Name: facilitating cerebral organoid culture via lateral soft light illumination
Uploaded: 2022-06-06T14:00:00
Description: Watch this Scientific Journal Video about Facilitating Cerebral Organoid Culture via Lateral Soft Light Illumination at JoVE.com

本研究由广东省自然科学基金（批准号：2020A0505100062）、广州市科技重点课题项目（第201904020025号）、国家自然科学基金项目（31872800号、32070582、82101937）和广州市博士后科研资助项目（陈邦珠）资助。

该议定书是在《赫尔辛基宣言》之后进行的。经广州医科大学第三附属医院伦理委员会批准（医德伦理审查[2021]022号）。在实验之前，每种培养基都是根据Juergen A. Knoblich的配方12 （补充表1-4）制备的，或者使用市售的脑类器官试剂盒（见 材料表）。本研究中使用的iPSC先前由我们的实验室建立，并已获得知情豁免。SCA3-iPSCs由一名31岁的女性脊髓小脑共济...

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大脑类器官为医学研究开辟了新的途径。该技术的许多有用应用才刚刚开始探索28.本研究发现，遗传性疾病性脑类器官与正常脑类器官的转录组测序结果可以反映疾病与健康之间的差异。例如，RNA-seq数据分析结果（图3B）与许多报道的SCA3疾病29，30，31，32的研?...

与二维（2D）培养系统相比，三维（3D）培养系统具有几个优点，包括某些器官的复杂结构的真实复制和有效复制1。因此，脑类器官是人脑发育与疾病2、药物筛选、细胞治疗等研究领域的重要辅助方法之一。
通过旋转悬浮法培养脑类器官有利于它们的发育和成熟3.虽然大脑类器官培养系统取得了巨大的成功，但它们仍然面临着限制其应用的关键挑战。例如，手工栽培涉及复杂的操作步骤，并给实现大规模应用带来了障碍。此外，在脑类器官培养的每个发育阶段，需要改变不同的培养基和细胞因子4.然而，在早期阶段，类器官或EB具有微小的尺寸（约200μm至300μm），并且在没有适当的设备的情况下几乎无法在视觉上进入。不可避免地，当介质发生变化时，一定量的珍贵类器官样品被冲走。已经探索了许多技术来克服其他类型的类器官培养物中的这一点，一些例子包括将整个类器官芯片浸入培养基中3天而不进行干预5;在旧介质被吸收后，使用吸水纸5通过盖玻片添加新鲜介质;或应用复杂的微流体管道进行流体交换6，7，8。在类器官培养的早期阶段遇到的另一个障碍是难以用肉眼进行直接观察，这可能导致操作不良，导致类器官移植步骤中的类器官损伤和丢失。因此，有必要建立一个更可行的方案，促进介质变化和类器官转移以产生类器官。
为了克服这些问题，提出了一种基于工程原理的相应优化操作，这显着且方便地促进了许多类器官手术。在自然界中，当太阳透过窗户缝隙照射到房屋中时，肉眼可以看到光束中舞动的灰尘。由于太阳光对尘埃的漫反射，一些光线被折射到眼球中以产生视觉图像。受9，10现象原理的启发，本研究制作了柔和的光灯并横向照亮了EB。研究发现，EB可以在不影响观察范围的情况下在视觉上清晰可见。由于涡流11，通过旋转培养板在液体中产生指向中心的二次流。原本分散的EB积聚在板的中心。基于此，针对类器官沉降速度快于细胞的现象，提出一种无需离心即可进行介质更换和类器官转移的简便操作方法。通过这种转移操作，培养基中的类器官可以有效地与游离细胞和死细胞碎片分离。
这里提出了一种易于操作的方案，从人多能干细胞中产生脑类器官。通过应用工程原理优化操作技术，使3D文化中的操作与2D文化中的操作一样简单可行。改进的液体交换方法和类器官转移操作也有助于其他类型的类器官培养和自动培养机的设计。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 &#956;m Filter</td>
			<td>NEST Biotechnology, China</td>
			<td>331001</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#956;L wide-bore pipette tip</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>9405163</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#956;L wide-bore pipette tip</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>9405020</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Merck, Germany</td>
			<td>8057400005</td>
			<td></td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde</td>
			<td>Servicebio, China</td>
			<td>G1101</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well low adhesion plate</td>
			<td>NEST Biotechnology, China</td>
			<td>703011</td>
			<td>It is used for EBs suspension cultures</td>
		</tr>
		<tr>
			<td>Aaccute cell detachment solution</td>
			<td>STEMCELL Technologies, Canada</td>
			<td>07920</td>
			<td>It is used to digest iPSC into single cells.</td>
		</tr>
		<tr>
			<td>AggreWell800 24-well</td>
			<td>STEMCELL Technologies, Canada</td>
			<td>34811</td>
			<td>Microporous culture plate for EBs preparation.</td>
		</tr>
		<tr>
			<td>Anti-Adherence Rinsing Solution</td>
			<td>STEMCELL Technologies, Canada</td>
			<td>07010</td>
			<td>Rinsing solution for cultureware to prevent cell adhesion</td>
		</tr>
		<tr>
			<td>B27-vit. A supplement</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>12587010</td>
			<td></td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>Peprotech, USA</td>
			<td>GMP100-18B</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Beyotime Biotechnology, China</td>
			<td>ST025</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf, Germany</td>
			<td>5810 R</td>
			<td>It can be used for centrifugation of various types of centrifuge tubes, reagent bottles and working plates.</td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>Shitai Laboratory Equipment, China</td>
			<td>10212020C</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Beyotime Biotechnology, China</td>
			<td>C1002</td>
			<td>Used for nuclear staining. After DAPI was combined with double stranded DNA, the maximum excitation wavelength was 364nm and the maximum emission wavelength was 454nm.</td>
		</tr>
		<tr>
			<td>DMEM-F12</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>11330032</td>
			<td></td>
		</tr>
		<tr>
			<td>ES-quality FBS</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>10270106</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll Paque</td>
			<td>General Electric Company, USA</td>
			<td>17-5442-02</td>
			<td>Isolate the peripheral blood mononuclear cells according to Ficoll-Paque method.</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sangon Bioteach, China</td>
			<td>A609764</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax supplement</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax supplement</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Chicken IgY&#160; secondary antibody</td>
			<td>Abcam, UK</td>
			<td>ab150171</td>
			<td>Goat anti-Chicken IgG. Conjugation: Alexa Fluor 647. Ex: 652 nm, Em: 668 nm. Use at 1:500 dilution.</td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG secondary antibody</td>
			<td>Abcam, UK</td>
			<td>ab150120</td>
			<td>Goat anti-Mouse IgG. Conjugation: Alexa Fluor 594. Ex: 590 nm, Em: 617 nm. Use at 1:500 dilution.</td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG secondary antibody</td>
			<td>Abcam, UK</td>
			<td>ab150077</td>
			<td>Goat Anti-Rabbit IgG. Conjugation: Alexa Fluor 488. Ex: 495 nm, Em: 519 nm. Use at 1:500 dilution.</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Merck, Germany</td>
			<td>H3149</td>
			<td></td>
		</tr>
		<tr>
			<td>Horizontal shaker</td>
			<td>Servicebio, China</td>
			<td>DS-H200</td>
			<td>Relative centrifugal force (RCF) of 0.11808 x g is more appropriate, according to the manufacturer INFORS HT (Switzerland).</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Merck, Germany</td>
			<td>I9278-5ML</td>
			<td></td>
		</tr>
		<tr>
			<td>KOSR</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>10828028</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning, USA</td>
			<td>354277</td>
			<td>Matrigel will solidify in the environment higher than 4 &#176;C, so it should be sub packed at low temperature.</td>
		</tr>
		<tr>
			<td>MEM-NEAA</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR1</td>
			<td>STEMCELL Technologies, Canada</td>
			<td>85850</td>
			<td>iPSC culture medium</td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT4 primary antibody</td>
			<td>Abcam, UK</td>
			<td>ab19857</td>
			<td>Host: Rabbit. Dissolve with 500 &#956;L PBS. Use at 1:200 dilution.</td>
		</tr>
		<tr>
			<td>Pathological frozen slicer</td>
			<td>Leica, Germany</td>
			<td>Leica CM1860</td>
			<td></td>
		</tr>
		<tr>
			<td>PAX6 primary antibody</td>
			<td>Abcam, UK</td>
			<td>ab78545</td>
			<td>Host: Mouse. Use at 1:100 dilution.</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>STEMCELL Technologies, Canada</td>
			<td>37350</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>PSC dissociation solution</td>
			<td>Beijing Saibei Biotechnology, China</td>
			<td>CA3001500</td>
			<td>Enzyme free dissociation solution can be used for iPSC digestion and passage.</td>
		</tr>
		<tr>
			<td>Sendai Reprogramming Kit</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>A16518</td>
			<td>Establish iPSC according to the protocol of Sendai Reprogramming Kit.</td>
		</tr>
		<tr>
			<td>Slide Glass</td>
			<td>Shitai Laboratory Equipment, China</td>
			<td>188105W</td>
			<td></td>
		</tr>
		<tr>
			<td>Soft light lamp</td>
			<td>NUT</td>
			<td>NUT</td>
			<td>A simple self made device, refer to supplementary figure 2 for preparation.</td>
		</tr>
		<tr>
			<td>STEMdiff Cerebral Organoid Kit</td>
			<td>STEMCELL Technologies, Canada</td>
			<td>8570</td>
			<td>Contain: 1. EB Formation Medium; 2. Induction Medium; 3. Expansion Medium; 4. Maturation Medium.</td>
		</tr>
		<tr>
			<td>STEMdiff Cerebral Organoid Maturation Kit</td>
			<td>STEMCELL Technologies, Canada</td>
			<td>8571</td>
			<td>Maturation Medium</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sangon Bioteach, China</td>
			<td>A502792</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Merck, Germany</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>TUJ1 primary antibody</td>
			<td>Abcam, UK</td>
			<td>ab41489</td>
			<td>Host: Chicken. Use at 1:1000 dilution.</td>
		</tr>
		<tr>
			<td>Vaseline</td>
			<td>Sangon Bioteach, China</td>
			<td>A510146</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>STEMCELL Technologies, Canada</td>
			<td>72302</td>
			<td>Prepare a 5 mM stock solution in PBS, resuspend 1 mg in 624 &#181;L of PBS.</td>
		</tr>
		<tr>
			<td>Weblink</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Raw sequencing data</td>
			<td>Genome Sequence Archive (Genomics, Proteomics &#38; Bioinformatics 2021) in National Genomics Data Center (Nucleic Acids Res 2022), China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences</td>
			<td>GSA-Human: HRA002430</td>
			<td>https://ngdc.cncb.ac.cn/gsa-human/</td>
		</tr>
	</tbody>
</table>

facilitating cerebral organoid culture via lateral soft light illumination

目前，脑类器官培养技术操作仍复杂，难以大规模应用。有必要找到一个简单而实用的解决方案。因此，本研究提出了一种更可行的脑类器官方案。为了解决早期介质变化和类器官转移中不可避免的不便，目前的研究通过应用工程原理优化了操作技术。采用柔和的光灯横向照亮胚体（EB）样品，通过增强的漫反射效果，肉眼可以看到EB。利用旋转产生的二次流动原理，类器官向孔中心聚集，这有利于介质变化或类器官转移的操作。与分散的细胞相比，胚状体在移液器中沉降得更快。利用这种现象，可以以简单的方式有效地去除大多数游离细胞和死细胞碎片，防止EB因离心而受到损害。本研究促进脑类器官培养的操作，并有助于促进脑类器官的应用。

本研究诱导iPSCs（图2B）进入脑类器官（图2C）。早期培养的EB表示OCT4标记（图2A），表明良好的多能性。在后期阶段，EBs发育成成熟的脑类器官（图2D）。该研究将iPSC从正常健康个体和SCA3患者培养成脑类器官（图3A）。SCA3，也称为马查多- 约瑟夫病（MJD），是由ATXN3基因25<...

Watch this Scientific Journal Video about Facilitating Cerebral Organoid Culture via Lateral Soft Light Illumination at JoVE.com

Facilitating Cerebral Organoid Culture via Lateral Soft Light Illumination

脑类器官为研究器官发育和人类疾病病理学提供了前所未有的机会。虽然脑类器官培养系统取得了巨大成功，但在应用这项技术方面仍然存在操作困难。本方案描述了一种促进介质改变和类器官转移的脑类器官程序。

通过侧向柔光照明促进脑类器官培养

At present, cerebral organoid culture technology is still complicated to operate and difficult to apply on a large scale. It is necessary to find a simple ...

该协议有助于脑类器官培养的操作，并有助于促进脑类器官的应用。早期类器官可以通过使用横向柔光灯用肉眼看到，这对于许多操作来说很方便。改进的培养基更换方法和类器官转移操作也有助于其他类型的类器官培养和自动培养机的设计。首先，将带有mTeSR1培养基的iPSC培养在覆盖有35毫米培养皿的基质中，并每天更换培养基。iPSC生长密度不得超过75%，然后从iPSC中移出mTeSR1培养基，并用一毫米PBS洗涤两次。移出PBS后，加入300微升细胞分离溶液，在37摄氏度下将iPSC消化到单个细胞中三到四分钟。然后，将细胞重悬于两毫米的EB形成培养基中，并以300倍g离心样品五分钟。用每孔500微升的抗粘附冲洗溶液处理专门的24孔板五分钟。现在，将细胞重悬于含有Rho激酶抑制剂Y27632的EB形成培养基中。除去抗粘附冲洗液后，用两毫升EB形成介质冲洗每个孔。接下来，将细胞以3乘以10的密度将细胞加入专门的24孔板中，并在室温下以100倍g离心板两分钟。将细胞均匀地分布在板底部的每个腔室中。将样品在37摄氏度和5%二氧化碳下孵育24小时。如果在上一步中未使用离心，请孵育36小时并保持样品稳定。使用厚度为0.3至0.5厘米的透明丙烯酸板，大小为A5纸，并在丙烯酸板的正面和背面粘贴白色垫。接下来，在板的边缘安装一排LED白光灯，使灯可以从亚克力板的侧面进入，然后平行射出。现在，准备一个新的六孔低粘接板，并向每孔中加入两毫升EB形成培养基。使用1000微升宽孔移液器吸头将EB与培养基一起除去后，将每孔约100个EB转移到六孔低粘附板中。为了每天用相同体积的新鲜培养基替换EB形成培养基，通过沿圆形轨道旋转培养皿来诱导涡流。由于通过旋转产生的二次流动，EB或类器官会聚到井的中心。通过缓慢地将移液到孔的边缘来吸入旧介质。不要吸吮得太用力，否则EB会一起被移除。然后，添加新鲜介质以重悬 EB。对于神经诱导，制备一个新的六孔低粘附板，每孔有三毫升神经诱导介质。打开侧向柔光灯并关闭其他室内光源。现在，通过使用宽口移液器吸头吸出EB和培养基，将EB转移到具有添加神经诱导培养基的六孔板中。然后，将移液器竖直。EBs将在重力作用下逐渐下沉，并收敛到移液器吸头的口。当移液器吸头的口再次接触液体表面时，由于液体表面张力，EBs会迅速沉入介质中。将样品在37摄氏度和5%二氧化碳下孵育24小时。将膜基质放入四摄氏度的冰箱中溶解60分钟。打开侧向柔光灯后，关闭控制台顶部的光源，并将膜基质保持在冰上以防止凝固。将少量EB转移到含有新鲜膨胀培养基的60毫米培养皿中，以便更容易去除单个EB。使用200微升宽口径移液器吸头，每次吸出含有约10微升培养基的单个EB，然后通过将其添加到六孔板的底部来产生液滴。每孔使用五个液滴。将15微升的膜基质加入每个含有EB的液滴中。快速混合EB球并将其嵌入液滴的中心。通过将六孔板放入37摄氏度的培养箱中30分钟，使含有EB的液滴固化。然后，向每个孔中加入三毫升膨胀培养基，轻轻吹起嵌入的基质EB以悬浮它们并孵育三天。对于类器官成熟，轻轻除去原始培养基，并向每个孔中加入三毫升成熟培养基。将类器官板放在37摄氏度培养箱中的水平振荡器上，并继续水平旋转培养物。每两到三天更换一次新鲜成熟培养基。由于重力和相对高于介质的密度，重悬的EB将逐渐下沉。因此，可以方便地转移EB。与EBs相比，游离细胞和死细胞片段的下沉速度更慢。因此，可以去除大多数游离细胞和死细胞片段。早期培养的EBs表达了OCT4标记，表明多能性良好。在后期阶段，EBs发育成成熟的脑类器官。成熟脑类器官对顶端祖细胞标志物PAX6和神经元特异性标志物微管蛋白J1呈阳性。来自健康个体和SCA3患者的iPSC被培养成类器官。正常脑类器官组和SCA3脑类器官组的基因表达谱在神经递质转运、突触形成和调节等通路上存在显著差异。当用基底膜基质嵌入EB时，操作时间不应太长，以避免EB变干。建议快速保护。

Research

JoVE Journal

Bioengineering

Facilitating Cerebral Organoid Culture via Lateral Soft Light Illumination

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

用于细胞培养应用光介导的形成和构图的水凝胶

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

科研

生物工程

Soft Lithographic Procedure for Producing Plastic Microfluidic Devices with View-ports Transparent to Visible and Infrared Light

生产塑料微流控装置与查看端口对可见光和红外透明软光刻过程

Infrared (IR) spectro-microscopy of living biological samples is hampered by the absorption of water in the mid-IR range and by the lack of suitable ...

Production of Extracellular Matrix Fibers via Sacrificial Hollow Fiber Membrane Cell Culture

牺牲纤维膜细胞培养生产细胞外基质纤维

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure ...

Handling and Assessment of Human Primary Prostate Organoid Culture

人原发性前列腺组织培养的处理与评价

This paper describes a detailed protocol for three-dimensional (3D) culturing, handling, and evaluation of human primary prostate organoids. The process ...

Applications for Open Source Microplate-Compatible Illumination Panels

开源微孔板兼容照明面板的应用

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well ...

Bioinspired Soft Robot with Incorporated Microelectrodes

生物灵感软机器人与合并微电极

Bioinspired soft robotic systems that mimic living organisms using engineered muscle tissue and biomaterials are revolutionizing the current biorobotics ...

Building a Simple and Versatile Illumination System for Optogenetic Experiments

为光遗传学实验构建简单通用的照明系统

Controlling biological processes using light has increased the accuracy and speed with which researchers can manipulate many biological processes. Optical ...

Experimental and Data Analysis Workflow for Soft Matter Nanoindentation

Nanoindentation refers to a class of experimental techniques where a micrometric force probe is used to quantify the local mechanical properties of soft ...

Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5&#8242;-Phosphate

Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5&#8242;-Phosphate

通过可见光光解 核 黄素-5′-磷酸灭活病原体

Riboflavin-5&#39;-phosphate (or flavin mononucleotide; FMN) is sensitive to visible light. Various compounds, including reactive oxygen species (ROS), can ...

Reconstituting Cytoarchitecture and Function of Human Epithelial Tissues on an Open-Top Organ-Chip

在开顶器官芯片上重建人上皮组织的细胞结构和功能

Nearly all human organs are lined with epithelial tissues, comprising one or multiple layers of tightly connected cells organized into three-dimensional ...