作者感谢梁美芬 l. 杨和 Burri 哈佛医学院的帮助与初步传播和量化的 ZIKV。我们还要感谢纳撒尼尔 d 帕特里克的成像支持。K.E. 得到了斯坦利精神病研究中心和哈佛干细胞研究所的支持。

1. 从 iPSC/干细胞2D 区域性创建脑 Organoids (天 0) 注意: 此协议假定在委员会介质中使用受精或 Geltrex 进行干细胞 (SC) 维护基.如果使用一种替代性的高蛋白干细胞培养培养基, 在启动化形成之前, 宜将 SC 培养物转变为委员会至少两个通道。该协议尚未通过 feeder-based SC 培养方法进行测试. 
<ol> <li> 使用常规 SC 维护方法 25 , 26 , 将区域性带到 50%-70% 汇合处。这通常是3-4 天后传代, 虽然这可能是依赖于文化方法和细胞线. 
	 注意: 大约2口井从一个6井板材是需要的生产一个充分的96井板材 organoids. </li>
	<li> 通过明显微镜检查培养基的 10 x-20 x 放大倍数, 以确保健康的菌落形态, 无可察觉的分化. </li>
	<li> 将委员会带到37和 #176; 在热水浴中进行加热, 并解冻50和 #181; 50 毫米 Y-27632 (岩石抑制剂) 和2毫升酶分离试剂到室温. </li>
	<li> 准备一个超低附着 (ULA) U 底96井板, 多通道 P200 吸管和25毫升试剂库. </li>
	<li> 分45毫升的委员会在50毫升锥形管, 并添加45和 #181; L 50 毫米 Y-27632。由研制彻底混合. </li>
	<li> 真空抽吸两个含 SCs 的井, 并使用 P1000 吸管将1毫升无酶的分离试剂迅速添加到每个油井。在37和 #176 中孵育盘; C 孵化4分钟. </li>
	<li> 真空抽吸处理好的井, 并使用 P1000 吸管将1毫升的酶分离试剂添加到每个井中。
	<ol> <li> 在37和 #176 中孵化板; C 孵化器为额外的5分钟 </li>
		<li> 卸下板并检查处理过的井。轻轻拍打盘子以分解聚集的细胞。如果大的细胞簇仍然可见, 在37和 #176 孵育板2额外的分钟; c. 重复此步骤, 直到肉眼看不到簇. </li>
	</ol> </li> <li> 一旦适当分离细胞, 将1毫升的委员会添加到每个井中, 以解除离解酶。池4毫升的细胞悬浮在一个50毫升锥管, 并采取小分细胞计数。
	<ol> <li> 将维修板中的任何未处理的井返回到37和 #176; C 恒温箱. </li>
	</ol> </li> <li> 在计数时, 将单元格悬浮在 300 x g 处离心5分钟 </li>
	<li> 确定单元格的总数, 并计算实现6万单元/mL 悬浮液所需的委员会 + Y-27632 解决方案的数量. </li>
	<li> 小心吸真空吸取上清, 并增加委员会 + Y-27632 溶液的计算量。混合5-10 次5毫升吸管, 确保一个统一的细胞悬浮. </li>
	<li> 立即将电池悬浮液转移到试剂库, 并将吸管150和 #181; L 插入 ULA U 底96孔板的每一个井中, 使用多通道 P200 吸管。这将产生96个单独的 organoids, 每个最初由9000单元组成. </li>
	<li> 离心板在 150 x g 为 1 min. </li>
	<li> 将板放入37和 #176; C 恒温箱, 请勿干扰板48小时. </li>
</ol> 2。脑化维护 (2 天) <ol> <li> 准备17毫升委员会与17和 #181; L 50 毫米 Y-27643 在50毫升锥形管。将委员会 + Y-27632 溶液加热到37和 #176; C 在热水浴中. </li>
	<li> 从孵化箱中取化板, 并使用多通道 P200 吸管设置为75和 #181; L, 从第一排的井边慢慢画出介质。一旦培养基被抽出, 迅速将培养基排出回井中, 以释放出松散的细胞和 organoids。对每行重复此操作. 
	 注意: 这项技术, 以后称为 #34;d ispersion, #34; 将细胞碎片和化吊起。化迅速下沉到井的底部, 而较小的残骸仍然悬浮, 允许它被去除与媒介变动。这有助于防止化在文化中的细胞残骸中休息。
	<ol> <li> 在所有井都被分散后, 等待十五年代, 以确保 organoids 已下沉到每口井的底部. </li>
	</ol> </li> <li> 缓慢而小心地吸入75和 #181; 从每个井中的培养基中, 使用多通道 P200 吸管并分配到一个废物库. 
	 注意: 大多数用户在常规的 feed 中偶尔会抽化。这可能发生大约1% 的时间。请相应地计划, 以确保足够的 organoids 生存到实验时间点. </li>
	<li> 将委员会 + Y-27632 溶液转移到试剂库中, 并将150和 #181; L 使用多通道 P200 吸管进入每个化井. </li>
	<li> 将板材放回 37 #176; C 恒温箱. </li>
</ol> 3。脑化维护 (3 天) <ol> <li> 温暖17毫升的委员会到37和 #176; C, 此时 没有 Y-27643. </li>
	<li> 用多通道 P200 吸管设置为100和 #181; L, 分散化板的所有井 (见步骤 2.2)。等待十五年代, 以确保 organoids 已沉没到他们的井底. </li>
	<li> 准备一个废弃的水库, 并将温热的委员会转移到一个源油藏中. </li>
	<li> 小心地抽吸125和 #181; 从每个井中吸出培养基 l, 用150和 #181 取代; 用多通道 P200 吸管代替新鲜委员会. </li>
	<li> 将板材放回 37 #176; C 恒温箱. </li>
</ol> 4。脑化维护 (4 天 +) 注意: 每隔一天进行一次定期维护, 直到感染为止. 
<ol> <li> 在开始4天的提要之前, 准备神经感应 (NI) 培养基。
	<ol> <li> 解冻250和 #181; 分2毫克/毫升肝素, 以及一个5毫升的 100X N-2 补充剂。添加250和 #181; 5 毫升的 100X N-2, 5 毫升的100X 记忆 NEAA 到500毫升的 DMEM/F12+GlutaMAX。无菌过滤器的混合解决方案在一个500毫升过滤器. 
		 注意: 不使用时, 将 NI 保持在4和 #176;在新培养基准备好之前, NI 可持续两周. </li>
	</ol> </li> <li> 热17毫升 NI 培养基到37和 #176; C. </li>
	<li> 用多通道 P200 吸管设置为100和 #181; L, 分散化板的所有井 (见步骤 2.2)。等待十五年代, 以确保 organoids 已沉没到他们的井底. </li>
	<li> 准备一个废弃的水库, 并将温热的 NI 转移到一个源油藏中. </li>
	<li> 小心地抽吸125和 #181; 从每个井中抽出培养基, 用125和 #181 替换; 用多通道 P200 吸管代替新鲜镍. </li>
	<li> 将板材放回 37 #176; C 恒温箱. </li>
</ol> 5。化感染与 ZIKV (~ 天 24) 注意: 在约3周的差异, 有 wi我将是大型上皮结构和花环可见的化, 将高度敏感 ZIKV 感染. 
<ol> <li> 将厄尔和 #39 的1X 平衡盐溶液 (EBSS)、1X PBS 和 NI 到37和 #176; C 在热水浴中. </li>
	<li> 稀释 ZIKV 的已知浓度在 1X EBSS 到目标感染多重感染 (莫伊). 
	 注意: 建议滴定不同的 MOIs, 以达到病毒剂量反应。这可能因病毒株而异;对于 ATCC VR-1838 和 ATCC VR-1843, 典型的莫伊值范围介于0.1 和10之间). </li>
	<li> 使用单通道 P200 吸管小心地从化中取出所有介质。快速添加200和 #181; L 1X PBS 每井使用 P200 多通道吸管清洗 organoids. </li>
	<li> 使用单通道 P200 吸管小心地从化中取出所有介质。快速添加50和 #181; L 1X EBSS 只 (模拟感染) 或 ZIKV 病毒解决方案, 并确保化完全淹没。重复每一个化, 将被感染的研究. </li>
	<li> 将板材放回37和 #176; C 型恒温箱2小时. 
	 注意: 与未受干扰的 organoids 相比, 在这段时间内孵化模拟感染的 organoids 不应显著影响其生长. </li>
	<li> 在病毒孵化完成前约30分钟, 分25毫升的 NI 成50毫升的锥形管, 并带到37和 #176; C 在热水澡. </li>
	<li> 在病毒暴露完成后, 用200和 #181 清洗 organoids; 每个井的 1X pbs, 允许 organoids 定居十五年代, 然后使用单一通道 P200 吸管删除 1X pbs. </li>
	<li> 添加200和 #181; 每个井都有新鲜的 NI, 然后放回孵化器。
	<ol> <li> 可选: 一天0时间点, organoids 可能会在感染后一小时被成像. </li>
	</ol> </li> <li> 通过替换125和 #181 继续进行区域性操作; 我每隔一天使用色散方法 (请参见步骤 4)。一定要正确处理废弃的介质和提示, 因为这包含有传染性的 ZIKV 粒子. </li>
</ol>

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作者声明他们没有竞争的金融利益。

在使用人脑 organoids 研究 ZIKV 感染时, 有几个注意事项需要考虑。一个重要的考虑是, 化的形成和结构是高度依赖于干细胞线, 从它们形成。在比较细胞系时要小心, 包括基因的工程线;最好的结论是使用多个 subclones, 理想的是多条干细胞系。此外, 由于这种差异的细胞线, 试点实验, 以确保适当的差异是发生在 organoids 的建议。虽然上皮结构是可见的明显微镜, 也建议进行 qRT PCR 或 cryosectioning 实验寻找神经分化标记, 如 PAX6 或磷-波形。
在同一细胞系内, 也应预测 organoids 之间的相当大的变异性。由于协议的 unpatterned 性质, 上皮结构的数量和大小在个体 organoids 之间会有所不同。通常, 你可以期望至少两个大 (&gt; 200 µm 直径由 D24) 神经花环每化。因此, 纵向研究, 如观察化大小的变化对感染, 尤其有启发意义。批次之间的可变性也可能发生, 其中最可能的原因是不准确的细胞计数或播种。建议进行多次计数, 并确保在 ULA U 底96孔板上播种前, 细胞悬浮液混合良好。
当培养 organoids 在 ULA U 底 96-井板, 计划看到可观的蒸发作用在板材的边缘附近。这是理想的填充这些井与 PBS 和只工作与中心60井。由于蒸发, organoids 在外部井将暴露在不同的条件比那些在中心, 将可能影响他们的成长和分化。在计划需要进行实验的 organoids 的数量时, 请考虑这一点。另外, organoids 将开始满96井格式在大约30天以后, 将是可看见的由于文化媒介的变色。如果计划采取 organoids 过去的30天, 建议将 organoids 转移到一个 ULA 24-井板。要进行转移, 使用剪刀削减 P1000 小费, 使开幕式比 organoids 本身大得多, 并转移 organoids 的移。
人脑 organoids 在促进该领域对神经和疾病的认识方面具有巨大的潜力。鼓励研究人员在必要时修改该协议。例如, 你可以实施模式的因素, 以提高分化效率, 以特定的细胞类型, 使他们能够探索病毒影响的某些解剖区域。ZIKV 的神经发育效应仍未得到很好的理解, 但这种新的方法将使研究人员有一个可访问、快速和高通量的方法来调查感染机制。

Zika 病毒 (ZIKV) 迅速蔓延在密克罗尼西亚, 法属波利尼西亚和美洲, 最近被证明跨越胎盘屏障1,2感染发育中的胎儿大脑, 导致神经发育疾病, 如作为小头3,4,5,6 。长期影响这些患者的生活, 目前缺乏治疗, 导致研究人员和临床医生, 以争夺更好地了解的机制背后的 ZIKV 感染和复制。以往的研究已经检查了 ZIKV 感染的体外系统在各种生理相关的细胞类型7,8,9, 以及在体内10与免疫和免疫缺陷小鼠11,12,13和非人类灵长类动物141516。除了这些更传统的技术, 几个小组已经实施干细胞衍生脑 organoids, 以了解更多关于 ZIKV 感染的发展中的人脑。这些组已利用人脑 organoids 确认小头表型17,18, 调查与病毒条目相关的受体19, 检查对感染的生理反应20,21, 潜在的筛选药物候选者22。这里描述了一种快速产生和感染干细胞衍生脑 organoids 的技术, 如前所示19, 以提高对发展中人脑的 ZIKV 感染的认识。
由于 organoids 是由标准多能干细胞 (PSC) 培养而成的, 因此这种技术可以对发展中的大脑的病毒感染问题进行各种科学的解答。例如, CRISPR 工程可用于在化感染之前以更快的速度修改这些 PSC 线, 而不是大多数在体内遗传研究19。另外, 与标准二维 (2D) 分化培养不同, organoids 展示了复杂的细胞结构, 这对 corticogenesis 是至关重要的, 最近的研究表明, 这种体系结构可能在感染时被破坏。21。最后, 相对于体内模型而言, 生成 organoids 的成本较低, 可以进行更高的吞吐量试验和筛选。另一方面, organoids 在 ZIKV 研究中的运用也存在一些弊端。虽然 organoids 比2D 文化更具生物学意义, 但由于其三维 (3D) 的性质, 在 organoids 的评估中还有其他的挑战。organoids 的成像和离解往往涉及更多的设备和比标准2D 文化更大的资源投资。此外, organoids 缺乏的血管和免疫成分存在于体内模型, 因此, 研究人员对这些方面的病毒感染建议寻求一个替代协议。
在文化中有许多技术来形成人类的 organoids 和球, 它们通常属于图案或unpatterned类别。图案化方法实现了调节 Wnt、BMP、TGFβ和其他信号通路的因素, 以将分化推向特定的血统23。Unpatterned 方法, 如这里描述的, 利用诱导的多能干细胞 (干细胞) 和人类胚胎干细胞 (干细胞) 的倾向, 以区别于神经血统的默认24。经过大约三周的分化, 由此产生的 organoids 包括大型, 仿生上皮结构, 包含几个细胞类型, 在早期发育的大脑中观察到。
提出了从标准、无饲养的 PSC 文化中生产 organoids 的技术, 并将这些 organoids 与 ZIKV 进行了充分的研究。关于无馈线公司所需的培养方法的指导, 请参阅以前的方法出版物25,26。此外, 为了在多个实验中应用一致的病毒数量, 重要的是要提前计算语言教学。这是通过进行感染的维罗细胞, 其次是治疗的叠加培养基, 孵化, 和免疫。此技术的描述和方法以前已被描述为19,27。
一旦 PSC 文化达到 50%-70% 的目标汇流, 细胞就会被离解并聚集到超低附着的 96-井板中。细胞被维护3天在无异的干细胞维护媒介 (委员会), 然后转换成神经归纳媒介为文化的剩下的人。一旦 organoids 有分化3周, 感染可以进行。通过在感染后一周内例行拍摄图像, 研究人员将观察化细胞的渐进性死亡和破坏。研究人员也可能在这个时候分离 organoids 进行转录或蛋白质组分析。Cryosectioning 和 lightsheet 方法是建议的成像, 研究人员可以期望看到高水平的感染和病毒复制, 特别是在神经祖细胞 (NPC) 的人口在 organoids。最终, 这项技术使研究人员能够迅速检测到低成本和有限设备的人脑病毒感染的机制。

<table><tbody><tr><td>Enzyme-Free Detachment Reagent, ReLeSR</td><td>StemCell Technologies</td><td>5873</td><td></td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D2650-100ML</td><td></td></tr><tr><td>Glutamate-supplemented DMEM/F12 (1X)</td><td>Gibco</td><td>10565-018</td><td>(+)2.438 g/L sodium bicarbonate, (+)sodium pyruvate</td></tr><tr><td>N-2 Supplement (100X)</td><td>Gibco</td><td>17502-048</td><td></td></tr><tr><td>MEM Non-Essential Amino Acids (MEM-NEAA)</td><td>Gibco</td><td>11140-050</td><td></td></tr><tr><td>Earle&#039;s Balanced Salt Solution (EBSS)</td><td>GE Healthcare Life Sciences</td><td>SH30029.02</td><td></td></tr><tr><td>Stericup Filter (500mL)</td><td>Millipore</td><td>220009-140</td><td></td></tr><tr><td>Costar Ultra-Low Attachment Plate, 96-Well, Round Bottom</td><td>Corning</td><td>7007</td><td></td></tr><tr><td>Low Residual Volume Reagent Reservoir (25mL)</td><td>Integra</td><td>4312</td><td></td></tr><tr><td>Pipet-Lite Multi Pipette L12-200XLS+</td><td>Rainin</td><td>17013810</td><td></td></tr><tr><td>Large Centrifuge, Culture Plate-Compatible</td><td>Thermo Scientific</td><td>75007210</td><td></td></tr><tr><td>Culture Plate Centrifuge Rotor</td><td>Thermo Scientific</td><td>75005706</td><td></td></tr><tr><td>Vero Cells</td><td>ATCC</td><td>CCL-81</td><td>For virus expansion, if necessary</td></tr><tr><td>Zika Virus (Uganda Strain)</td><td>ATCC</td><td>VR-1838</td><td>Other strains may be used</td></tr><tr><td>Zika Virus (Puerto Rico Strain)</td><td>ATCC</td><td>VR-1843</td><td>Other strains may be used</td></tr><tr><td>phospho-vimentin</td><td>MBL</td><td>D076-3</td><td>mouse polyclonal,&amp;nbsp; 1:250 dilution</td></tr><tr><td>TBR2</td><td>Abcam</td><td>ab23345</td><td>rabbit polyclonal, 1:300 dilution</td></tr><tr><td>MAP2</td><td>Novus</td><td>NB300-213</td><td>chicken polyclonal, 1:1500 dilution</td></tr><tr><td>GFAP</td><td>Cell Signaling Technologies</td><td>CST12389S</td><td>rabbit polyclonal, 1:200 dilution</td></tr><tr><td>D1-4G2 flavivirus envelope protein</td><td>EMD Millipore</td><td>MAB10216</td><td>mouse monoclonal, 1:500 dilution</td></tr><tr><td>cleaved caspase-3</td><td>Cell Signaling Technologies</td><td>9661</td><td>rabbit polyclonal, 1:200 dilution</td></tr><tr><td>488 anti-mouse IgG</td><td>Thermo Fisher</td><td>A-11001</td><td>goat polyclonal, 1:1000</td></tr><tr><td>594 anti-rabbit IgG</td><td>Thermo Fisher</td><td>A-11037</td><td>goat polyclonal, 1:1000 dilution</td></tr><tr><td>647 anti-chicken IgY</td><td>Thermo Fisher</td><td>A-21449</td><td>goat polyclonal, 1:10&lt;span class=&quot;font0&quot;&gt;00 dilution&lt;/span&gt;</td></tr></tbody></table>

modelling zika virus infection of the developing human brain in vitro using stem cell derived cerebral organoids

最近 Zika 病毒 (ZIKV) 在易感人群中的出现导致了新生儿小头和其他神经发育状况的突然增加。虽然蚊子是病毒传播的主要途径, 但它也被证明通过性接触和垂直 mother-to-fetus 传输传播。在后一种情况下的传输, 由于独特的病毒取向的 ZIKV, 该病毒被认为是主要目标的神经祖细胞 (npc) 发展的大脑。
这里描述了 ZIKV 感染的建模方法, 以及当人类大脑 organoids 暴露于活 ZIKV 时产生的小头。organoids 在其神经祖群中显示高水平的病毒, 并随着时间的推移表现出严重的细胞死亡和小头。这个三维脑化模型允许研究人员进行物种配对实验, 观察和潜在干预与发展中的人脑的 ZIKV 感染。该模型提供了改进的相关性比标准 two-dimensional 方法, 并包含了人类特有的细胞结构和蛋白表达, 是不可能的动物模型。

在化形成的那一天, SC 文化应该在日志生长阶段, 在其中他们是 50%-70% 汇合, 如图 1(A-c)所示。区域性应形成具有不同边缘的圆形菌落, 并且井应明确区分图 1(D-E)。如果发生差异, 建议在创建 organoids 前至少一天内进行 pick-to-remove, 以清理板块内部受影响的区域。如果文化的大部分被区分, 可能有必要进行额外的通道, 解冻新的小瓶, 或改变干细胞培养技术, 以确保 organoids 是由纯干细胞培养形成。
由无酶和酶分离试剂的结合处理的离解应该导致多数单细胞, 虽然可能仍然有小聚集细胞观察在计数期间。小团聚体的存在不会破坏化的形成, 尽管它可能会影响计数精度。建议每个样本进行多次计数, 如果计数变化超过 20%, 则可能需要进一步增加离 singularize 细胞的离解时间。
一旦细胞被播种并在一个 ULA 的 U 底96井板 (图 2a) 中旋转下来, 它们就会在每个井的底部显示为扁平的、致密的细胞板。细胞在接下来的两天内会慢慢聚集。最好不要在这48小时内扰乱板块, 因为机械力会扰乱松散形成的骨料。在前10天, 化将保持主要球形, 并将增长从〜300µm 到600-800 µm (图 2B)。在10天和20天 (图 2C-E)之间的化中, 更可观察到的结构开始出现。在24天, 研究人员应该能够通过明显微镜观察玫瑰样的结构 (图 2F)。在继续进行区域性的同时, 这些上皮结构可能会在数天内增加数量和大小 (图 2G)。在喂食过程中, 研究者会注意到在每一个井的底部收集大量的碎片, 特别是在最初的2周的文化中。步骤2.2 中描述的色散技术去除了大部分的细胞碎片, 以防止凋亡的碎片堆积在多个源上 (图 3A)。这些碎片可以干扰成像, 它可以使它更具挑战性的观察和量化感染诱导细胞死亡, 它可能提供不对称信号的邻近化, 可能会影响分化。如果对色散技术进行了正确的处理, 则应减少此方法 (图 3B-C, 3 d-E)。
建议进行 cryosections 或 lightsheet 成像, 以检查在 organoids 内形成的皮层结构。在25天 organoids, 莲座丛结构和心室区在 DAPI (图 4A) 和磷-波形 (图 4B) 染色中清晰可见。TBR2 (图 4C) 的存在表明中间的祖细胞正在形成, 其形态学表明向外迁移。MAP2+ (图 4D) 神经元填充每个莲座丛的外部区域。从这些蛋白质, 你可以可视化的心室区 (VZ), 脑室区 (SVZ), 中间区 (伊兹), 皮质板 (CP) 在每个花环 (图 4E,4E ")。你能继续培养这些 organoids 为了观察发展的以后阶段。虽然皮质分层在这种类型的化不突出, 自然开关到 gliogenesis 确实发生了很像它在体内。这可以在108天的 organoids 中观察到, 其中很大一部分细胞表示 MAP2 或胶质蛋白 (图 4F-I)。
用户可能希望在 ZIKV 感染后的大约3天内看到化大小的差异, 具体取决于应用于 organoids (图 5A-b) 的病毒语言。在这3天之后, 与模拟井相比, 受感染水井中的细胞碎片也会增加。化大小的这种差异将在接下来的一周内增加, 直到感染的 organoids 开始分离 (图 5C-d)。在这一点上可以使用多种化验方法来调查感染机制, 包括病毒 RNA 提取和 cryosectioning (在材料表中报告的推荐抗体)。在 cryosectioning, 用户将观察到在上皮结构的顶端区域有大量的病毒存在, 这表明神经祖细胞 (npc) 对 ZIKV 感染的敏感性 (图 6)。免疫荧光切片 organoids 也将显示在被感染的 organoids 的 caspase-3 表达增加 (图 7)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56404/56404fig1.jpg"> 
图 1: 在化形成之前, 干细胞菌落形态. 殖民地应该是 50%-70% 汇合在分离时产生大量的一致 organoids。(A)稀疏、最近播种的区域性尚未达到日志增长阶段, 不会产生许多 organoids。(B)一旦细胞到达适当的汇合处, 它们就可以分离。同样重要的是, 这些殖民地的检查, 以确保它们是明确的分化。(C)太远的殖民地将达到 over-confluence, 不建议化形成。这种状态下的区域性更可能包含区分单元格。(D)菌落边缘应是不同的, 中心的圆角单元格具有一致的细胞形态学, 边缘上有稍拉长的细胞。(E)在区域性中应存在最小差异。如果在菌落的中心或边缘 (白色箭头) 观察到更大的扁平细胞, 并构成超过大约1% 的培养物, 建议 pick-to-remove 或其他通道进行, 以形成一个均匀的干细胞种群。<a href="//ecsourc href=""></a href="//ecsourc>文件/ftp_upload/56404/56404fig1large. jpg "目标 =" _blank ">> 请点击这里查看这个数字的一个更大的版本。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56404/56404fig2.jpg"> 
图 2: 脑化随时间增长. 
(a)从2D 公司中维护的 organoids 的生产图, 无异、无馈线介质。(B)在最初的8天内, organoids 将仍然主要是球形, 很少有可检测的特征。化的直径将范围从大约300µm 到500µm. (C-E)在10天, 更清晰的区域将开始被探测周围的 organoids, 他们将失去他们的球形形状。它们的大小将继续增加到直径约1毫米, 这主要取决于用于生产 organoids 的细胞线。(F)在大约20天的差异化后, 用户将观察上皮结构 (黑色箭头) 在大多数 organoids 的清晰外围区域的形成。这些有环形的形态学, 顶端和基底结构模仿那些发展中的皮层。(G)这些上皮结构将在大约20-30 天的时间内继续增长并增加大小。<a href="//ecsource.jove.com/files/ftp_upload/56404/56404fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56404/56404fig3.jpg"> 
图 3: 通过分散饲料技术清除细胞碎片. 在化生长期间, 大量的细胞死亡是预期的, 并可能导致堆积的细胞碎片周围的基地的化。(A)所描述的色散技术允许研究人员在每次进食时移除大部分碎片。要做到这一点, 使用多通道 P200 吸管, 慢慢地绘制使用的培养基, 并迅速排出, 以解除化和细胞碎片悬浮。化将迅速下落到井的底部, 在这个时候可以进行常规饲料。在(B)和(C)后的6天化的例子中, 分散喂养显示了大量的碎片减少。这将持续数周, 如在(D)之前的18天化和(E)分散喂养之后显示。<a href="//ecsource.jove.com/files/ftp_upload/56404/56404fig3large.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/56404/56404fig4.jpg"> 
图 4: 通过免疫组织化学表征脑 organoids. Lightsheet 脑 organoids 的图像显示, 提供一个细胞结构的例子, 形成。在25天, DAPI (A)和磷光-波形(B)分别表示单独的花环和心室区。(C) TBR2 标签正向外迁移的中间祖和 MAP2 (D)标签在皮质板中形成的神经元。(e, e ')这些蛋白质的结合清楚地显示了脑化模型中皮质发育的重述。(F-I)在108天, gliogenesis 已经发生, 与混合的胶质蛋白 + 和 MAP2+ 细胞填充大部分的化。VZ = 心室区, SVZ = 脑室区, 伊兹 = 中间区, CP = 皮质板。<a href="//ecsource.jove.com/files/ftp_upload/56404/56404fig4large.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/56404/56404fig5.jpg"> 
图 5: 感染的 organoids 的降解. 
(A)在莫伊 = 0.1 和10中模拟和 ZIKV 感染的脑 organoids 的明亮场图像。图像被采取后立即感染 (天 0), 以及3和6天感染。(B-c)明周围的细胞碎片的图像(B)模拟和(C) ZIKV 感染在莫伊 = 10 脑 organoids 3 天感染。<a href="//ecsource.jove.com/files/ftp_upload/56404/56404fig5large.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/56404/56404fig6.jpg"> 
图 6: 感染 organoids 的 Cryosection 和免疫荧光. 24天 organoids 被暴露在 ZIKV 在莫伊 = 0.1, 和 cryosectioned 6 天后。在这一阶段, 有明显的病毒存在于心室, 脑室, 和中间区的化, 其中神经祖细胞和径向胶质的驻留。(A)通过核染色, 这些上皮区域显然是可见的, 因为 (白色箭头) 到核的高密度, 形成环形结构的顶端表面周围。(B)通过对病毒包络进行染色, 可以观察到这些莲座丛结构 (白色箭头) 中病毒的相对较高的存在。注意, 在各种不明的外围细胞中存在少量的病毒。(C-D)MAP2 或其他神经元特异的污渍表明, 有神经元存在的文化, 似乎没有感染的病毒时, 覆盖的病毒染色。<a href="//ecsource.jove.com/files/ftp_upload/56404/56404fig6large.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/56404/56404fig7.jpg"> 
图 7: caspase-3 感染化的免疫荧光. 感染后, 人们可以使用凋亡指标来研究 organoids 内发生的细胞死亡的机制。在这个例子中, 24 天 organoids 被暴露在 ZIKV 在莫伊 = 0.1, 和 cryosectioned 6 天后。(A)未感染的 organoids 显示没有病毒包膜 (4G2 蛋白), 以及在组织中发生的自我平衡的细胞凋亡的少量 caspase-3。(B)受感染的花环开始显示细胞凋亡水平的升高。(C)通过4G2 染色显示严重感染的花环也倾向于在这些区域表现出高水平的劈开 caspase-3。感染的严重性与被感染的花环内的 caspase-3 表达有直接关系。<a href="//ecsource.jove.com/files/ftp_upload/56404/56404fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids at JoVE.com

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

利用干细胞衍生脑 Organoids体外培养人脑组织 Zika 病毒感染的模拟研究

本协议描述了一种用于开发人脑的 Zika 病毒感染模型的技术。利用野生或工程化的干细胞系, 研究人员可以利用这种技术来发现可能影响早期脑部感染的各种机制或治疗方法, 并在 Zika 病毒感染的胚胎中产生小头。

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions ...

modelling-zika-virus-infection-developing-human-brain-vitro-using

Research

JoVE Journal

Neuroscience

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

10.4K 次观看.  Novartis Institutes for BioMedical Research. 本协议描述了一种用于开发人脑的 Zika 病毒感染模型的技术。利用野生或工程化的干细胞系, 研究人员可以利用这种技术来发现可能影响早期脑部感染的各种机制或治疗方法, 并在 Zika 病毒感染的胚胎中产生小头。

视频: Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that limits the translation of basic brain research into clinical application. While induced pluripotent stem cells (iPSCs) have replaced the ethically questionable human embryonic stem cells, iPSC-based neuronal differentiation studies remain descriptive at the cellular level but fail to adequately provide the details that could be derived from a complex, 3D human brain tissue. 
This gap is now filled through the application of iPSC-derived, 3D brain organoids, "Brains in a dish," that model many features of complex human brain development. Here, a method for generating iPSC-derived, 3D brain organoids is described. The organoids can help with modeling autosomal recessive primary microcephaly (MCPH), a rare human neurodevelopmental disorder. A widely accepted explanation for the brain malformation in MCPH is a depletion of the neural stem cell pool during the early stages of human brain development, a developmental defect that is difficult to recreate or prove in vitro. 
To study MCPH, we generated iPSCs from patient-derived fibroblasts carrying a mutation in the centrosomal protein CPAP. By analyzing the ventricular zone of microcephaly 3D brain organoids, we showed the premature differentiation of neural progenitors. These 3D brain organoids are a powerful in vitro system that will be instrumental in modeling congenital brain disorders induced by neurotoxic chemicals, neurotrophic viral infections, or inherited genetic mutations.

Modeling human brain development has been hindered due to the unprecedented complexity of neural epithelial tissue. Here, a method for the robust generation of brain organoids to delineate early events of human brain development and to model microcephaly in vitro is described.

源自iPSC的人脑组织体的一代新型早期神经发育障碍

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that ...

科研

发育生物学

Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental neurobiology. Rather than use an animal model or genetically modified induced pluripotent cells, human neural stem cells provide an effective in vitro system to examine the effects of treatments, screen drugs, or examine individual differences. Here, we provide the detailed protocols for methods used to expand human fetal brain neural stem cells in culture with serum-free media, to differentiate them into various neuronal subtypes and astrocytes via different priming procedures, and to freeze and recover these cells. Furthermore, we describe a procedure of using human fetal brain neural stem cells to study Zika virus infection.

This article details the methods that are used to expand human fetal brain neural stem cells in culture, as well as how to differentiate them into various neuronal subtypes and astrocytes, with an emphasis on the use of neural stem cells to study Zika virus infection.

人胎脑神经干细胞 Zika 病毒感染的免疫细胞化学分析

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental ...

Brain Organoid Generation from Induced Pluripotent Stem Cells in Home-Made Mini Bioreactors

The iPSC-derived brain organoid is a promising technology for in vitro modeling the pathologies of the nervous system and drug screening. This technology has emerged recently. It is still in its infancy and has some limitations unsolved yet. The current protocols do not allow obtaining organoids to be consistent enough for drug discovery and preclinical studies. The maturation of organoids can take up to a year, pushing the researchers to launch multiple differentiation processes simultaneously. It imposes additional costs for the laboratory in terms of space and equipment. In addition, brain organoids often have a necrotic zone in the center, which suffers from nutrient and oxygen deficiency. Hence, most current protocols use a circulating system for culture medium to improve nutrition.
Meanwhile, there are no inexpensive dynamic systems or bioreactors for organoid cultivation. This paper describes a protocol for producing brain organoids in compact and inexpensive home-made mini bioreactors. This protocol allows obtaining high quality organoids in large quantities.

Here we describe a protocol for generating brain organoids from human induced pluripotent stem cells (iPSCs). To obtain brain organoids in large quantities and of high quality, we use home-made mini bioreactors.

在自制迷你生物反应器中从诱导多能干细胞中生成脑类器官

The iPSC-derived brain organoid is a promising technology for in vitro modeling the pathologies of the nervous system and drug screening. This technology ...

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV is an RNA virus of the Flaviviridae family. ZIKV is mainly transmitted by mosquitoes, but can also be spread by maternal to fetal vertical transmission as well as sexual contact. To date, there are no reliable treatment or vaccine options available to protect those infected by the virus. The development of a reproducible, effective Zika virus infectious cell culture system is critical for studying the molecular mechanisms of ZIKV replication as well as drug and vaccine development. In this regard, a protocol describing a mammalian cell-based in vitro Zika virus culture system for viral production and growth analysis is reported here. Details on the formation of plaques by Zika virus on a cell monolayer and plaque assay for measuring viral titer are presented. Viral genome replication kinetics and double-stranded RNA genome replicatory intermediates are determined. This culture platform was utilized to screen against a library of a small set of cytokines resulting in the identification of interferon-&#945; (IFN-&#945;), IFN-&#946; and IFN-&#947; as potent inhibitors of Zika viral growth. In summary, an in vitro infectious Zika viral culture system and various virological assays are demonstrated in this study, which has the potential to greatly benefit the research community in elucidating further the mechanisms of viral pathogenesis and the evolution of viral virulence. Antiviral IFN-alpha can further be evaluated as a prophylactic, post-exposure prophylactic, and treatment option for Zika virus infections in high-risk populations, including infected pregnant women.

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV), an emerging pathogen, is linked to fetal developmental abnormalities and microcephaly. The establishment of an effective infectious cell culture system is crucial for studies of ZIKV replication as well as vaccine and drug development. In this study, various virological assays pertaining to ZIKV are illustrated and discussed.

兹卡病毒感染细胞培养系统和<em&gt;体外</em&gt;干扰素的预防作用

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV ...

免疫与感染

灵长类动物大脑类器官的基因改造

Targeted Microinjection and Electroporation of Primate Cerebral Organoids for Genetic Modification

The cerebral cortex is the outermost brain structure and is responsible for the processing of sensory input and motor output; it is seen as the seat of higher-order cognitive abilities in mammals, in particular, primates. Studying gene functions in primate brains is challenging due to technical and ethical reasons, but the establishment of the brain organoid technology has enabled the study of brain development in traditional primate models (e.g., rhesus macaque and common marmoset), as well as in previously experimentally inaccessible primate species (e.g., great apes), in an ethically justifiable and less technically demanding system. Moreover, human brain organoids allow the advanced investigation of neurodevelopmental and neurological disorders.
As brain organoids recapitulate many processes of brain development, they also represent a powerful tool to identify differences in, and to functionally compare, the genetic determinants underlying the brain development of various species in an evolutionary context. A great advantage of using organoids is the possibility to introduce genetic modifications, which permits the testing of gene functions. However, the introduction of such modifications is laborious and expensive. This paper describes a fast and cost-efficient approach to genetically modify cell populations within the ventricle-like structures of primate cerebral organoids, a subtype of brain organoids. This method combines a modified protocol for the reliable generation of cerebral organoids from human-, chimpanzee-, rhesus macaque-, and common marmoset-derived induced pluripotent stem cells (iPSCs) with a microinjection and electroporation approach. This provides an effective tool for the study of neurodevelopmental and evolutionary processes that can also be applied for disease modeling.

作者聚焦:灵长类动物脑类器官的靶向显微注射和电穿孔以进行基因改造  

The electroporation of primate cerebral organoids provides a precise and efficient approach to introduce transient genetic modification(s) into different progenitor types and neurons in a model system close to primate (patho)physiological neocortex development. This allows the study of neurodevelopmental and evolutionary processes and can also be applied for disease modeling.

灵长类动物脑类器官的靶向显微注射和电穿孔用于基因修饰

The cerebral cortex is the outermost brain structure and is responsible for the processing of sensory input and motor output; it is seen as the seat of ...

<meta charset="utf8"></head><body>使用 iPSC 衍生的 HPC 生成具有驻留小胶质细胞的人脑类器官

Derivation of a Human Brain Organoid with Microglia Development

Three-dimensional (3D) brain organoid cultures derived from induced pluripotent stem cells (iPSC) provide an important alternative in vitro tool&#160;for studying human brain development and pathogenesis of neurological diseases. However, the lack of incorporation of microglia in the human brain organoids is still a major hurdle for 3D models of neuroinflammation. Current approaches include either the incorporation of fully differentiated microglia into mature brain organoids or the induction of microglial differentiation from the early stage of iPSC-derived embryoid bodies (EBs). The first approach misses the stage when microglial differentiation interacts with the adjacent neural environment, and the later approach is technically challenging, resulting in inconsistency among the final organoids in terms of the quantity and quality of microglia. To model brain organoids with microglia to study the early interactions between microglial and neuronal development, highly pure hematopoietic progenitor cells (HPC) differentiated from human iPSCs were incorporated&#160;into iPSC-derived EBs to make brain organoids. Using immunostaining and single-cell RNA sequencing (sc-RNA-seq) analysis, we confirmed that HPCs were incorporated into the 3D organoids, which eventually developed into brain organoids with both microglia and neurons. Compared to brain organoids without HPCs, this approach produces significant microglial incorporation in the brain organoids. This novel 3D organoid model, which consists of both microglial and neural development properties, can be used to study the early interactions between innate immune and nervous system development and potentially as a model for neuroinflammation and neuroinfectious disorders.

<meta charset="utf8"></head><body>小胶质细胞发育的人脑类器官的衍生

We present a protocol to generate a human brain organoid with resident microglia by incorporating Induced pluripotent stem cell (iPSC)-derived hematopoietic progenitor cells (HPCs) into organoid development.

小胶质细胞发育的人脑类器官的衍生

Three-dimensional (3D) brain organoid cultures derived from induced pluripotent stem cells (iPSC) provide an important alternative in vitro tool&#160;for ...

利用干细胞衍生脑 Organoids体外培养人脑组织 Zika 病毒感染的模拟研究

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源自iPSC的人脑组织体的一代新型早期神经发育障碍

人胎脑神经干细胞 Zika 病毒感染的免疫细胞化学分析

在自制迷你生物反应器中从诱导多能干细胞中生成脑类器官

兹卡病毒感染细胞培养系统和

灵长类动物脑类器官的靶向显微注射和电穿孔用于基因修饰

灵长类动物脑类器官的靶向显微注射和电穿孔用于基因修饰

灵长类动物脑类器官的靶向显微注射和电穿孔用于基因修饰

小胶质细胞发育的人脑类器官的衍生

小胶质细胞发育的人脑类器官的衍生

小胶质细胞发育的人脑类器官的衍生