/56615
, , , , ,
1Department of Psychiatry,University of Alberta, 2Alberta Innovates-Health Solutions Interdisciplinary Team in Smart Neural Prostheses (Project SMART),University of Alberta, 3Department of Chemical and Materials Engineering,University of Alberta, 4Division of Physical Medicine and Rehabilitation,University of Alberta, 5Centre for Neuroscience,University of Alberta

Summary

在此, 我们为大鼠脑源性胶质细胞的3D 培养提供了一项协议, 包括星状细胞、小胶质和突。我们展示了原细胞培养, methacrylated 透明质酸 (哈马) 水凝胶合成, HAMAphoto 聚合和细胞封装, 和样品处理共聚焦和扫描电子显微成像。

Abstract

在中枢神经系统, 许多急性损伤和神经退行性疾病, 以及植入设备或生物材料的设计, 以提高功能的结果相同的结果: 过量炎症导致胶质, 细胞毒性, 和/或形成一个共同加剧伤害或防止健康恢复的神经胶质瘢痕。为了建立一个系统来塑造神经胶质瘢痕形成和研究炎症过程, 我们已经产生了一个3D 细胞支架, 能够容纳原代培养的胶质细胞: 小胶质组织, 调节异物反应, 并启动炎症事件, 星形胶质细胞的反应形成一个纤维状的疤痕, 和突, 通常易受炎症损伤。本工作提供了一个详细的分步方法, 对透明质酸基3D 水凝胶支架与大鼠脑源性胶质细胞的制备、培养和显微表征。此外, 通过共焦免疫荧光和扫描电子显微镜对细胞包封和水凝胶支架的特性进行了研究, 并展示了用生物活性基质修饰支架的能力, 以及将商业基板混合物加入到改进的细胞整合中。

Introduction

中枢神经系统 (cns) 的炎症一直被认为是急性 (如: 如, 缺血性中风, 外伤性脑和脊髓损伤) 和慢性 (如: 老年痴呆, 帕金森氏症, 亨廷顿氏病) 中枢神经损伤的标志,但越来越被认为是神经退行性疾病和精神障碍的原因。持续或不适当的炎症可能导致神经损伤和鞘 (如多发性硬化), 并对大脑发育产生负面影响 (如:如、精神分裂症、自闭症) 和心境状态 (如、抑郁症、焦虑, 躁郁症)。此外, 使用植入设备的新的治疗策略 (e. g., 脑-计算机-接口1,2,3, 深脑刺激4,5, 椎管内microstimulation6,7,8,9,10在设备和 CNS 之间的接口上生成可预知的炎症反应, 从而导致保护组织的反应, 可能导致在植入物的生命周期的失效或设备故障11。中枢神经系统的炎症通常是由小胶质细胞发起的, 它作为中枢神经系统的常驻免疫单元, 负责组织监测和安装异物反应 (已被审查的12)。根据侮辱的严重性, 小胶质细胞信号和招募额外的单元类型到受伤地点。具体来说, 小胶质细胞激活星形细胞, 这反过来充当继发性炎症单元和形成一个密集的保护屏障, 以包含伤害网站13,14。小胶质细胞也可以在外周免疫系统中启动一个活动级联, 这可能导致 BBB 的分解以允许免疫浸润 (在参考文献15中进行了回顾)。

对于植入中枢神经系统的设备, 由于设备插入而导致的组织损伤以及异物的持续存在, 可能会引发一种称为胶质瘢痕的过程。在此过程中, 小胶质细胞迁移到损伤部位并增殖。他们还开始释放炎症因子, 以抵消潜在的威胁, 并招募更多的胶质细胞。随后, 激活的星形胶质细胞变得肥大, 开始封装植入的设备形成连续的纤维屏障16。炎症信号还有助于促进从种植体附近撤出神经元过程, 并最终招募成纤维细胞, 以加强发育中的神经胶质瘢痕17。突, 负责鞘内的神经元, 以提高电导率, 不生存这个过程和遥远的细胞被分割从植入物的疤痕18。胶质瘢痕极大地降低了植入装置的功能和寿命, 特别是用于记录电极, 并最终限制了神经接口19的功能。

在 CNS20、21、22、23中, 已利用多种方法提高植入设备的生物相容性和界面活性。对这些神经接口的生物相容性设计24进行了广泛的回顾。最突出的策略包括围绕电极与相容涂层, 如 polyelthyleneglycol (PEG), 聚乳酸-乙醇酸 (PLGA) 的25, 或增强电极与导电聚合物, 如聚乙烯 (乙烯dioxythiophene) (PEDOT) 和聚吡咯 (吡)26,27,28,29,30,31。生物活性涂层也被用于提供神经组织生长的线索, 使用来自细胞外基质的配体, 包括胶原蛋白、fibronectins 和透明质酸32,33,34 ,35,36,37。这些涂层的生物活性已进一步探索与生长因子释放系统模拟自然细胞分泌物30,38,39,40,41,42,43,44,45,46,47,48,49,50. 同时, 一些研究小组选择了重塑电极的几何形状、柔韧性和成分, 以减少设备和组织之间的机械不匹配51,52,53 ,54,55,56,57。总之, 这些策略已经导致了下一代神经界面设备的许多有希望的改进, 但是长期的兼容性是一个持续的问题, 而且在复杂和耗时的体内模型中可能会阻碍进展。.

以动物模型为基础的方法可以限制实验的吞吐量, 增加测试电极生物相容性的成本。体外使用常规细胞培养技术的方法提供了一个更经济高效的替代方案, 但无法重述设备和组织之间的交互的复杂程度58。特别是, 使用2D 细胞培养的表面涂层的测试限制了电极几何的建模以及机械不匹配和微思想对生成导致设备故障的主机响应的影响59,60。

为了克服与2D 细胞培养有关的问题, 水凝胶培养的神经细胞已经开发了广泛的各种应用, 药理学研究61, 以指导神经细胞分化62, 以了解疾病路径63,64, 或在与其他单元格类型的 co 区域性中进行分层, 以模拟单元格迁移、神经保护或模型组织环境61。水凝胶易于形成不同的大小和几何可以包含多种类型的主要或永生细胞文化, 并高度适合分析的常用技术, 如共用荧光显微镜。为了建立一个模型的胶质瘢痕的过程, 我们最近开发和特征的透明质酸基3D 水凝胶系统的高通量测试的胶质反应植入电极 (图 1)65。该系统有几个明显的优点: 1) 原发胶质细胞 (小胶质、星形胶质和突) 被封装在一个由透明质酸聚合物组成的3D 基质中, 它是一种内源性细胞外基质成分;2) 矩阵刚度可进行 “调谐”, 以再现脑或脊髓组织的力学特性;和 3) 细胞可以封装在矩阵的快速台阶顶部的方法使用聚合与绿色光, 限制毒性在封装。该系统可实现体内生物相容性的关键功能: 设备以与组织相类似的方式插入到水凝胶中, 对植入设备的细胞反应进行监测, 以实现范围广泛的参数65。这些包括设备与各种结构的水凝胶涂层和电刺激脉冲之间的机械不匹配。该系统还包括胶质和相关的前体, 它们经常存在并在胶质瘢痕中被吸收。小胶质细胞的损伤、死亡和吞噬作用是炎症性损伤的高度表现, 作为一种模型可以减少疤痕或恢复, 他们有能力证明神经元重新鞘66。

本文介绍了一种合成和形成混合透明质酸水凝胶的方法, 并结合商用基底膜配方, 以改善细胞合并。此外, 我们将展示的主要培养的神经胶质细胞 (小胶质, 星形胶质, 和突) 和分析的文化生长, 使用细胞化学和共聚焦显微镜。

Protocol

1天的大鼠幼崽的脑组织提取协议, 被斩首的安乐死, 得到了阿尔伯塔大学动物护理和使用委员会的批准。 1. 小胶质细胞和星形胶质细胞隔离67,68 注: 所有用于隔离和细胞培养的介质在水浴中预热至37° c。汉克的平衡盐溶液 (HBSS) 有1% 青霉素-链霉素 (PS)。所有 Dulbecco 的改良鹰的媒体与火腿的 F12 营养混合物 (DMEM/F12) ?…

Representative Results

为了在高吞吐量下对神经组织宿主反应和胶质瘢痕进行建模,体外系统需要一个具有生物相容的基质材料的3D 细胞支架, 在原位形成过程中不会发生细胞毒事件, 并且可以修改用生物活性成分指导仁慈的反应。为此, 我们创建了一个基于透明质酸的3D 细胞支架系统, 并封装了一个主要的混合胶质细胞群体, 以研究细胞间的相互作用和胶质 bioreactivity。对细胞和支架…

Discussion

为了建立一个3D 的培养系统来塑造神经胶质 bioreactivity 和胶质瘢痕的过程, 我们开发了一个能支持原代培养的小胶质细胞、星形胶质细胞和突的系统, 并能使细胞膜的强健特性形态学和细胞间的相互作用。从显微显示, 每个细胞类型的形态学是明显不同的 2D, 3 d-哈马, 和3D 哈马基板混合平台。在2D 系统中, 形态学在表面的平面上明显偏倚, 但与3D 的哈马相比, 小胶质细胞和星状体在母体内普遍较小, 但…

Declarações

The authors have nothing to disclose.

Acknowledgements

作者感谢 NSERC、CFI、AIHS、艾伯塔省卫生服务机构以及戴维大脑研究基金会的资助。

Materials

1. Materials for HAMA synthesis and photopolymerization
Hyaluronic acid (HA) Sigma-Aldrich 53747-10G Streptococcus equi, MW: 1.5 – 1.8 X 10^6
Methacrylic anhydride (MA) Sigma-Aldrich 275585-100ML
Sodium hydroxide (NaOH) Sigma-Aldrich 221465-25G
Ethanol (EtOH) Commerical Alcohols Inc. Anhydrous
Phosphate buffered saline (pH 7.4) tablets Fisher Scientific 18912014
Triethanolamine (TEA) Sigma-Aldrich 90279-100ML
1-Vinyl-2pyrrolidinone (NVP) Sigma-Aldrich V3409-5G
EosinY (EY) Sigma-Aldrich E6003-25G
Polydimethylsiloxane (PDMS) Sylgard 184 Silicone Elastomer Kit Dow Corning
3-(Trimethoxysilyl)propyl methacrylate Sigma-Aldrich 440159-100ML
Beaker (100 mL) Corning 1000-100
Beaker (500 mL) Corning 1000-600
pH paper (Labstick) Sigma-Aldrich 9580
Name Company Catalog Number Comments
2. Materials for glial cell isolation and cell culture
P1-2 Sprague Dawley rat pups Charles River CD Sprague Dawley rat strain code 001
Dissector scissors – slim blades (small) Fine Science Tools 14081-09
Surgical scissors – Toughcut (large) Fine Science Tools 14130-17
Fine forceps (Dumont #5) Fine Science Tools 11521-10
Curved fine forceps (Dumont #7) Fine Science Tools 11271-30
Hank's balanced salt solution (HBSS) Gibco 14170-112
Dulbecco's modified Eagle's medium and Ham's nutrient mixture F-12 (DMEM/F12) Gibco 11320-033
Penicillin-streptomycin (PS) Gibco 15140-122
Fetal bovine serum (FBS) Gibco 12483-020
0.25% Trypsin-ethylenediaminetetraacetic acid (EDTA) Gibco 25200-072
Poly-L-lysine (PLL) Sigma-Aldrich P-6282
50 mL conical centrifuge tube Fisher Scientific 05-539-13
15 mL conical centrifuge tube Fisher Scientific 05-539-5
12 well Tissue culture treated plates (Cellstar) Greiner Bio-One 665 108
10 mL serological pipette Fisher Scientific 13-676-10F
25 mL serological pipette Fisher Scientific 12-676-10K
Petri dish (60 mm X 15 mm) Fisher Scientific FB0875713A
Petri dish (100 mm X 15 mm) Fisher Scientific FB0875712
Microscope Coverslip (18 mm) Fisher Scientific 12-545-100 18CIR
Name Company Catalog Number Comments
3. Materials for microscopy (confocal and scanning electron microscopy)
Mouse monoclonal anti-CNPase abcam ab6319
Rabbit anti-Iba1 Wako Laboratory Chemicals 019-17741
Chicken anti-GFAP abcam ab4674
Hoechst 33342 Fisher Scientific 62249
Fluoromount-G Fisher Scientific 00-4958-02
Formalin Sigma Aldrich HT501128-4L Buffered (10%)
Triton X-100 Fisher Scientific BP151-500
Horse Serum Gibco 16050-122
Paraformaldehyde Electon Microscopy Sciences 157-8 Buffered (8%)
Guteraldehyde Electon Microscopy Sciences 16019 Buffered (8%)
Osmium tetraoxide Electon Microscopy Sciences 19152 Buffered (2%)
Hexamethyldilazane (HMDS) Electon Microscopy Sciences 16700
Ethanol (EtOH) Electon Microscopy Sciences 15055 Anhydrous
Microscope Slide (25 X 75 X 1 mm) VWR International 48311-703
DOWNLOAD MATERIALS LIST

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3D Cell CultureGlial CellsMicrogliaAstrocytesOligodendrocytesNeuroinflammationGlial ScarHydrogelHyaluronic AcidIn Vitro ModelCell EncapsulationConfocal MicroscopySEM

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Koss, K. M., Churchward, M. A., Jeffery, A. F., Mushahwar, V. K., Elias, A. L., Todd, K. G. Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation. J. Vis. Exp. (130), e56615, doi:10.3791/56615 (2017).
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