财政支持由国立卫生研究院提供 [U01-NS094340 (卡伦) & F31-NS090746 (Katiyar)], 迈克尔 j ·福克斯基金会 [治疗管道计划 #9998 (卡伦)], 佩恩医学神经科学中心试点奖 (卡伦),国家科学基金会 [研究生研究金 DGE-1321851 (Struzyna)], 退伍军人事务部 [RR & D 值得审查 #B1097 I (卡伦)] 和美国陆军医学研究和装备司令部 [#W81XWH-13-207004 (卡伦) &W81XWH-15-1-0466 (卡伦)]。

作者没有什么可透露的。

与三七总皂苷的支持环境相比, 中枢神经系统在处理神经和神经的有害后果方面特别有限。在对哺乳动物中枢神经系统的严重侮辱后, 形成了一个神经胶质瘢痕, 由纤维化和炎症细胞的核心组成, 由一个致密网状的无条理的反应性星形胶质组织所包围, 分泌轴突生长抑制蛋白14。此疤痕作为一个物理和生物化学的障碍, 反对再生轴突结束12。此外, 成年哺乳动物中枢?...

中枢神经系统 (CNS) 的能力有限, 以抵消的损失和/或功能障碍的神经元和轴突通路, 伴随的条件, 如创伤性脑损伤 (脑外伤), 中风, 脊髓损伤 (SCI), 和神经变性疾病1 ,2,3,4,5。中枢神经系统的神经再生仅限于大脑中的有限区域, 从而阻碍了丢失神经元的恢复6,7。此外, 由于缺乏定向引导, 存在的生长抑制剂, 和反应 astrogliosis 在神经组织损伤后的中枢神经系统失去的轴突通路的再生是不够的2,8, 9,10。星形胶质细胞在帮助神经元的离子稳态、神经递质清除、突触形成和神经血管耦合方面具有不同的功能11。然而, 随着对神经组织的轻度损伤, 星形胶质细胞在转变为肥厚状态11时, 可能会发生分子、结构和功能上的变化。为了应对严重的神经, 这些变化导致形成了一个包含迁移反应性星形胶质细胞的半影和一个病变的核心, 包括从破裂的血脑屏障 (BBB), 小胶质细胞漏出的白血球,突, 和成纤维细胞11,12,13。这些反应性星形胶质细胞获得丝状的形态学, 无序的过程, 并表现出增加的表达中间丝蛋白和硫酸软骨素蛋白 (CSPGs), 这阻碍神经再生12。尽管胶质瘢痕最初有助于恢复血脑屏障的完整性, 避免对周围健康组织的炎症反应的传播, 它作为一个物理和生物化学的屏障, 反对轴突再生12,14 ,15,16。例如, 遇到胶质瘢痕的轴突显示球状不良生长锥和发育迟缓的生长12。此外, 损伤后星过程的解体阻碍了再生轴突的扩展17。这些抑制特征的结果表现在经常永久的身体和神经损伤, 病人遭受严重的神经后, 包括创伤和 SCI。
不管在中枢神经系统的功能再生面临的外在挑战, 轴突已经被证明具有内在的再生能力。例如, 与胶质瘢痕接触的不良生长锥的动态性质表明, 这些结局保留了它们扩展12的能力。因此, 人们认为, 轴突再生的主要障碍是后中枢神经系统的抑制环境, 并通过减少胶质瘢痕和/或提供跨越疤痕的再生桥来提供更宽松的环境有利.事实上, 先前的研究表明, 中枢神经系统神经元能够通过利用周围神经移植作为桥梁的病变来延长轴突, 这为轴突再生提供了一个更有利的环境12,18, 19。还采取了其他一些战略来利用这种退化的再生能力。例如, 在各种损伤模型中对细胞生长信号通路的操纵导致轴突再生和胶质瘢痕减少10,20,21。此外, 研究表明, 酶 ABC, 克里夫斯 CSPGs 的大部分糖链的治疗, 减少反应性星形胶质细胞分泌 CSPGs 的抑制作用22。尽管取得了令人鼓舞的结果, 但这些方法并没有提供对生长锥的定向指导, 这可能导致异常再生12, 也不能解释神经元的丢失。细胞方法已经被用来克服胶质瘢痕的影响, 并补充丢失的神经细胞, 特别是神经元。有些组有分化活性星形胶质细胞进入神经细胞, 而另一些则将神经祖细胞移植到中枢神经系统病变, 以重新填充损伤区并促进轴突再生23,24,25. 然而, 仅靠干细胞移植是由于存活率低、集成度差和在受损的组织中保持适度的5。此外, 这些细胞的策略无法恢复长距离轴突, 特别是以控制的方式。因此, 生物材料与其他方法的结合正在探索作为各种神经和祖细胞的运载工具和生长因子26。Biomaterial-based 的方法具有高度的设计控制, 以产生模仿特定的物理, haptotaxic, 和 chemotaxic 提示存在于目标主机组织的三维 (3D) 微环境中的构造,27, 28,29,30,31,32,33,34。这些环境信号的再现是最重要的移植细胞呈现本机样的形态学, 增殖, 迁移和信号, 在其他神经生物学特征29。尽管这些优越的性能, 超越传统的细胞种子生物材料支架的进步需要同时促进定向长距离轴突再生和取代丢失的神经元。
一个有前途的替代方法是基于神经组织工程 "活支架", 这是不同于其他细胞的方法, 由于存在的活神经细胞与预制细胞, 模拟本地神经和/或发展机制, 以促进有针对性的更换, 重建和再生神经回路4,35。生活支架设计的考虑因素包括神经细胞的表型和来源, 以及由任何伴随的生物材料的组成所决定的机械/物理特性和生物化学信号35。在制作体外后, 这些活体支架可以植入体内, 以呈现细胞黏附分子和趋和神经营养信号, 以积极调节神经细胞迁移和轴突生长, 这取决于状态和再生过程的进展35。胶质细胞可以作为生活支架工程细胞的基础, 因为这些细胞介导各种发育机制在体内。在大脑发育过程中, 新的神经元依赖于由心室区的径向胶质细胞向发育中的皮质板延伸的基底过程, 作为定向迁移的活体支架36,37。此外, 扩展生长锥被证明是自我定位, 通过传感路标细胞诱发的诱人和排斥信号, so-called "先驱" 轴突被建议通过沿 pre-patterned 神经胶质的延伸来达到正确的目标。支架35,38,39。因此, 神经胶质细胞是必要的先驱轴突的指导, 后来作为 axon-based "活支架", 以指导投影的 "追随者" 轴突。此外, 胶质细胞介导的生长机制已被证明坚持后天, 因为细胞跟随侧迁徙流 (RMS), 从脑室区 (SVZ), 在成年大脑中少数残存的神经再生领域之一, 以导航嗅球 (OB)40。这些细胞在 RMS 中迁移到胶质管内 (图 1A-1), 它由纵向排列的星过程组成, 通过直接的细胞-细胞粘连和局部可溶性因子37,41. 最后, 虽然在哺乳动物中枢神经系统损伤导致中断星过程安排形成了一个神经胶质瘢痕, 物理上阻碍轴突再生17, 许多哺乳体系缺乏形成一个有害的胶质瘢痕。相反, 哺乳种类的胶质细胞维持更有条理的、对齐的图案, 这些模式在受伤区域17、42、43中用作参考线。例如, 在哺乳的 SCI 模型中, 轴突被证明与通过病变的神经胶质桥紧密结合生长, 这表明有组织的胶质支架作为促进轴突再生和功能恢复的基质的重要作用 (图 1A-2)42,44,45. 上述神经特征和发展/再生机制的重述可能会产生一种新的工程 glial-based 活体支架, 可同时驱动未成熟的神经元迁移和轴突寻通过其他允许环境, 从而有可能减轻神经元和轴突道变性与中枢神经系统损伤和疾病相关的影响。
我们的研究小组以前曾设计过多种类型的活体支架, 可通过微组织工程化神经网络 (微 TENNs) 和组织对中枢神经系统和周围神经系统 (三七总皂苷) 的轴突重建和再生。工程神经移植 (雄姿), 分别为27,46,47,48。这两种策略都是基于仿生学的。微 TENNs 是解剖学上的启发结构设计, 以结构和功能取代轴突, 连接不同的神经元群体的大脑。雄姿利用轴突促进轴突再生的发育机制, 以 "从动杆" 轴突生长为例, 以 "先锋" 轴突为目标, 实现有针对性的主机轴突再生35,46,48。我们最近利用了生活支架技术的多功能性, 使用了类似的装箱方案作为微 TENNs, 并寻求启发从 glia-based 机制在整个发展中呈现。在这里, 我们开发的结构组成的星束跨越的胶原管的水凝胶微柱49。这些星活体支架的研制首先是用液体琼脂糖来填充毛细管针总成, 以创建一个中空的圆柱形水凝胶, 外径和内径 (ID) 对应的直径管和针分别。在琼脂糖凝胶和提取的水凝胶微柱从毛细管管, 空心内涂层与 i 型胶原蛋白, 以提供一个环境纵容星形胶质细胞粘附和排列束形成 (图 1B-1)。之后, 腔内的大脑皮质星形胶质细胞从产后大鼠幼崽中分离 (图 1B-2)。与 two-dimensional (2D) 的对准技术, 依赖于电场、micropatterned 槽和细胞外基质 (ECM) 蛋白的应用, 活支架技术中的星形胶质星对准依赖于自组装根据可控变量, 如基底曲率 (列 ID), 细胞密度, 和胶原蛋白浓度50,51,52。星形胶质细胞收缩并重塑胶原蛋白, 并获得与自然支架类似的双极性、纵向排列的形态学观察在体内(图 1B-3)。事实上, 我们正积极地使用这些类似电缆的结构作为物理基底, 用于定向迁移未成熟神经元, 以及通过受损的中枢神经系统的不利环境促进轴突再生, 特别是哺乳动物胶质瘢痕 (图 1C)。本文将介绍星微柱的详细制作方法、相衬和预期细胞的免疫荧光图像, 并对当前的局限性和今后的发展方向进行了全面的讨论。技术.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55848/55848fig1.jpg"> 图 1: 对齐的星网络的启发、制造协议和建议的应用程序.(A) 神经生物学灵感: (1) 来源于神经源性脑室区 (SVZ) 的细胞, 利用侧洄游流 (RMS) 中的纵向对中的胶质管向嗅球定向迁移 (OB);(2) 非哺乳动物, 如两栖动物和鱼类可以维持再生后, 神经组织损伤部分由于形成了一个胶质桥连接的两端病变 (如断脊髓), 并作为一个脚手架的指导再生轴突。(B) 制作概述: (1) 微米、空心水凝胶微柱与管腔内涂敷 ECM, (2) 生后大鼠幼崽的初生皮质星形胶质细胞的播种, (3) 自组装的纵向捆绑在文化, 和 (4) 从生物材料装箱的捆绑提取为未来的植入研究。(C)在体内应用程序: (1) 这些活体支架可作为神经源性神经中心定向神经元迁移到重新填充神经元缺陷区域的神经胶质管。(2) non-mammals 的发展机制的重述和神经胶质桥的再生机制, 可以赋予这些星支架的能力, 以引导轴突再生横跨允许哺乳动物胶质瘢痕的环境。<a href="//cloudflare.jove.com/files/ftp_upload/55848/55848fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

<table><tbody><tr><td>Acupuncture needle (300 &amp;micro;m diameter)</td><td>Lhasa Medical</td><td>HS.30x40</td><td>The diameter may be varied according to the desired size for the micro-column lumen.</td></tr><tr><td>Petri dish</td><td>Fisher</td><td>08772B</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate buffered saline (DPBS)</td><td>Invitrogen</td><td>14200075</td><td></td></tr><tr><td>Polystyrene disposable serological pipet</td><td>Fisher</td><td>13-678-11D</td><td></td></tr><tr><td>Agarose</td><td>Sigma</td><td>A9539-50G</td><td></td></tr><tr><td>Microliter glass capillary tube (701 &amp;micro;m)</td><td>Fisher</td><td>21-170J</td><td>The diameter may be varied according to the desired size for the micro-column shell.</td></tr><tr><td>Microcap bulb dispenser</td><td>Fisher</td><td>21-170J</td><td>Bulb comes with the microcap tubes.</td></tr><tr><td>Hot plate</td><td>Fisher</td><td>SP88857200</td><td></td></tr><tr><td>Magnetic bar</td><td>Fisher</td><td>1451352</td><td></td></tr><tr><td>Micropipette</td><td>Sigma</td><td>Z683884-1EA</td><td></td></tr><tr><td>25 mm gauge needle</td><td>Fisher</td><td>14-826-49</td><td></td></tr><tr><td>Microscalpel</td><td>Roboz Surgical</td><td>RS-6270</td><td></td></tr><tr><td>Scissors</td><td>Fine Science Tools</td><td>14081-09</td><td></td></tr><tr><td>Forceps</td><td>World Precision Instruments</td><td>501985</td><td></td></tr><tr><td>Hot bead sterilizer</td><td>Sigma</td><td>Z378550-1EA</td><td></td></tr><tr><td>Stereoscope</td><td>Nikon</td><td>SMZ800N</td><td></td></tr><tr><td>Micro-spatula</td><td>Fisher</td><td>S50821</td><td></td></tr><tr><td>Rat tail type I collagen</td><td>Corning</td><td>354236</td><td>Maintain at 4 &amp;ordm;C and remove only when needed. Use ice to preserve its temperature when in use.</td></tr><tr><td>Microcentrifuge tube</td><td>Fisher</td><td>02-681-256</td><td></td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>Fisher</td><td>SS2661</td><td></td></tr><tr><td>Hydrochloric acid (HCl)</td><td>Fisher</td><td>SA48-1</td><td></td></tr><tr><td>Litmus paper</td><td>Fisher</td><td>09-876-18</td><td></td></tr><tr><td>Hank&#039;s balanced salt solution (HBSS)</td><td>Invitrogen</td><td>14170112</td><td>Store at 4 &amp;ordm;C.</td></tr><tr><td>0.25% Trypsin-EDTA</td><td>Invitrogen</td><td>25200056</td><td>Store at -20 &amp;ordm;C and warm at 37 &amp;ordm;C before use.</td></tr><tr><td>Bovine pancreatic deoxyribonuclease (Dnase) I</td><td>Sigma</td><td>10104159001</td><td>Store at -20 &amp;ordm;C and warm at 37 &amp;ordm;C before use.</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM) with Ham&#039;s F-12 Nutrient Mixture</td><td>Gibco</td><td>11330-032</td><td>Store at 4 &amp;ordm;C.</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Atlanta Biologicals</td><td>S11195</td><td>Store at -20&amp;ordm;C.</td></tr><tr><td>Postnatal day 0 or day 1 Sprague Dawley rat pups</td><td>Charles River</td><td>Strain 001</td><td></td></tr><tr><td>Neurobasal embryonic neuron basal medium</td><td>Invitrogen</td><td>21103049</td><td>Store at 4&amp;ordm;C and warm at 37 &amp;ordm;C before use.</td></tr><tr><td>B-27 serum free supplement</td><td>Invitrogen</td><td>12587010</td><td>Store at -20 &amp;ordm;C and warm at 37 &amp;ordm;C before use.</td></tr><tr><td>L-glutamine</td><td>Invitrogen</td><td>35050061</td><td>Store at -20 &amp;ordm;C and warm at 37 &amp;ordm;C before use.</td></tr><tr><td>G5 astrocytic supplement</td><td>Invitrogen</td><td>17503012</td><td></td></tr><tr><td>Sprague Dawley embryonic day 18 rats</td><td>Charles River</td><td>Strain 001</td><td></td></tr><tr><td>Pasteur pipette</td><td>Fisher</td><td>22-042816</td><td></td></tr><tr><td>15 mL centrifuge tube</td><td>EMESCO</td><td>1194-352099</td><td></td></tr><tr><td>Vortex</td><td>Fisher</td><td>02-215-414</td><td></td></tr><tr><td>Centrifuge</td><td>Fisher</td><td>05-413-115</td><td></td></tr><tr><td>Hemocytometer</td><td>Fisher</td><td>02-671-6</td><td></td></tr><tr><td>Incubator</td><td>Fisher</td><td>13 998 076</td><td></td></tr><tr><td>Formaldehyde 40%</td><td>Fisher</td><td>F77P-4</td><td>Formaldehyde is a toxic compound known to be carcinogenic, and must be disposed of in a separate container.</td></tr><tr><td>Glass cover slip</td><td>Fisher</td><td>12-548-5M</td><td></td></tr><tr><td>Nail polish</td><td>Electron Microscopy Sciences (EMS)</td><td>72180</td><td></td></tr><tr><td>Fluoromont mounting medium</td><td>Southern Biotech</td><td>0100-01</td><td></td></tr><tr><td>Poly-L-lysine</td><td>Sigma</td><td>P4707</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Fisher</td><td>BP3994</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>Normal horse serum</td><td>Gibco</td><td>16050-122</td><td></td></tr><tr><td>Rabbit anti-glial acidic fibrillary protein (GFAP) primary antibody</td><td>Millipore</td><td>AB5804</td><td>Store at -20&amp;ordm;C.</td></tr><tr><td>Mouse anti-beta-tubulin III primary antibody</td><td>Sigma</td><td>T8578</td><td>Store at -20&amp;ordm;C.</td></tr><tr><td>Rabbit anti-collagen I primary antibody</td><td>Abcam</td><td>ab34710</td><td>Store at -20&amp;ordm;C.</td></tr><tr><td>Rabbit anti-vimentin</td><td>Millipore</td><td>AB3400</td><td>Store at -20&amp;ordm;C.</td></tr><tr><td>Mouse anti-nestin</td><td>Millipore</td><td>AB5326</td><td>Store at -20&amp;ordm;C.</td></tr><tr><td>Donkey anti-mouse 568 secondary antibody</td><td>Invitrogen</td><td>A10037</td><td>Store at 4&amp;ordm;C.</td></tr><tr><td>Donkey anti-rabbit 568 secondary antibody</td><td>Invitrogen</td><td>A10042</td><td>Store at 4&amp;ordm;C.</td></tr><tr><td>Donkey anti-rabbit 488 secondary antibody</td><td>Invitrogen</td><td>A21206</td><td>Store at 4&amp;ordm;C.</td></tr><tr><td>Hoechst 33342, Trihydrochloride</td><td>Invitrogen</td><td>H3570</td><td>Store at 4&amp;ordm;C. Hoechst is a known mutagen that should be treated as a carcinogen. Therefore, it must be disposed of in a separate container.</td></tr><tr><td>Calcein AM</td><td>Sigma</td><td>C1359</td><td>4 mM in anhydrous DMSO</td></tr><tr><td>Ethidium homodimer-1</td><td>Life Technologies</td><td>E1169</td><td>2 mM in DMSO/H2O 1:4 (v/v)</td></tr><tr><td>Dimethyl sulfoxane (DMSO)</td><td>Sigma</td><td>276855</td><td></td></tr><tr><td>A1RSI Laser Scanning Confocal Microscope</td><td>Nikon</td><td></td><td>Used for taking the confocal reconstructions of immunolabeled constructs.</td></tr><tr><td>Eclipse Ti-S Microscope</td><td>Nikon</td><td></td><td>Used for taking the phase-contrast images. With digital image acquisition using a QiClick camera interfaced with Nikon Elements Basic Research software (4.10.01).</td></tr></tbody></table>

three-dimensional tissue engineered aligned astrocyte networks to recapitulate developmental mechanisms and facilitate nervous system regeneration

神经和神经退行性疾病往往导致持久的神经缺损, 由于中枢神经系统 (CNS) 的能力有限, 以取代失去的神经元和再生轴突通路。然而, 在神经系统的发育过程中, 神经元的迁移和轴突的扩展通常发生在其他细胞形成的通路上, 称为 "活支架"。为了模拟这些机制, 并设计一个绕过中枢神经系统抑制环境的策略, 本手稿提出了一种制造组织工程 astrocyte-based "活支架" 的协议。为了创建这些结构, 我们采用了一种新的生物材料装箱方案, 诱导星形胶质细胞组装成稠密的三维双极纵向排列的胞和过程的束。首先, 对中空水凝胶微柱进行了组装, 内腔表面涂有胶原细胞基质。然后将游离的大脑皮质星形胶质细胞传送到圆柱形微柱的腔内, 并在 < 350 µm 的临界内径上自发对准, 并收缩产生由稠密束组成的长纤维状电缆。星形胶质细胞的过程和胶原纤维的测量 < 150 µm 的直径, 但延长了几个厘米的长度。这些工程化的活体支架展示了 > 97% 细胞的生存能力, 实际上完全由星形胶质细胞、胶质纤维酸性蛋白、波形和巢组成。这些对齐的星形胶质细胞网络被发现为神经元附着和对突起扩展提供了一个可容许的基底。此外, 这些结构保持完整性和对齐时, 提取从水凝胶装箱, 使其适合中枢神经系统植入。这些预制结构模仿自然发生的 glial-based "活体支架"在体内的关键细胞元素。因此, 这些工程化的活体支架可以作为试验床, 用于研究神经发育机制体外或促进 neuroregeneration, 方法是引导神经元迁移和/或轴突寻继中枢神经系统变性后体内.

最初, 用相衬显微镜监测星形胶质细胞黏附和束形成的进展, 以及细胞的整体稳定性作为时间的函数。在1小时的电镀后, 星形胶质细胞在微柱的腔内被发现球形形态 (图 2A)。在5小时, 星形胶质细胞开始扩展过程和收缩, 而由 8 h 电池展示了一个完整的过程-轴承形态, 并形成了电缆状结构, 沿纵向轴线的微柱 (图 2A)。值得注意的是, ?...

Watch this Scientific Journal Video about Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration at JoVE.com

三维组织工程对齐星形胶质细胞网络可概括发育机制并促进神经系统再生 |生物工程|视频

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

我们展示了自组装的发展, 三维束纵向排列星胞和过程内的新型生物材料装箱。这些被设计的 "活脚手架", 显示微米尺度的直径, 但长度延长厘米, 可作为试验床研究神经发育机制或促进 neuroregeneration 通过引导神经元迁移和/或轴突寻.

三维组织工程化排列的星形胶质细胞网络, 以概括发展机制和促进神经系统再生

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

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14.3K 次观看.  University of Pennsylvania. 我们展示了自组装的发展, 三维束纵向排列星胞和过程内的新型生物材料装箱。这些被设计的 "活脚手架", 显示微米尺度的直径, 但长度延长厘米, 可作为试验床研究神经发育机制或促进 neuroregeneration 通过引导神经元迁移和/或轴突寻.

视频: Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

极化神经组织工程三维蚕丝胶原蛋白为基础的模式

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

科研

生物工程

Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying the human brain. One emerging technology to address this issue is the use of three dimensional (3D) neural cell cultures, termed &#39;organoids&#39; or &#39;spheroids&#39;, for long term preservation of intercellular interactions including extracellular adhesion molecules. However, these culture systems are time consuming and not systematically generated. Here, we detail a method to rapidly and consistently produce 3D cocultures of neurons and astrocytes from human pluripotent stem cells. First, pre-differentiated astrocytes and neuronal progenitors are dissociated and counted. Next, cells are combined in sphere-forming dishes with a Rho-Kinase inhibitor and at specific ratios to produce spheres of reproducible size. After several weeks of culture as floating spheres, cocultures (&#39;asteroids&#39;) are finally sectioned for immunostaining or plated upon multielectrode arrays to measure synaptic density and strength. In general, it is expected that this protocol will yield 3D neural spheres that display mature cell-type restricted markers, form functional synapses, and exhibit spontaneous synaptic network burst activity. Together, this system permits drug screening and investigations into mechanisms of disease in a more suitable model compared to monolayer cultures.

In this protocol, we aim to describe a reproducible method for combining dissociated human pluripotent stem cell derived neurons and astrocytes together into 3D sphere cocultures, maintaining these spheres in free floating conditions, and subsequently measuring synaptic circuit activity of the spheres with immunoanalysis and multielectrode array recordings.

3D Cocultures 星形胶质细胞与人多潜能干细胞神经元的突触电路建模

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying ...

发育生物学

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

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

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the gold-standard experimental model to study basic neurophysiological mechanisms involved in the formation and maintenance of neuronal cell assemblies. However, in vitro studies have been limited to the recording of the electrophysiological activity generated by bi-dimensional (2D) neural networks. Nonetheless, given the intricate relationship between structure and dynamics, a significant improvement is necessary to investigate the formation and the developing dynamics of three-dimensional (3D) networks. In this work, a novel experimental platform in which 3D hippocampal or cortical networks are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are realized by seeding neurons in a scaffold constituted of glass microbeads (30-40 &#181;m in diameter) on which neurons are able to grow and form complex interconnected 3D assemblies. In this way, it is possible to design engineered 3D networks made up of 5-8 layers with an expected final cell density. The increasing complexity in the morphological organization of the 3D assembly induces an enhancement of the electrophysiological patterns displayed by this type of networks. Compared with the standard 2D networks, where highly stereotyped bursting activity emerges, the 3D structure alters the bursting activity in terms of duration and frequency, as well as it allows observation of more random spiking activity. In this sense, the developed 3D model more closely resembles in vivo neural networks.

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

In this work, a novel experimental model in which 3D neuronal cultures are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are built by seeding neurons in a scaffold made up of glass microbeads on which neurons grow and form interconnected 3D structures.

接口3D工程神经细胞微电极阵列:一种创新<em&gt;体外</em&gt;实验模型

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the ...

神经科学

Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory environment and the limited capacity for neurogenesis. We are developing&#160;a strategy to simultaneously address&#160;neuronal and axonal pathway loss within the damaged CNS. This manuscript presents the fabrication protocol for micro-tissue engineered neural networks (micro-TENNs), implantable constructs consisting of neurons and aligned axonal tracts spanning the extracellular matrix (ECM) lumen of a preformed hydrogel cylinder hundreds of microns in diameter that may extend centimeters in length. Neuronal aggregates are delimited to the extremes of the three-dimensional encasement and are spanned by axonal projections. Micro-TENNs are uniquely poised as a strategy for CNS reconstruction, emulating aspects of brain connectome cytoarchitecture and potentially providing means for network replacement. The neuronal aggregates may synapse with host tissue to form new functional relays to restore and/or modulate missing or damaged circuitry. These constructs may also act as pro-regenerative&#160;&#34;living scaffolds&#34; capable of exploiting developmental mechanisms for cell migration and axonal pathfinding, providing synergistic structural and soluble cues based on the state of regeneration. Micro-TENNs are fabricated by pouring liquid hydrogel into a cylindrical mold containing a longitudinally centered needle. Once the hydrogel has gelled, the needle is removed, leaving a hollow micro-column. An ECM solution is added to the lumen to provide an environment suitable for neuronal adhesion and axonal outgrowth. Dissociated neurons are mechanically aggregated for precise seeding within one or both ends of the micro-column. This methodology reliably produces self-contained miniature constructs with long-projecting axonal tracts that may recapitulate features of brain neuroanatomy. Synaptic immunolabeling and genetically encoded calcium indicators suggest that micro-TENNs possess extensive synaptic distribution and intrinsic electrical activity. Consequently, micro-TENNs represent a promising strategy for targeted neurosurgical reconstruction of brain pathways and may also be applied as biofidelic models to study neurobiological phenomena in vitro.

This manuscript details the fabrication of micro-tissue engineered neural networks: three-dimensional micron-sized constructs comprised of long aligned axonal tracts spanning aggregated neuronal population(s) encased in a tubular hydrogel. These living scaffolds can serve as functional relays to reconstruct or modulate neural circuitry or as biofidelic test-beds mimicking gray-white matter neuroanatomy.

解剖学启发的三维微组织工程神经网络用于神经系统重建，调制和建模

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory ...

3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also respond to neural injuries and act to protect the tissue from degenerative events. In vitro studies of astrocytes&#39; functionality are important to elucidate the mechanisms involved in such events and contribute to developing therapies to treat neurological disorders. This protocol describes a method to biofabricate a neural-like tissue structure rich in astrocytes by 3D bioprinting astrocytes-laden bioink. An extrusion-based 3D bioprinter was used in this work, and astrocytes were extracted from C57Bl/6 mice pups&#39; brain cortices. The bioink was prepared by mixing cortical astrocytes from up to passage 3 to a biomaterial solution composed of gelatin, gelatin-methacryloyl (GelMA), and fibrinogen, supplemented with laminin, which presented optimal bioprinting conditions. The 3D bioprinting conditions minimized cell stress, contributing to the high viability of the astrocytes during the process, in which 74.08% &#177; 1.33% of cells were viable right after bioprinting. After 1 week of incubation, the viability of astrocytes significantly increased to 83.54% &#177; 3.00%, indicating that the 3D construct represents a suitable microenvironment for cell growth. The biomaterial composition allowed cell attachment and stimulated astrocytic behavior, with cells expressing the specific astrocytes marker glial fibrillary acidic protein (GFAP) and possessing typical astrocytic morphology. This&#160;reproducible protocol provides a valuable method to biofabricate 3D neural-like tissue rich in astrocytes that resembles cells&#39; native microenvironment, useful to researchers that aim to understand astrocytes&#39; functionality and their relation to the mechanisms involved in neurological diseases.

Here we report a method of 3D bioprinting murine cortical astrocytes for biofabricating neural-like tissues to study the functionality of astrocytes in the central nervous system and the mechanisms involving glial cells in neurological diseases and treatments.

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

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also ...

iPSC 来源的神经元和星形胶质细胞的 3D 生物打印共培养物

Three-Dimensional Bioprinting of Human iPSC-Derived Neuron-Astrocyte Cocultures for Screening Applications

For a cell model to be viable for drug screening, the system must meet throughput and homogeneity requirements alongside having an efficient development time. However, many published 3D models&#160;do not satisfy these criteria. This therefore, limits their usefulness in early drug discovery applications. Three-dimensional (3D) bioprinting is a novel technology that can be applied to the development of 3D models to expedite development time, increase standardization, and increase throughput. Here, we present a protocol to develop 3D bioprinted coculture models of human induced pluripotent stem cell (iPSC)-derived glutamatergic neurons and astrocytes. These cocultures are embedded within a hydrogel matrix of bioactive peptides, full-length extracellular matrix (ECM) proteins, and with a physiological stiffness of 1.1 kPa. The model can be rapidly established in 96-well and 384-well formats and produces an average post-print viability of 72%. The astrocyte-to-neuron ratio in this model is shown to be 1:1.5, which is within the physiological range for the human brain. These 3D bioprinted cell populations also show expression of mature neural cell type markers and growth of neurite and astrocyte projections within 7 days of culture. As a result, this model is suitable for analysis using cell dyes and immunostaining techniques alongside neurite outgrowth assays. The ability to produce these physiologically representative models at scale makes them ideal for use in medium-to-high throughput screening assays for neuroscience targets.

作者聚焦:推进 3D 细胞建模 – 一种用于神经共培养的高通量方法 

Here, we present a protocol to produce 3D-bioprinted cocultures of iPSC-derived neurons and astrocytes. This coculture model, generated within a hydrogel scaffold in 96- or 384-well formats, demonstrates high post-print viability and neurite outgrowth within 7 days and shows the expression of maturity markers for both cell types.

人 iPSC 衍生的神经元-星形胶质细胞共培养物的三维生物打印用于筛选应用

For a cell model to be viable for drug screening, the system must meet throughput and homogeneity requirements alongside having an efficient development ...

Generating 3D Co-culture Spheres of Astrocytes and Neurons to Induce Synapse Formation

Source: Cvetkovic, C., et al. <a href="https://app.jove.com/v/58034">Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells.</a> J. Vis. Exp. (2018).
This video demonstrates a procedure for co-culturing the astrocytes and neurons to form 3D spheres. These 3D spheres provide a distinct platform for examining synapse formation and neural network integration, thereby serving as a potential model for research in developmental neuroscience, neurodegenerative diseases, and neuropharmacological interventions.

生成星形胶质细胞和神经元的 3D 共培养球体以诱导突触形成

1. Preparation and Maintenance of 3D Sphere Cocultures

	Dissociate hAstros from 3D aggregates in suspension.&#8203;
	
		Gently dissociate human astrocyte ...

三维组织工程化排列的星形胶质细胞网络, 以概括发展机制和促进神经系统再生

摘要

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极化神经组织工程三维蚕丝胶原蛋白为基础的模式

3D Cocultures 星形胶质细胞与人多潜能干细胞神经元的突触电路建模

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

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