Name: generation of human brain organoids for mitochondrial disease modeling
Uploaded: 2021-06-21T14:00:00
Description: Watch this Scientific Journal Video about Generation of Human Brain Organoids for Mitochondrial Disease Modeling at JoVE.com

我们感谢Miriam Bünning的技术支持。我们感谢德国联邦医疗协会（DFG）（PR1527/5-1至AP）、Spark和柏林卫生研究院（BIH）（BIH验证基金至AP）、联合线粒体疾病基金会（UMDF）（利综合征国际财团资助AP）、杜塞尔多夫大学医院（Forschungskommission UKD至AP）和德国联邦教育与研究部（BMBF）（e：生物年轻研究员向AP授予AZ 031L0211）。C.R.R.实验室的工作得到了DFG的支持（FOR 2795"压力下的突触"，Ro 2327/13-1）。

注意：使用人类 iPSC 可能需要获得伦理批准。本研究中使用的iPSCs来自当地伦理批准后的健康对照个体（#2019-681）。所有细胞培养程序必须在无菌细胞培养罩下进行，在转移到罩下之前仔细消毒所有试剂和耗材。用于分化的人类iPSC的传代数应低于50，以避免在广泛培养时可能发生的潜在基因组畸变。细胞的多能状态应在类器官生成之前进行验证，例如，通过监测多能性相关标志物（如NANOG或OCT4）?...

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I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+iPSC-derived+cerebral+organoids+model+features+of+Leigh+Syndrome+and+reveal+abnormal+corticogenesis.">Human iPSC-derived cerebral organoids model features of Leigh Syndrome and reveal abnormal corticogenesis.</a> bioRxiv. , (2020).</li><li>Inak, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+metabolic+programming+impairs+early+neuronal+morphogenesis+in+neural+cultures+and+an+organoid+model+of+Leigh+syndrome.">Defective metabolic programming impairs early neuronal morphogenesis in neural cultures and an organoid model of Leigh syndrome.</a> Nature Communications. 12 (1), 1929 (2021).</li><li>Falk, M. 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本文描述了人类iPSC衍生的脑类器官的可重复生成及其在线粒体疾病建模中的应用。此处描述的协议是根据以前发表的 work20 进行修改的。本方案的一个主要优点是它不需要手动将每个类器官嵌入到支架基质中。实际上，基质溶液只是简单地溶解到细胞培养基中。此外，无需使用昂贵的生物反应器，因为类器官可以在标准组织培养物中培养6孔板，放置在培养箱内的轨道振荡器上。...

线粒体疾病是最大的一类先天性代谢缺陷1。它们是由破坏不同线粒体过程的基因突变引起的，包括氧化磷酸化（OXPHOS）2，呼吸链组装，线粒体动力学和线粒体DNA转录或复制3。有能量需求的组织尤其受线粒体功能障碍的影响4。因此，患有线粒体疾病的患者通常会出现早发性神经系统表现。
目前尚无针对线粒体疾病患儿的治疗方法5。线粒体疾病药物开发的一个主要障碍是缺乏概括人类疾病过程的有效模型6。目前研究的几种动物模型没有表现出患者中存在的神经缺陷7。因此，线粒体疾病神经元病理学的潜在机制仍未完全清楚。
最近的研究从受线粒体疾病影响的患者中产生了iPSCs，并使用这些细胞来获得患者特异性神经元细胞。例如，已发现与线粒体疾病Leigh综合征相关的遗传缺陷可导致细胞生物能量学8，9，蛋白质合成10和钙稳态9，11的畸变。这些报告为线粒体疾病中发生的神经元损伤提供了重要的机制线索，为这些不治之症的药物发现铺平了道路12。
然而，二维（2D）文化无法调查3D器官的建筑复杂性和区域组织13。为此，使用源自患者特异性iPSCs14 的3D脑类器官可能使研究人员能够获得额外的重要信息，从而有助于剖析线粒体疾病如何影响神经系统的发育和功能15。使用iPSC衍生的脑类器官来研究线粒体疾病的研究开始揭示线粒体疾病的神经发育成分。 
脊髓类器官携带与线粒体疾病、线粒体脑病、乳酸性酸中毒和卒中样发作综合征（MELAS）相关的突变，显示神经发生缺陷和运动神经元分化延迟16。来自线粒体疾病Leigh综合征患者的皮质类器官显示尺寸减小，神经上皮芽生成缺陷和皮质结构丧失17。来自Leigh综合征患者的脑类器官表明，疾病缺陷始于神经祖细胞的水平，其不能致力于线粒体代谢，导致异常神经元分支和形态发生18。因此，神经祖细胞可能代表线粒体疾病的细胞治疗靶点，促进其线粒体功能的策略可能支持神经系统的功能发育。
脑类器官的使用可能有助于揭示线粒体疾病的神经发育成分。线粒体疾病主要被认为是早发性神经变性5。然而，受线粒体疾病影响的患者也存在神经发育缺陷，包括发育迟缓和认知障碍19。患者特异性脑类器官可能有助于解决这些方面，并阐明线粒体疾病如何影响人脑发育。线粒体功能障碍也可能在其他更常见的神经系统疾病中起致病作用，例如阿尔茨海默病，帕金森病和亨廷顿病4。因此，使用脑类器官阐明线粒体缺陷对神经发育的影响也可能有助于研究这些疾病。本文描述了一种用于生成可重复脑类器官的详细方案，可用于进行线粒体疾病的疾病建模。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Gibco</td>
			<td>31350-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Affinity Designer</td>
			<td>Serif (Europe) Ltd</td>
			<td></td>
			<td>Layout software; Vector graphics editor</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 donkey anti-guinea pig</td>
			<td>Sigma Aldrich</td>
			<td>SAB4600033-250UL</td>
			<td>1:300</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 donkey anti-mouse</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-31571</td>
			<td>1:300</td>
		</tr>
		<tr>
			<td>Antimycin A</td>
			<td>Sigma Aldrich</td>
			<td>1397-94-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-&#946;-Tubulin III (TUJ-1)</td>
			<td>Sigma Aldrich</td>
			<td>T8578</td>
			<td>1:2000</td>
		</tr>
		<tr>
			<td>Argon Laser</td>
			<td>Melles Griot</td>
			<td></td>
			<td>Any other Laser, e.g., diode lasers emitting 488 is fine, too</td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Sigma</td>
			<td>A92902</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 with Vitamin A</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Agar</td>
			<td>Becton Dickinson</td>
			<td></td>
			<td>3% in PBS, store solution at -20 &#176;C</td>
		</tr>
		<tr>
			<td>BDNF</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-0811</td>
			<td></td>
		</tr>
		<tr>
			<td>cAMP</td>
			<td>Sigma</td>
			<td>D0627</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Star cell culture 6 well plate</td>
			<td>Greiner-Bio-One</td>
			<td>657160</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemically Defined Lipid Concentrate</td>
			<td>Gibco</td>
			<td>11905031</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope C1</td>
			<td>Nikon Microscope Solutions</td>
			<td></td>
			<td>Modular confocal microscope system</td>
		</tr>
		<tr>
			<td>Corning Matrigel Growth Factor Reduced (GFR) Basement membrane matrix, Phenol Red-free, LDEV-free</td>
			<td>Corning</td>
			<td>356231</td>
			<td>Matrix component</td>
		</tr>
		<tr>
			<td>CyQUANT Cell Proliferation Assay Kit</td>
			<td>Thermo Fisher</td>
			<td>C7026</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>ThermoFisher</td>
			<td>31330038</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D2660-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-goat Cy3</td>
			<td>Merck Millipore</td>
			<td>AP180C</td>
			<td>1:300</td>
		</tr>
		<tr>
			<td>Donkey anti-mouse Cy3</td>
			<td>Merck Millipore</td>
			<td>AP192C</td>
			<td>1:300</td>
		</tr>
		<tr>
			<td>Donkey anti-rabbit Cy3</td>
			<td>Merck Millipore</td>
			<td>AP182C</td>
			<td>1:300</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14190250</td>
			<td></td>
		</tr>
		<tr>
			<td>DS-Q1Mc camera</td>
			<td>Nikon Microscope Solutions</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eclipse 90i upright widefield microscope</td>
			<td>Nikon Microscope Solutions</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eclipse E 600FN upright microscope</td>
			<td>Nikon Microscope Solutions</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eclipse Ts2 Inverted Microscope</td>
			<td>Nikon Microsope Solutions</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EZ-C1 Silver Version 3.91</td>
			<td>Nikon Microscope Solutions</td>
			<td></td>
			<td>Imaging software for confocal microscope</td>
		</tr>
		<tr>
			<td>FCCP</td>
			<td>Sigma Aldrich</td>
			<td>370-86-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>GDNF</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-291</td>
			<td></td>
		</tr>
		<tr>
			<td>Glasgow MEM</td>
			<td>Gibco</td>
			<td>11710-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Pasteur pipette</td>
			<td>Brand</td>
			<td>747715</td>
			<td>Inverted</td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Helium-Neon Laser</td>
			<td>Melles Griot</td>
			<td></td>
			<td>Every other Laser, e.g., diode lasers emitting 594 is fine, too</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Merck</td>
			<td>H3149-25KU</td>
			<td></td>
		</tr>
		<tr>
			<td>HERACell 240i CO2 Incubator</td>
			<td>Thermo Scientific</td>
			<td>51026331</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Invitrogen</td>
			<td>H3570</td>
			<td>1:2500</td>
		</tr>
		<tr>
			<td>Image J 1.53c</td>
			<td>Wayne Rasband National Institute of Health</td>
			<td></td>
			<td>Image processing Software</td>
		</tr>
		<tr>
			<td>Injekt Solo 10 mL/ Luer</td>
			<td>Braun</td>
			<td>4606108V</td>
			<td></td>
		</tr>
		<tr>
			<td>Knockout Serum Replacement</td>
			<td>Gibco</td>
			<td>10828010</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser (407 nm)</td>
			<td>Coherent</td>
			<td></td>
			<td>Any other Laser, e.g., diode lasers emitting 407 is fine, too</td>
		</tr>
		<tr>
			<td>Map2</td>
			<td>Synaptic Systems</td>
			<td>No. 188004</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>Maxisafe 2030i</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MEM NEAA</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR Plus</td>
			<td>Stemcell Technology</td>
			<td>85850</td>
			<td>iPSC medium</td>
		</tr>
		<tr>
			<td>Multifuge X3R Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>10325804</td>
			<td></td>
		</tr>
		<tr>
			<td>MycoAlert Mycoplasma Detection Kit</td>
			<td>Lonza</td>
			<td># LT07-218</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 Supplement</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle for single usage (23G x 1&#8221; TW)</td>
			<td>Neoject</td>
			<td>10016</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS-Elements Aadvanced Research 3.2</td>
			<td>Nikon</td>
			<td></td>
			<td>Imaging software</td>
		</tr>
		<tr>
			<td>Oligomycin A</td>
			<td>Sigma Aldrich</td>
			<td>75351</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital Shaker Heidolph Unimax 1010</td>
			<td>Heidolph</td>
			<td>543-12310-00</td>
			<td></td>
		</tr>
		<tr>
			<td>PAP Pen</td>
			<td>Sigma</td>
			<td>Z377821-1EA</td>
			<td>To draw hydrophobic barrier on slides.</td>
		</tr>
		<tr>
			<td>Papain Dissociation System kit</td>
			<td>Worthington</td>
			<td>LK003150</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Merck</td>
			<td>818715</td>
			<td>4% in PBS, store solution at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Pasteur pipette 7mL</td>
			<td>VWR</td>
			<td>612-1681</td>
			<td>Graduated up to 3 mL</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Plan Apo VC 20x / 0.75 air DIC N2&#160; &#8734;/0.17 WD 1.0</td>
			<td>Nikon Microscope Solutions</td>
			<td></td>
			<td>Dry Microscope Objective</td>
		</tr>
		<tr>
			<td>Plan Apo VC 60x / 1.40 oil DIC N2 &#8734;/0.17 WD 0.13</td>
			<td>Nikon Microscope Solutions</td>
			<td></td>
			<td>Oil Immersion Microscope Objective</td>
		</tr>
		<tr>
			<td>Polystyrene Petri dish (100 mm)</td>
			<td>Greiner Bio-One</td>
			<td>664161</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene round-bottom tube with cell-strainer cap (5 mL)</td>
			<td>Falcon</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Roth</td>
			<td>6781.1</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Glass Antifade Moutant</td>
			<td>Invitrogen</td>
			<td>P36980</td>
			<td></td>
		</tr>
		<tr>
			<td>Qualitative filter paper</td>
			<td>VWR</td>
			<td>516-0813</td>
			<td></td>
		</tr>
		<tr>
			<td>Rock Inhibitior</td>
			<td>Merck</td>
			<td>SCM075</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>Sigma</td>
			<td>83-794</td>
			<td></td>
		</tr>
		<tr>
			<td>S100&#946;</td>
			<td>Abcam</td>
			<td>Ab11178</td>
			<td>1:600</td>
		</tr>
		<tr>
			<td>SB-431542</td>
			<td>Cayman Chemical Company</td>
			<td>13031</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blades</td>
			<td>Heinz Herenz Hamburq</td>
			<td>1110918</td>
			<td></td>
		</tr>
		<tr>
			<td>SMI312</td>
			<td>Biolegend</td>
			<td>837904</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Merck/Sigma</td>
			<td>31437-1kg-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Roth</td>
			<td>3957</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dihydrogen phosphate</td>
			<td>Applichem</td>
			<td>131965</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Gibco</td>
			<td>11360070</td>
			<td></td>
		</tr>
		<tr>
			<td>SOX2</td>
			<td>Santa Cruz Biotechnology</td>
			<td>Sc-17320</td>
			<td>1:100</td>
		</tr>
		<tr>
			<td>StemPro Accutase Cell Dissociation Reagent</td>
			<td>Gibco/StemPro</td>
			<td>A1110501</td>
			<td>Reagent A</td>
		</tr>
		<tr>
			<td>Super Glue Gel</td>
			<td>UHU</td>
			<td>63261</td>
			<td>adhesive gel</td>
		</tr>
		<tr>
			<td>SuperFrost Plus</td>
			<td>VWR</td>
			<td>631-0108</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe for single usage (1 mL)</td>
			<td>BD Plastipak</td>
			<td>300015</td>
			<td></td>
		</tr>
		<tr>
			<td>TB2 Thermoblock</td>
			<td>Biometra</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TC Plate 24 Well</td>
			<td>Sarstedt</td>
			<td>83.3922</td>
			<td></td>
		</tr>
		<tr>
			<td>TC Plate 6 Well</td>
			<td>Sarstedt</td>
			<td>83.392</td>
			<td></td>
		</tr>
		<tr>
			<td>TGFbeta3</td>
			<td>Miltenyi Biotec</td>
			<td>130-094-007</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Hood</td>
			<td>ThermoFisher</td>
			<td>51032711</td>
			<td></td>
		</tr>
		<tr>
			<td>TOM20</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-11415</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>Triton-X</td>
			<td>Merck</td>
			<td>X100-5ML</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure 0.5M EDTA</td>
			<td>Invitrogen</td>
			<td>15575020</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome Microm HM 650 V</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>Production terminated, any other adjustable microtome is fine, too.</td>
		</tr>
		<tr>
			<td>Vibratome Wilkinson Classic Razor Blade</td>
			<td>Wilkinson Sword</td>
			<td>70517470</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman Benchkote</td>
			<td>Merck/Sigma</td>
			<td>28418852</td>
			<td></td>
		</tr>
		<tr>
			<td>Wnt Antagonist I</td>
			<td>EMD Millipore Corp</td>
			<td>3378738</td>
			<td></td>
		</tr>
		<tr>
			<td>XF 96 extracellular flux analyser</td>
			<td>Seahorse Bioscience</td>
			<td>100737-101</td>
			<td></td>
		</tr>
		<tr>
			<td>XF Assay DMEM Medium</td>
			<td>Seahorse Bioscience</td>
			<td>103680-100</td>
			<td></td>
		</tr>
		<tr>
			<td>XF Calibrant Solution</td>
			<td>Seahorse Bioscience</td>
			<td>100840-000</td>
			<td></td>
		</tr>
		<tr>
			<td>XFe96 FluxPak (96-well microplate)</td>
			<td>Seahorse Bioscience</td>
			<td>102416-100</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of human brain organoids for mitochondrial disease modeling

线粒体疾病是最大的一类先天性代谢缺陷，目前无法治愈。这些疾病导致神经发育缺陷，其潜在机制仍有待阐明。一个主要障碍是缺乏有效的模型来概括患者中早发性神经元损伤。诱导多能干细胞（iPSCs）技术的进步使三维（3D）脑类器官的产生成为可能，可用于研究疾病对神经系统发育和组织的影响。包括这些作者在内的研究人员最近引入了人脑类器官来模拟线粒体疾病。本文报告了人类iPSC衍生的脑类器官的强大生成及其在线粒体生物能量分析和成像分析中的应用的详细方案。这些实验将允许使用脑类器官来研究代谢和发育功能障碍，并可能为剖析线粒体疾病的神经元病理学提供关键信息。

这里描述的协议有助于圆类器官的稳健生成（图1A）。生成的类器官包含成熟的神经元，可以使用轴突（SMI312）和树突（微管相关蛋白2（MAP2））特异性的蛋白质标记物进行可视化（图1B） ）。成熟的类器官不仅含有神经元细胞（MAP2阳性），还含有神经胶质细胞（例如，星形胶质细胞标志物S100钙结合蛋白B（S100ß）...

Watch this Scientific Journal Video about Generation of Human Brain Organoids for Mitochondrial Disease Modeling at JoVE.com

Generation of Human Brain Organoids for Mitochondrial Disease Modeling

我们描述了产生人类诱导多能干细胞衍生脑类器官及其在线粒体疾病建模的详细方案。

用于线粒体疾病建模的人脑类器官的生成

Mitochondrial diseases represent the largest class of inborn errors of metabolism and are currently incurable. These diseases cause neurodevelopmental ...

该协议旨在使用脑类器官来研究线粒体疾病。它被开发用于产生可重复的脑类器官，以评估其线粒体特性。这种方法不需要昂贵的生物反应器和耗时的包埋程序。因此，它易于实施和升级。该协议使得产生可重复的脑类器官并测试其线粒体特性成为可能。这可以导致识别无法治愈的线粒体疾病的介入靶标。首先，在无饲养层条件下，在iPSC培养基中，在包被的六孔板上培养人iPSCs，并将其保存在37摄氏度和5%二氧化碳的加湿组织培养箱中。使用无酶分离培养基以80%汇合度通过iPSCs，比例范围从1×4到12。为了提高细胞存活率，每次分裂后加入10微摩尔rho相关蛋白激酶或ROCK抑制剂。在第 0 天解离 80% 的 iPSC。制备皮质分化培养基一或CDM1，并在将其添加到细胞之前在室温下预热。用PBS清洗含有iPSC的孔，以去除死细胞和碎屑。向每个孔中加入500微升预热试剂A，并在37摄氏度下孵育5分钟。在显微镜下检查以确保细胞脱离。加入一毫升iPSC培养基以稀释试剂A以中和其活性。使用1， 000微升移液器通过上下管道解离细胞，并将细胞悬浮液转移到15毫升离心管中。在室温下以125倍G轻轻离心iPSCs五分钟，然后小心地吸出上清液以避免干扰细胞沉淀。用一毫升CDM1重悬沉淀以获得单个细胞悬浮液并计数细胞数。在CDM1中每100微升用9， 000 iPSCs制备接种培养基，并补充20微摩尔ROCK抑制剂，三微摩尔WNT连环蛋白抑制剂或IWR-1和5微摩尔SB-431542。将每孔100微升的接种培养基加入96孔V底板中。将板保持在37摄氏度和5%二氧化碳的加湿组织培养箱中。为了产生神经球，在第一天，观察具有明确平滑边界的圆形细胞聚集体正在形成。注意聚集体周围的死细胞。继续在37摄氏度和5%二氧化碳的培养箱中培养。在第三天，通过敲击侧面三次以分离死细胞来搅拌板，然后向每个孔中加入100微升CDM1，其中补充有20微摩尔ROCK抑制剂，三个微摩尔IWR-1和五个微摩尔SB-431542。将板保持在37摄氏度和5%二氧化碳的培养箱中。在第六天，小心地从每个孔中取出80微升的上清液培养基。避免触摸井底。向每个孔中加入100微升CDM1，并补充有三个微摩尔IWR-1和五个微摩尔SB-431542并孵育板。每三天后重复上一步，直到第18天。在第18天，为了转移神经球，准备CDM2并向100毫米超低附着细胞培养板中加入10毫升。使用200微升移液器，尖端被切断，将圆形神经球从96孔板转移到100毫米超低附着细胞培养板。从含有神经球的板中取出五毫升培养基，并加入五毫升新鲜的CDM2。将板放在轨道振荡器上，在37摄氏度和5%二氧化碳的加湿组织培养箱内以每分钟70次旋转。每三天，小心地吸出上清液培养基，并用新鲜的CDM2替换。留下少量培养基，以防止神经球变干。在第35天，准备CDM3。从板中吸出培养基，加入10毫升冷CDM3。更换培养基后，将板放回轨道振荡器上，在37摄氏度和5%二氧化碳的加湿组织培养箱内以每分钟70次旋转。每三到五天更换培养基，具体取决于培养基颜色指示的生长速度。在第70天，准备CDM4。使用CDM4培养基，直到达到所需的类器官年龄。在此期间，将板保持在37摄氏度和5%二氧化碳的加湿组织培养箱内，每分钟旋转70次的轨道振荡器上。根据生长速度每三到五天更换一次培养基。准备4%PFA溶液并将其置于安全罩下。收集脑类器官，并用钝头的三毫升塑料巴斯德移液管轻轻地将它们转移到充满PFA的六孔板中。将类器官在室温下在PFA溶液中保存一小时。用三毫升塑料巴斯德移液器小心地去除PFA，并使用PBS洗涤固定的类器官三次。将固定的类器官储存在四摄氏度的PBS中，直到进一步使用。使用该协议生成的类器官包含成熟的神经元，可以使用轴突和树突特异性的蛋白质标记物进行可视化。成熟的类器官不仅含有神经元细胞，还含有神经胶质细胞。使用切片的脑类器官分析，可以监测SMI 312阳性轴突和MAP2阳性树突或S100β神经胶质细胞。此外，共聚焦图像有助于研究 SOX2 阳性神经祖细胞相对于 β-3 微管蛋白阳性神经元的详细分布和组织。对脑类器官进行染色以检测线粒体特异性标志物，例如外膜转座酶或TOM20。通过使用耗氧率（OCR）测量线粒体代谢和使用细胞外酸化速率或ECAR的糖酵解代谢来进行脑类器官的生物能量分析。寡霉素导致OCR谱下降，因此确定ATP生产所需的OCR。在寡霉素治疗后，ECAR也可能有代偿性增加，表明细胞可以上调糖酵解以防止代谢应激。当将神经球从96孔板转移到培养板或在固定过程中时，轻轻处理类器官以避免损坏它们是至关重要的。在脑类器官生成之后，多电极阵列或钙成像将是测试类器官功能的理想方法。

Research

JoVE Journal

Neuroscience

Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Preparation of Oligomeric ÃƒÅ½Ã‚Â²-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

低聚β-淀粉样蛋白的制备 1-42和突触可塑性损伤诱导海马脑片

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). ...

科研

神经科学

Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay

Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay

线粒体 Ca2 +保留容量检测和 Ca2 +触发的线粒体肿胀试验

The production of ATP by oxidative phosphorylation is the primary function of mitochondria. Mitochondria in higher eukaryotes also participate in ...

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

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

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

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

人类成人皮肤成纤维细胞诱导神经元高产培养的简单生成

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

在体内果蝇前运动神经末端的单分子跟踪

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. ...

Production, Purification, and Quality Control for Adeno-associated Virus-based Vectors

与腺相关病毒的向量的生产、纯化和质量控制

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), ...

Three-Dimensional Shape Modeling and Analysis of Brain Structures

脑结构三维形状建模与分析

Statistical shape analysis of brain structures has been used to investigate the association between their structural changes and pathological processes. ...

Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions

从多能干细胞生成人类神经元和少突胶质细胞，用于模拟神经元-少突胶质细胞相互作用

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it ...

Generation of Human Motor Units with Functional Neuromuscular Junctions in Microfluidic Devices

在微流体装置中生成具有功能性神经肌肉接头的人类运动单元

Neuromuscular junctions (NMJs) are specialized synapses between the axon of the lower motor neuron and the muscle facilitating the engagement of muscle ...

Flow Cytometric Analysis of Multiple Mitochondrial Parameters in Human Induced Pluripotent Stem Cells and Their Neural and Glial Derivatives

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential ...