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, 成年哺乳动物中枢神经系统拥有有限的新神经元来源, 以恢复创伤或神经后丢失的细胞, 因为神经源中心仅限于 SVZ, 海马齿状回, 和潜在的其他通常不活跃区域54。一个理想的策略将绕过中枢神经系统有限的疗养能力的两个方面, 目前的方法无法解决。因此, 我们的研究小组开发了 astrocyte-based 支架, 目的是为神经移植向神经元不足的区域提供生存途径, 并引导再生轴突生长锥的延伸。在琼脂糖微柱内的胶原内部排列的星束构成了这些星活支架。与常规的细胞和 biomaterial-based 策略, 用于中枢神经系统修复和再生的一个相当大的区别是, 这些结构的细胞完全预制体外模拟几个体内glia-based 指导和脚手架体系结构。例如, 在胚胎发育过程中, 桡神经胶质通路充当新神经元迁移到大脑皮层的活体支架, 而胶质细胞则介导先驱轴突的靶向延伸, 构成图案基底和/或路标细胞35 ,38,39。脑室区的胚胎桡神经胶质细胞也产生了形成纵向排列的胶质管的星形胶质, 细胞从 SVZ 到 OB 的41将后天从链式的形成中迁移。此外, 在一些 non-mammals 中已经证明, 在 CNS 损伤之后, 由于形成一个有组织的、对齐的星排列44, 可能会有更大程度的再生。例如, 成年斑马鱼能够再生他们的脊髓后, 不成熟的胶质细胞迁移到病变部位, 分化成两极形态, 并延长胶质的过程, 以弥合病变, 形成一条路径,引导再生轴突45。对齐的星束的工程构造可以综述这些机制, 并提供所需的结构和可溶性信号, 用于胶质细胞介导的神经元迁移和轴突再生35。此外, 由于包括活的星形胶质细胞, 这些结构可以主动提出所需的环境提示, 根据来自主机的反馈。
在我们的技术, 星形胶质细胞对齐是一个自组装过程的物理线索和细胞间的相互作用促进的微柱内直径, 胶原浓度, 和播种密度。这个方法的另一个诱人的特点是它的微米尺度大小, 这可能允许微创植入在受损的中枢神经系统的区域。此外, 本文的研究结果表明, 该协议所获得的结构具有很高的生存能力, 并表达了中间丝蛋白胶质、巢和波形, 而不考虑通道数所定义的星形胶质细胞年龄.在未成熟和成熟的星形胶质细胞中均表达胶质和波形, 巢主要限于未分化或未成熟的神经细胞, 成熟的胶质植入蛋白的表达和巢45,55. 以前的研究表明, 星形胶质细胞的行为随着年龄的变化而改变, 然而中间长丝蛋白的表达也有一致, 在一系列的通道数 (通道 4-12)56中也看到了捆绑。星支架也被证明达到了超, 厘米尺度的长度在体外, 这表明这项技术是适应不同的伤害长度和几何遇到的在体内。此外, 对齐的星束表现出卓越的结构完整性和稳定性, 因为它们的电缆状细胞在被从微柱中提取后仍然保持不变。Astrocyte-based 活体支架还支持神经元黏附和突起生长, 增强了这种技术的潜力, 促进定向神经元迁移和轴突延长的中枢神经修复。
对齐的星束包含在一个保护性管状水凝胶装箱的胶原内部。星支架的制作由微柱组装开始, 将针刺针插入毛细管中, 并将液体琼脂糖吸入其中。琼脂糖是所选择的生物材料, 因为它的惰性特性, 相容性, 易于控制的机械, 物理和生物化学的属性49。琼脂糖浓度的水凝胶可能是一个重要的变数, 因为细胞感觉机械刺激从他们的基板 (例如刚度), 并响应细胞内变化57。这些微柱的设计标准包括足够高的孔隙度, 以允许足够的质量运输, 但足够小的孔隙大小, 以禁止横向过程的生长;这些标准是满意的纳米级 open-porosity 目前在琼脂糖的浓度在本议定书中使用。该协议的琼脂糖浓度也选择了由于它的稳定性, 易于处理, 并在机械性能与大脑环境相似性58。一个值得注意的考虑是针头在毛细管内的中心位置。通常, 在过程中, 针可能会略微倾斜或移位, 导致在琼脂糖凝胶和从试管中去除微柱后, 管腔相对于外壁处于偏心。为了避免这种情况, 针应该是直的, 活塞管针总成应保持在垂直位置, 而琼脂糖正在绘制, 以保持针沿中心轴。精确集中的流明保护在琼脂糖壁之间排列的星形胶质细胞;腔体更容易破裂, 如果位于近边缘的圆筒的外缘。此外, 腔体与外壁之间的足够距离可在移植期间和术后提供保护性好处 (前提是星结构在植入前未从微柱上移除)。星脚手架制作的初始阶段对于技术的成功至关重要, 因为针径的选择对于正确的星形胶质细胞/过程对准和电缆自组装至关重要。我们发现, 星形胶质细胞的排列与微柱的 id 成反比, 有一个最佳的单元格对准 id 范围, 并形成了180到350µm (图 3)。同样, 先前的证明是神经元沿着最小衬底曲率的方向直接产生, 以减少过程弯曲59,60, 类似的机制可能在工作中影响星形胶质细胞系统49中的对齐方式。除了影响星形胶质细胞排列的曲率外, 我们还发现, 180 到350µm 的 id 范围导致了星状体形成双极形态的49, 与以1毫米的 id 或当星形胶质细胞在2D (图 3)。很可能, 这些深刻的形态学变化的双极星形胶质细胞形态改变他们的生理特征, 以及;例如, 这些星形胶质细胞可能表达替代的 secretable 因子或细胞表面标记, 可能有利于再生, 但这值得进一步定性。总的来说, 我们的研究结果表明, 适当选择的物理线索, 如表面曲率, 是必要的模拟本地细胞的星生活脚手架。
星支架的建造得益于将胶原蛋白 ECM 溶液和皮质星形胶质细胞依次纳入微柱的空心内部。微柱的内腔被涂上胶原蛋白, 以支持星形胶质细胞的存活、附着和排列束的形成, 因为琼脂糖无法独立支持突起的生长61。选择了1毫克/毫升的胶原蛋白, 因为先前的研究确定, 低浓度和高含量导致 ECM 缺乏黏附力和不稳定性, 分别为49。因此, 如预期的, 胶原浓度有戏剧性的影响, 该技术的有效性。此外, ECM 解决方案只包括 i 型胶原蛋白。与2D 星培养的层粘连蛋白和基质涂层基质相比, 在 i 型胶原蛋白上镀的星形胶质细胞表现出最佳的附着力和网络形成62。在细胞播种之前, 胶原聚合的孵育时间也是协议中的一个重要步骤。我们以前发现, 当神经元同时与 unpolymerized ECM 的琼脂糖微柱, 减少突起的副产物和轴突束被视为47, 这是可能的, 这些发现将扩展到星形胶质细胞也。由于星形胶质细胞粘附和束形成需要胶原蛋白, 因此在整个结构中存在 ECM, 且缺乏气泡或空口袋, 应以显微技术进行可视化和确认。
该协议的结论与星形胶质细胞电镀, 一个孵化期, 以确保细胞膜黏附胶原蛋白, 和短期的文化形成的目标细胞。星形胶质细胞之间的通道数4-12 使用, 因为他们表现出强大的星捆绑和胶原蛋白改革。这些星形胶质细胞也呈现出成熟的表型, 包括 > 95% 纯文化63,64 , 几乎没有任何神经元污染。与普通的星形胶质细胞不同的是, 它使用含血清的培养基 (图 3A,左), 我们的技术利用无血清培养基来允许胶质细胞采用过程轴承形态 (图 3A,右) , 并在微列62、65、66中形成对齐的胞和进程束。在细胞播种浓度方面, 高浓度的 9.0-12.0 x 105细胞/毫升, 与 ID 为350µm, 导致形成紧密包装捆绑伴随胶原重塑, 而星形胶质细胞更分散, 但在使用较低浓度时仍然对齐 (图 3C)。因此, 播种浓度对细胞间相互作用的影响影响星形胶质星的排列程度和观察到的索状结构的形成。在腔内注入细胞溶液通常是用立体来进行可视化, 以确保在微柱长度上均匀传递, 这对于连续束形成是至关重要的。播种后, 与培养基进行水淹的前期潜伏期是保证星形胶质细胞在 ECM 中的锚固的必要条件。如果星形胶质细胞从构造中逸出, 其持续时间可能会被修改, 前提是该结构足够湿润以防止干燥。星支架的相衬图像作为时间的函数显示, 在 8 h, 一个排列的星束网络正在形成的过程中。此外, 当构造被标记为胶原蛋白和胶质胶时, 也观察到腔壁的 ECM 脱离、随之而来的收缩以及细胞与收缩的胶原蛋白之间的紧密联系, 如图 8所示。这一现象可能是星收缩力从墙上分离胶原蛋白的结果, 这是基于先前关于胶质细胞成纤维细胞样能力的报道, 产生扭曲挠性基底和基质重塑的力。星形胶质细胞只与神经元的能力 co-cultures28,49,67,68。具体来说, 星形胶质细胞运动的后果之一和整合素介导的与胶原纤维的相互作用是产生足够的力量来收缩纤维的17,69。总体而言, 所描述的协议的实施将产生活支架, 由致密、对齐的星电缆状结构的自组装网络组成, 它重述在 CNS 发展的几个阶段中存在的物理基底。
除了在整个协议中可能出现的个别问题外, 星脚手架技术还有可能影响其修复中枢神经系统能力的其他障碍。例如, 这项技术的最终成功取决于神经系统的可塑性和星支架与宿主电路的功能整合。与这些支架的植入有关的是可能的炎症反应, 可以抵消星束的 pro-regenerative 属性。所有的 transplant-based 技术都有能力破坏血脑屏障, 并导致炎症细胞浸润, 因为在组织47中神经元和毛细血管的接近。在这种情况下, 水凝胶微柱或其他装箱方案的星支架可作为一种工具的控制释放的抗炎因子, 以减轻炎症反应。最初, 对齐的星束在微柱内不稳定, 因为迫使细胞对准和基质重塑的力最终导致了在培养1-2 天后所需细胞的崩溃。然而, 额外的收缩和捆绑崩溃的阻碍, 通过提供额外的加强, 在两端的微柱通过添加较高浓度的胶原蛋白, 以保持捆绑到位。一个额外的策略可能是采用二次封装, 据此, 新的水凝胶涂层适用于对齐的星束, 因为它们是从微型柱拉。很可能, 将定向的星形胶质细胞网络传送到大脑中, 也会稳定由于宿主之间细胞间的粘连和构建整个长度的细胞而形成的结构, 尽管这需要在今后的研究中直接进行测试。
因此, 今后的努力将着眼于开发一种有效的移植方法, 确保在体内和之后的内星束的稳定和保护, 从而使神经元迁移、修复和在受伤的中枢神经系统再生。我们先前已经成功地植入了神经元/axonal-based 微 TENNs, 它分享了星构造的装箱方案, 在 corticothalamic 通路中, 导致生存和与宿主的结构集成组织至少直到1月46,47。因此, 可以利用从微柱上提取的电缆的鲁棒性。在这种情况下, 琼脂糖凝胶将作为最初的培养床, 以诱导自组装, 然后星电缆将从装箱和直接注射到大脑中使用薄壁针。在水凝胶微柱萃取过程中, 由于机械力的作用而引起的星形胶质细胞变形没有被观察到。以前的工作研究了机械变形对星形胶质细胞反应性的影响表明, 任何结果的变形都不能迅速地应用于诱导 astrocytosis 或过程伸长62,70。为了尽量减少对脑组织的损伤和可能的炎症反应, 在水凝胶内的束可以使用以前为微 TENNs 而开发的无针技术进行植入。在这种方法中, 水凝胶被涂上一层薄薄的羧, 可以减少针头进入大脑, 从而降低植入47的破坏性和发炎的足迹。之后, 可以进行移植研究, 评估这些支架的生存能力, 与宿主组织结合, 促进神经迁移和轴突引导。特别是, 这些结构可能被植入的一端, 从神经原区, 如 SVZ, 以访问它的神经元干细胞池和细胞, 在直接模仿 RMS, 和另一端连接到受伤现场, 以潜在的驱动神经迁移, 并以计量的方式重新填充该区域。
在移植协议优化后, 该技术可以在其他领域得到改进。微柱可能是专门与未成熟的星形胶质细胞 (例如径向胶质细胞, RMS 类型单元) 或成熟的表型, 后来分化, 以增加这些构造的再生诱导能力,49。在体外胶质瘢痕模型中, 成熟的星形胶质细胞没有形成轴突生成通路的能力71。相比之下, 未成熟的星形胶质细胞被证明促进突起的生长在体外和轴突再生在体内当移植伴随与酶 ABC, 以解决高水平的 CSPGs 在神经胶质瘢痕71,72. 为了配合个性化药物的问世, 自体星微柱可由诱导多能干细胞 (干细胞) 和人神经干细胞 (hNSCs) 等来源的干细胞培养而成。这一修改将允许这些支架作为个体化和特定通路修复和再生的导管。此外, 这些细胞来源的使用可能会绕过可能的免疫反应, 星形胶质细胞植入可能导致。尽管免疫原性因 iPSC 细胞系而异, 但这些细胞在理论上能够避免或减少移植时的免疫应答, 这是由于 iPSC 衍生器官后缺乏有害反应的建议植入小鼠73,74。此外, 自体 hNSCs 及其星衍生物抑制异基因免疫应答75。因此, 用自体干细胞制备这些对齐的星形胶质细胞网络可能是一个关键的未来步骤, 使这项技术更容易在临床环境中应用。
除了作为中枢神经系统修复和再生导管的应用外, 星活支架还可作为试管内试验床, 根据特定测试范例的目标, 使用或不含水凝胶装箱。例如, 这些星构造可用于研究星形胶质细胞介导的神经元迁移和突起扩展等神经生物学现象, 利用活体支架的可控特性和3D 环境作为模型在体内条件。本文所讨论的协议详述了在凝胶微柱内的胶原基质中自组装对准星束的制备。通过综述脑神经、发育/再生机制和 neuroblast 迁移通路的特征, 这些植入的星支架有可能为定向神经迁移和轴突提供支持途径。引导在其他抑制环境中受损的中枢神经系统。

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.remotexs.ntu.edu.sg/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 &#181;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&#39;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 &#181;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 &#186;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&#39;s balanced salt solution (HBSS)</td>
			<td>Invitrogen</td>
			<td>14170112</td>
			<td>Store at 4 &#186;C.</td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Invitrogen</td>
			<td>25200056</td>
			<td>Store at -20 &#186;C and warm at 37 &#186;C before use.</td>
		</tr>
		<tr>
			<td>Bovine pancreatic deoxyribonuclease (Dnase) I</td>
			<td>Sigma</td>
			<td>10104159001</td>
			<td>Store at -20 &#186;C and warm at 37 &#186;C before use.</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM) with Ham&#39;s F-12 Nutrient Mixture</td>
			<td>Gibco</td>
			<td>11330-032</td>
			<td>Store at 4 &#186;C.</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Atlanta Biologicals</td>
			<td>S11195</td>
			<td>Store at -20&#186;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&#186;C and warm at 37 &#186;C before use.</td>
		</tr>
		<tr>
			<td>B-27 serum free supplement</td>
			<td>Invitrogen</td>
			<td>12587010</td>
			<td>Store at -20 &#186;C and warm at 37 &#186;C before use.</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Invitrogen</td>
			<td>35050061</td>
			<td>Store at -20 &#186;C and warm at 37 &#186;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&#186;C.</td>
		</tr>
		<tr>
			<td>Mouse anti-beta-tubulin III primary antibody</td>
			<td>Sigma</td>
			<td>T8578</td>
			<td>Store at -20&#186;C.</td>
		</tr>
		<tr>
			<td>Rabbit anti-collagen I primary antibody</td>
			<td>Abcam</td>
			<td>ab34710</td>
			<td>Store at -20&#186;C.</td>
		</tr>
		<tr>
			<td>Rabbit anti-vimentin</td>
			<td>Millipore</td>
			<td>AB3400</td>
			<td>Store at -20&#186;C.</td>
		</tr>
		<tr>
			<td>Mouse anti-nestin</td>
			<td>Millipore</td>
			<td>AB5326</td>
			<td>Store at -20&#186;C.</td>
		</tr>
		<tr>
			<td>Donkey anti-mouse 568 secondary antibody</td>
			<td>Invitrogen</td>
			<td>A10037</td>
			<td>Store at 4&#186;C.</td>
		</tr>
		<tr>
			<td>Donkey anti-rabbit 568 secondary antibody</td>
			<td>Invitrogen</td>
			<td>A10042</td>
			<td>Store at 4&#186;C.</td>
		</tr>
		<tr>
			<td>Donkey anti-rabbit 488 secondary antibody</td>
			<td>Invitrogen</td>
			<td>A21206</td>
			<td>Store at 4&#186;C.</td>
		</tr>
		<tr>
			<td>Hoechst 33342, Trihydrochloride</td>
			<td>Invitrogen</td>
			<td>H3570</td>
			<td>Store at 4&#186;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, 与最初分散在微柱上的细胞的存在相比, 星形细胞似乎已经收缩形成了内部的稠密束网络 ( 图 2B)。在最初的细胞对齐后, 星形胶质网通常沿着纵轴拉在一起, 类似于拉链的闭合, 在微柱的中心形成一个稠密的束 (图 2C)。这一捆绑架构的形成是由于一个自组装过程, 部分取决于选择的微柱 ID 和细胞密度。星形胶质细胞排列并获得了一个双极形态, 当种子在微列的 ID 小于350µm (图 3B, 顶部和中间, 和3 维 3 f)49。相反, 当使用 1 mm 的 ID (图 3B, 底部) 时, 这些单元格显示了随机的方向性, 这与2D 星形胶质细胞在无血清、共培养培养基 (图 3A, 右) 的外观相似。此外, 即使星形胶质细胞在低播种密度下仍然显示对准 (2.0-3.0 x 105细胞/毫升或 2.0-3.0 x 102单元格/mm3) (图 3C), 稠密网络只在高密度的密度下形成 (9.0-12。0x 105单元格/mL 或 9.0-12.0 x 102单元格/mm3) 在类似维度的圆柱形微列中 (见图 3B, 顶部), 如协议49中所建议的那样。星丛中的生存能力是用活/死法来量化的, 这是因为荧光绿 (素 am) 与荧光绿 (素) 和红色 (EthD-1) 的细胞总面积的比值。星束的可行性为 97.4%, 这与2D 星形胶质细胞培养控制的98.2% 种生存能力相似 (图 4A-4B), 证明微列内的环境适合星状胶质细胞的生命力 (图 4E)。星支架中活和死细胞的3D 重建也显示了构造中的整体单元格对齐 (图 4C-4D)。此外, 还开发了超星微柱, 研究了具有相当长的星形胶质细胞束的稳定性。即使在3.5 厘米的非凡距离, 对齐, 致密, 和收缩的星束的细胞始终保持在整个微列的长度 (图 5A), 清楚地看到在放大地区微列 (图 5B-5C)。
一旦形成, 包是固定和染色的免疫细胞化学技术的中间丝蛋白的特点, 如胶质纤维, 巢, 和波形。共焦图像确定了星形胶质细胞的过程-轴承形态和过程的纵向对准 (图 6和图 7)。中间长丝蛋白的表达可以在2D 星形胶质细胞培养 (图 7A) 中看到, 以及微柱内的星状胶质细胞束 (图 6,图 7B-7D)。此外, 胶原蛋白染色证实了这种 ECM 蛋白在微柱中的存在。在2D 环境 (图 8A) 中, 星形胶质细胞在培养时并没有形成一个连续的胶原结构, 而星形胶质细胞在微柱中被培养时, 似乎坚持并重塑了腔内存在的胶原蛋白, 从而使用于细胞收缩和束形成 (图 8B)。胶质细胞和胶原蛋白通道的叠加表明, 星形胶质细胞电缆由与纵向胶原纤维紧密相连的过程 (图 8B) 组成。因此, 除了提供锚地, 胶原纤维也可能提供额外的方向性线索, 使星形胶质细胞形成致密束后收缩。在某些情况下, 星形胶质细胞-胶原收缩持续, 导致在形成后 12-24 h 的排列的捆绑的崩溃 (图 9A)。为了稳定水凝胶微柱内的索状星形胶质细胞结构, 在一个完全成形的束的两端添加了额外的胶原基质, 以抑制进一步的收缩和塌陷。这种增强技术使所需的电缆体系结构至少能存活4天, 并且需要更长的稳定性窗口 (图 9B)。此外, 有代表性的星束从水凝胶微柱, 安装, 并固定在玻璃片4% 甲醛, 以评估其鲁棒性时, 暴露在紧张的力量和外部环境。即使在提取和物理操作到不同形状 (图 10A、 10B) 之后, 星胞和进程仍保持一个对齐的、捆绑的微尺度形态学 (图 10C), 突出显示对准星支架的弹性, 一旦形成, 即使水凝胶微柱的结构支持缺席。
原发皮层神经元也共与星形胶质细胞内的微柱, 以探索是否能够煽动神经元粘附和突起的生长。胶质细胞和微管蛋白β-蛋白 III 被分别用作星形和神经元的特异标记。图 11A显示, 具有皮质神经元的星形胶质细胞 co-seeded 也能表现出过程和形成排列的束。神经元似乎有优先附着的星形胶质细胞束, 因为所有阳性β-蛋白 III 染色 co-localized 与胶质细胞 (覆盖在图 11B-11C)。为了证明星结构的再生特性, 皮层神经元 (图 12B) 在星形胶质细胞被镀后2天内被镀在水凝胶微柱上 (图 12A)。神经元附着在星形胶质细胞丛上, 并沿星突起直接延伸。进一步的检查表明, 突起的生长方向的星电缆, 而在地区的星形胶质细胞的不存在, 突起被视为以无序的方式生长 (图 12C)。这些观察表明, 该协议的结果是一致的, 可行的, 健全的星网络, 有潜力作为植入式生存途径促进神经元粘附和突起生长。

<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55848/55848fig2.jpg"> 图 2: 明显微镜显示了随着时间的推移, 水凝胶微柱内的星形胶质细胞束的形成.(A) 星形胶质细胞形态作为电镀后时间的函数: (1 h) 胶质细胞呈球形, 分布于整个结构;(5 h) 星形胶质细胞启动过程扩展和收缩;(8 h) 星形胶质细胞排列并收缩。缩放条形图 = 100 µm. (B) 随着时间的推移在一个额外的代表性脚手架形成: (1 h) 星形胶质细胞最初是球形的, 不表现过程;(24 h) 形成了密集的星过程。缩放条形图 = 300 µm (左) 和500µm (右)。(C) 在电镀后完全形成星束24小时, 显示 "zippering" 效果, 其中电缆两端附着在管腔壁的不同端部。缩放条 = 1000 µm。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55848/55848fig3.jpg"> 图 3: 微柱内径和细胞密度影响双极星形胶质细胞排列束的自组装.(A) 2D 的星形胶质细胞具有含血清的培养基 (左) 和无血清共培养培养基 (右) 分别显示非和多的轴承形态。缩放条形图 = 100 µm. (B) 在高密度 (9.0-12.0 x 105细胞/mL) 中的星形胶质细胞在微列中以180和350µm 的 id 与纵轴形成对齐的束, 而 1 mm 的 id 则导致星形胶质细胞显示倍数,齐过程贯穿管腔的直径。缩放条形图 = 100 µm (顶部、中部) 和150µm (底部)。(C) 在低密度 (2.0-4.0 x 105单元/mL) 中, 在350µm ID 微柱上播种的星形胶质细胞对齐, 但不捆绑。缩放条 = 100 µm. (D) 当在低或中密度的350µm ID 微柱上镀时, 星形胶质细胞获得双极形态。缩放条形图 = 300 µm. (E) 框在D中显示在水凝胶微柱内形成的双极星形胶质细胞链的放大。缩放条 = 300 µm. (F) 单个双极星形胶质细胞的例子。缩放栏 = 50 µm.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55848/55848fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55848/55848fig4.jpg"> 图 4: 在水凝胶微柱内形成束层后, 星形胶质细胞仍然可行.(A-B), (C-D) 3D 共焦重建, 显示整个平面区域性和对齐的星丛中的细胞的生存能力, 分别以素 AM/溴 homodimer 染色 (绿活细胞;红死细胞)。(E) 在2D 表面和微柱上进行培养时, 活的和死细胞的量化结果与活/死星形胶质细胞的比例相似。误差线表示标准偏差。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55848/55848fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55848/55848fig5.jpg"> 图 5: 具有代表性的超长不结盟星束在水凝胶微柱内的相衬图像.(a) 束形成于整个长度为3.5 厘米的微柱。缩放栏 = 1000 µm. (B-C) 更高的放大倍率显示星形胶质细胞对齐在整个构造的各个区域, 对应于A中的方框。缩放栏 = 500 µm.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55848/55848fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/55848/55848fig6.jpg"> 图 6: 星支架的 Immunolabeling 显示了星过程的纵向对准.(A) 星束的共焦重建, 显示染色为胶质纤维 (红色; 左) 和核 (赫斯特; 中间) 和两个通道 (右) 的叠加。(B) 在A的盒装区域中, 星束的高放大倍数放大允许在微列内显示星形胶质细胞的细胞。缩放栏 = 50 µm.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55848/55848fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/55848/55848fig7.jpg"> 图 7: 通过免疫细胞化学对代表性星束进行表征.(A-b)在2D 表面培养的星形胶质细胞, 在水凝胶微柱上播种, 表达相似的星标记物 (红) 和波形 (绿色)。(a) 2D 平面星形胶质细胞培养的共焦重建, 显示分离和合并的通道。缩放条形图 = 100 µm (B) 星束的共焦图像, 确认标记的表达式并展示星形胶质细胞排列。缩放条 = 50 µm. (C-D) 在微柱内培养的星形胶质细胞还表达了中间丝蛋白巢 (红色) 和波形 (绿色)。(C) 星束的共焦图像。缩放条形图 = 100 µm (D) 更高的放大倍数, 放大C。缩放栏 = 50 µm.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55848/55848fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/55848/55848fig8.jpg"> 图 8: 对准束中的星形胶质细胞与收缩的、纵向的胶原基质相互作用, 与2D 文化中的胶原蛋白的不规则排列相对应.细胞被 immunolabeled 观察星形胶质蛋白, 细胞核 (赫斯特; 蓝色) 和胶原 (绿色)。(A) 2D 星形胶质细胞培养, 胶原蛋白在1毫克/毫升, 显示齐状胶质细胞和随机分布的胶原蛋白。缩放条 = 250 µm. (B) 胶原蛋白从微柱腔壁上被拉开, 并收缩成一个对齐的矩阵, 这是星束的一部分。缩放栏 = 150 µm.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55848/55848fig8large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/55848/55848fig9.jpg"> 图 9: 附加的胶原蛋白可用于稳定星电缆的结构.(A) 包在胶原蛋白涂层的水凝胶微柱内电镀星形胶质细胞后形成约6小时。一旦束形成, 高浓度 (3 毫克/毫升) 胶原蛋白被添加到两端的微柱, 以抑制收缩的星电缆。在结构不稳定与额外的胶原蛋白, 星胶原束已开始收缩18小时, 和星形胶质细胞电缆已完全崩溃 24 h。当捆绑用3毫克/毫升胶原蛋白强化时, 这种收缩没有被观察到。刻度条 = 1000 μ m。(B) 放大的折叠星束不加3毫克/毫升胶原蛋白。刻度条 = 500 μ m。(C) 放大胶原增强微柱的区域。星形胶质细胞束形态在整个微柱中保持完整。缩放条形图 = 500 μ m。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55848/55848fig9large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/55848/55848fig10.jpg"> 图 10: 从微柱上的星束的提取和在聚 l-赖氨酸镀膜玻璃上的安装片证明了该技术的鲁棒性。(A) 几个提取的星束的相衬图像 , 纵成拼写单词佩恩。(B) 相同提取的包的共聚焦重建染色的星标记胶质蛋白 (红色) 和细胞核 (赫斯特; 蓝色)。缩放条形图 = 100 µm . (C) 在B中的盒装区域的放大倍数显示 , 即使在纵为不规则形状的情况下 , 星支架仍保留一个对齐、双极形态和一个捆绑的细胞。缩放条形图 = 200 µm. 注意, 相位和共焦图像之间的任何结构差异很可能是由于在免疫细胞化学过程中进行冲洗而导致构造移位的伪影。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55848/55848fig10large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/55848/55848fig11.jpg"> 图 11: 神经元-星形胶质细胞共培养的支架上的神经细胞黏附并延伸突起.(a) 神经元种子星束的相衬图像。标尺 = 300 µm (B)-(C) 神经元被观察到附着在星束上, 沿电缆方向延伸突起 (赫斯特-蓝色;绿色的;β-蛋白 III-红色)。缩放条形图 = 200 和100µm, 分别为。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55848/55848fig11large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 12" class="xfigimg" src="/files/ftp_upload/55848/55848fig12.jpg"> 图 12: 星形胶质细胞电缆作为定向突起生长的活支架.共焦重建显示一个微柱的区域组成的神经元在丛形成后大约2天播种。(A) 胶质细胞阳性的星形胶质细胞束, (B) β-蛋白 III 阳性神经元, 和 (C) 合并所有通道, 包括赫斯特染色核 (蓝色)。注意, 当黏附在星形胶质细胞过程 (箭头) 的区域时, 神经元表现出突起对齐, 而不粘附在排列的神经元则出现随机 (即多向) 突起突起 (箭头)。(D) 区域的放大显示突起副产物的方向性。缩放条形图 = 100 μ m。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55848/55848fig12large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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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14.2K 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

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

平面和立体印刷的导电油墨

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

科研

生物工程

Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing

Advanced heart failure represents a major unmet clinical challenge, arising from the loss of viable and/or fully functional cardiac muscle cells. Despite optimum drug therapy, heart failure represents a leading cause of mortality and morbidity in the developed world. A major challenge in drug development is the identification of cellular assays that accurately recapitulate normal and diseased human myocardial physiology in vitro. Likewise, the major challenges in regenerative cardiac biology revolve around the identification and isolation of patient-specific cardiac progenitors in clinically relevant quantities. These cells have to then be assembled into functional tissue that resembles the native heart tissue architecture. Microcontact printing allows for the creation of precise micropatterned protein shapes that resemble structural organization of the heart, thus providing geometric cues to control cell adhesion spatially. Herein we describe our approach for the isolation of highly purified myocardial cells from pluripotent stem cells differentiating in vitro, the generation of cell growth surfaces micropatterned with extracellular matrix proteins, and the assembly of the stem cell-derived cardiac muscle cells into anisotropic myocardial tissue.

The generation of aligned myocardial tissue is a key requirement for adapting the recent advances in stem cell biology to clinically useful purposes. Herein we describe a microcontact printing approach for the precise control of cell shape and function. Using highly purified populations of embryonic stem cell derived cardiac progenitors, we then generate anisotropic functional myocardial tissue.

通过微接触印刷的定向功能性心肌组织的产生

Advanced heart failure represents a major unmet clinical challenge,  arising from the loss of viable and/or fully functional cardiac muscle  cells. ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

人体软骨组织制作采用三维喷墨打印技术

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

三维全层皮肤的生成等效和自动伤人

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

工程三维细胞化胶原凝胶的血管组织再生

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. 
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRP&#945; and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

定义人工程心脏组织建设探讨心肌细胞疗法的机理

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

用于组织工程应用的三维聚 (ε-内) 支架的熔体静电纺丝

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

三维肽支架中哺乳动物细胞的培养

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. Human-induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs), human cardiac fibroblasts (HCFs), and human umbilical vein endothelial cells (HUVECs) are isolated and used to generate a cell suspension with 70% iPSC-CMs, 15% HCFs, and 15% HUVECs. They are co-cultured in an ultra-low attachment "hanging drop" system, which contains micropores for condensing hundreds of spheroids at one time. The cells aggregate and spontaneously form beating spheroids after 3 days of co-culture. The spheroids are harvested, seeded into a novel mold cavity, and cultured on a shaker in the incubator. The spheroids become a mature functional tissue approximately 7 days after seeding. The resultant multilayered tissues consist of fused spheroids with satisfactory structural integrity and synchronous beating behavior. This new method has promising potential as a reproducible and cost-effective method to create engineered tissues for the treatment of heart failure in the future.

This protocol describes a net mold-based method to create three-dimensional scaffold-free cardiac tissues with satisfactory structural integrity and synchronous beating behavior.

基于网模的无支架三维心脏组织创建方法

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. ...

Three-dimensional Patterning of Engineered Biofilms with a Do-it-yourself Bioprinter

Biofilms are aggregates of bacteria embedded in a self-produced spatially-patterned extracellular matrix. Bacteria within a biofilm develop enhanced antibiotic resistance, which poses potential health dangers, but can also be beneficial for environmental applications such as purification of drinking water. The further development of anti-bacterial therapeutics and biofilm-inspired applications will require the development of reproducible, engineerable methods for biofilm creation. Recently, a novel method of biofilm preparation using a modified three-dimensional (3D) printer with a bacterial ink has been developed. This article describes the steps necessary to build this efficient, low-cost 3D bioprinter that offers multiple applications in bacterially-induced materials processing. The protocol begins with an adapted commercial 3D printer in which the extruder has been replaced with a bio-ink dispenser connected to a syringe pump system enabling a controllable, continuous flow of bio-ink. To develop a bio-ink suitable for biofilm printing, engineered Escherichia coli bacteria were suspended in a solution of alginate, so that they solidify in contact with a surface containing calcium. The inclusion of an inducer chemical within the printing substrate drives expression of biofilm proteins within the printed bio-ink. This method enables 3D printing of various spatial patterns composed of discrete layers of printed biofilms. Such spatially-controlled biofilms can serve as model systems and can find applications in multiple fields that have a wide-ranging impact on society, including antibiotic resistance prevention or drinking water purification, among others.

This article describes a method of transforming a low-cost commercial 3D printer into a bacterial 3D printer that can facilitate printing of patterned biofilms. All necessary aspects of preparing the bioprinter and bio-ink are described, as well as verification methods to assess the formation of biofilms.

工程生物膜的三维造型

Biofilms are aggregates of bacteria embedded in a self-produced spatially-patterned extracellular matrix. Bacteria within a biofilm develop enhanced ...