Name: maintaining human glioblastoma cellular diversity ex vivo using three-dimensional organoid culture
Uploaded: 2022-08-25T13:10:00
Duration: 7 min 11 s
Description: Watch this Scientific Journal Video about Maintaining Human Glioblastoma Cellular Diversity Ex vivo using Three-Dimensional Organoid Culture at JoVE.com

我们要感谢Justin Lathia博士的宝贵建议和持续支持。我们还要感谢Katrina Fife，Lisa Wallace和Maya Camhi的出色技术支持。

<ol>
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F., Onzi, G. R., Zong, H., Lenz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+conditions+defining+glioblastoma+cells+behavior:+what+is+the+impact+for+novel+discoveries.">Culture conditions defining glioblastoma cells behavior: what is the impact for novel discoveries.</a> Oncotarget. 8 (40), 69185-69197 (2017).</li><li>Lenin, S., 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=A+drug+screening+pipeline+using+2D+and+3D+patient-derived+in+vitro+models+for+preclinical+analysis+of+therapy+response+in+glioblastoma.">A drug screening pipeline using 2D and 3D patient-derived in vitro models for preclinical analysis of therapy response in glioblastoma.</a> International Journal of Molecular Sciences. 22 (9), 4322 (2021).</li><li>Rybin, M. J., Ivan, M. E., Ayad, N. 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作者没有需要披露的冲突。

GBM类器官是传统球体的互补培养方法，具有更大的细胞和微环境异质性4，22，30。虽然需要花费更多的时间和资源，但类器官培养可以为肿瘤内行为和耐药机制提供有价值的见解。
GBM由CSCs的5，31种群驱动，这些方法的开发是为了允许CSC种群的持续增长和自我更新。已知表皮生长因子（EGF）和成纤维细胞生长因子（FGF）可增强干细胞的维持和生长，并提供活性受体酪氨酸激酶（RTK）信号传导。GBM肿瘤内异质细胞群和不同肿瘤微环境的形成依赖于支持CSC行为。lrECM的选择模仿富含层粘连蛋白的大脑环境，并支持类器官培养中的细胞通过侵袭进行自我组织和迁移。尽管一些小组已经在不使用lrECM或EGF / FGF富集培养基24，28的情况下建立了类器官培养，这可能为这种培养方法提供了更省时的方式，并且更能选择致癌信号来驱动生长，但选择这些方法来优化前干细胞环境以最好地建立类器官的细胞异质性。脑类器官和GBM类器官在之前的文献中均已用lrECM制成21，32，33。尽管我们已经建立了有关类器官中发现的肿瘤群体和空间变异的数据，但对类器官中的非肿瘤群体以及它们从原始患者标本中存活多长时间知之甚少。某些IHC染色剂（如CD45）可能会提供这些数据，并且可能是未来类器官的一个有趣的研究点。
了解类器官培养的预期用途对于选择合适的方法非常重要。从原始标本建立类器官与为特定实验生长均匀类器官的程序略有不同。了解类器官如何成熟并在 lrECM 支架中视觉填充对于能够为类器官培养分配适当的时间和资源非常重要。类器官中细胞的稀疏区域将缓慢扩展和生长以填充lrECM，这可能需要2-8周的时间，具体取决于标本的行为。这种生长速度在某种程度上是每个标本固有的;它保存在不同批次的类器官中，并且与球体生长的相对速率相当一致。类器官可以维持1年以上，并在异种移植到小鼠中保持肿瘤形成能力;但是，建议以不同的目的种植它们，以免浪费实验室资源（材料和时间）4。类器官生长已在多种孔大小和形式中进行了测试，并表明 10 cm 板是保持最佳细胞活力的理想设置，其次是每孔34 个类器官的 6 孔板。与二维培养物相比，类器官消耗更多的培养基，并且使用较小的孔格式不会导致适当的维护。例如，相对于类器官的大小，96孔板的一个孔没有足够的空间或培养基体积来维持类器官生长。
正如类器官所确立的那样，观察力对于提高成功率很重要。最初，类器官会缓慢消耗培养基，但随着它们变得更密集和更成熟，它们会更快地消耗培养基。向培养基中添加酚红有助于作为培养基消耗的指标。当培养基更黄时，可能会提示我们调整进料模式，无论是更换更大体积的培养基，增加板中的总培养基量，在多个细胞培养板之间分配类器官以跟上其生长，甚至调整实验的时间线。 
在许多方面，类器官是进行癌症研究的低效方式。它们涉及较长的时间尺度，与GBM球体培养相比，价格昂贵且资源密集。然而，与患者来源的异种移植物（一种重建细胞和微环境多样性的替代方法）相比，它们更直接、更便宜且可控。选择何时最好地使用类器官对癌症研究人员很重要。它们不是为了取代传统的领域或追随者文化，也不是为了取代异种移植模式。当类器官应用于正确的科学问题时，可以结合这两种系统的好处，并可能允许我们观察肿瘤细胞生物学，否则这些生物学将保持隐藏。科学界才刚刚开始了解类器官提供的学习机会，但很明显，它们将成为未来理解GBM复杂生物学的宝贵工具。

胶质母细胞瘤 （GBM） 是最常见的原发性脑肿瘤，诊断后约 15 个月。在临床前研究中有效的治疗通常对患者效果不佳2，3。临床反应差归因于许多因素，包括GBM的微环境异质性和复杂的肿瘤内相互作用。这些很难在实验室环境中使用传统的贴壁或球体培养方法重现4。GBM中存在一部分自我更新的癌症干细胞（CSC）也可能导致这种复杂性5，6。CSC 对肿瘤增殖至关重要，并通过促进活性血管生成、癌症侵袭和对包括辐射在内的治疗的耐药性来维持肿瘤生长7，8，9。CSCs并非均匀分布在整个肿瘤中，而是富集在特定微环境中，包括血管周围生态位和会坏死区域，每个区域都为其细胞状态提供不同的分子调控10，11，12，13，14。CSC不是微环境线索的被动接受者，而是具有重塑自身微环境的能力7，15，16。CSC的微环境可以促进干细胞状态的维持，以响应营养稀缺，pH和缺氧等压力17，18，19，20，这表明这些条件在模型系统中的重要性。因此，概述肿瘤内不同的细胞微环境对于理解治疗耐药性和确定新疗法至关重要。
三维文化近年来越来越受欢迎21，22。类器官已被用于其他类型的癌症，维持细胞作为类器官的主要目标是允许异质细胞群的生长（其中许多细胞群通常在更均匀的球体培养中可能被竞争所击败）和空间多样性，被视为具有遗传特异性的区域肿瘤微环境4，23，24，25，26.癌细胞的三维培养方法有很多种，每种方法都有优点和缺点27，28，29。类器官培养不能替代传统的贴壁或球状培养。当存在细胞微环境与肿瘤细胞反应之间的相互作用至关重要的特定问题时，最好将其用作二维方法的补充技术。
本文介绍了从原发性患者样本或患者来源的培养物中生成GBM类器官的可靠且可重复的方法。我们解决了三维类器官培养的两个不同目标：（1）从原发性患者组织中建立类器官，无论均匀性如何，都具有最大的植入潜力，或（2）生长均匀类器官以进行更定量的实验使用。当将原代标本建立为类器官时，无需过滤单个细胞或计数细胞，因为保持最大细胞数量和类型以建立初始培养物是当务之急。然而，当为比较实验培养类器官时，需要单细胞过滤和细胞计数，以确保重复类器官具有实验一致性的可比性。该协议详细介绍了如何建立类器官培养物并创建均匀的类器官，以及包埋和保存类器官和标准细胞培养实验的改进方法，包括免疫组织化学，免疫荧光和GBM类器官中总细胞活力的评估。

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			<td>3,3-Diaminobenzidine (DAB) tablets</td>
		</tr>
		<tr>
			<td>96-well PCR plates</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td>96-well PCR plates</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>ThermoFisher Scientific</td>
			<td>SCR005</td>
			<td>Cell detachment solution</td>
		</tr>
		<tr>
			<td>Antibiotic-antimycotic</td>
			<td>ThermoFisher Scientific</td>
			<td>15240062</td>
			<td>Antibiotic-antimycotic</td>
		</tr>
		<tr>
			<td>B-27 supplement minus vitamin A</td>
			<td>ThermoFisher Scientific</td>
			<td>12587001</td>
			<td>B-27 supplement minus vitamin A (50x)</td>
		</tr>
		<tr>
			<td>GelCode Blue Stain Reagent</td>
			<td>ThermoFisher Scientific</td>
			<td>24590</td>
			<td>Bluing reagent</td>
		</tr>
		<tr>
			<td>Cell strainer (70 &#181;m)</td>
			<td>CellTreat Scientific</td>
			<td>229483</td>
			<td>Cell strainer (70 &#181;m)</td>
		</tr>
		<tr>
			<td>CellTiter-Glo 3D</td>
			<td>Promega</td>
			<td>G9681</td>
			<td>Luminescent cell viability assay</td>
		</tr>
		<tr>
			<td>Clarifier 2</td>
			<td>ThermoFisher Scientific</td>
			<td>7301</td>
			<td>Nuclear hematoxylin clarifying reagent</td>
		</tr>
		<tr>
			<td>Clear-Rite 3</td>
			<td>ThermoFisher Scientific</td>
			<td>6901</td>
			<td>xylene substitute</td>
		</tr>
		<tr>
			<td>Epredia Gill 2 Hematoxylin</td>
			<td>ThermoFisher Scientific</td>
			<td>72504</td>
			<td>Hematoxylin</td>
		</tr>
		<tr>
			<td>Glutamine in 0.85% NaCl</td>
			<td>ThermoFisher Scientific</td>
			<td>35050061</td>
			<td>Glutamine in 0.85% NaCl (200 mM)</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>ThermoFisher Scientific</td>
			<td>354234</td>
			<td>Laminin-enriched extracellular matrix</td>
		</tr>
		<tr>
			<td>Mini PAP pen</td>
			<td>ThermoFisher Scientific</td>
			<td>008877</td>
			<td>Hydrophobic barrier pen</td>
		</tr>
		<tr>
			<td>Mounting medium</td>
			<td>ThermoFisher Scientific</td>
			<td>22-050-102</td>
			<td>Mounting medium</td>
		</tr>
		<tr>
			<td>Neurobasal media minus phenol red</td>
			<td>ThermoFisher Scientific</td>
			<td>12349015</td>
			<td>Neurobasal media minus phenol red (500 mL)</td>
		</tr>
		<tr>
			<td>Normal donkey serum (NDS)</td>
			<td>Jackson ImmunoResearch</td>
			<td>017-000-121</td>
			<td>Normal donkey serum (NDS)</td>
		</tr>
		<tr>
			<td>Paraformaldehyde 4% in PBS</td>
			<td>ThermoFisher Scientific</td>
			<td>AAJ19943K2</td>
			<td>Paraformaldehyde 4% in PBS</td>
		</tr>
		<tr>
			<td>Phenol red</td>
			<td>Sigma</td>
			<td>P0290</td>
			<td>Phenol red (0.5%)</td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant with DAPI</td>
			<td>ThermoFisher Scientific</td>
			<td>P10144</td>
			<td>Liquid curing mountant</td>
		</tr>
		<tr>
			<td>Recombinant human EGF protein</td>
			<td>R&#38;D systems</td>
			<td>236-EG-01M</td>
			<td>Recombinant human EGF protein (250 &#181;g/mL)</td>
		</tr>
		<tr>
			<td>Recombinant human FGF basic</td>
			<td>R&#38;D systems</td>
			<td>4144-TC-01</td>
			<td>Recombinant human FGF basic (250 &#181;g/mL)</td>
		</tr>
		<tr>
			<td>SignalStain Antibody Diluent</td>
			<td>Cell Signaling</td>
			<td>8112</td>
			<td>Antibody diluent</td>
		</tr>
		<tr>
			<td>SignalStain Boost IHC Detection Reagent</td>
			<td>Cell Signaling</td>
			<td>8114</td>
			<td>Immunohistochemistry detection reagent</td>
		</tr>
		<tr>
			<td>SignalStain Citrate Unmasking Solution</td>
			<td>Cell Signaling</td>
			<td>14746</td>
			<td>Citrate unmasking solution</td>
		</tr>
		<tr>
			<td>Single edge razor blade</td>
			<td>Uline</td>
			<td>H-595B</td>
			<td>Single edge razor blade</td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>ThermoFisher Scientific</td>
			<td>11360070</td>
			<td>Sodium pyruvate (100 mM)</td>
		</tr>
		<tr>
			<td>Tissue-Tek Cryomolds</td>
			<td>VWR</td>
			<td>25608-916</td>
			<td>Disposable cryomolds</td>
		</tr>
		<tr>
			<td>Tissue-Tek O.C.T. Compound</td>
			<td>VWR</td>
			<td>25608-930</td>
			<td>Optimal cutting temperature compound</td>
		</tr>
		<tr>
			<td>Trypan Blue Stain</td>
			<td>ThermoFisher Scientific</td>
			<td>T10282</td>
			<td>Cell impermeant stain (0.4%)</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>ThermoFisher Scientific</td>
			<td>X3P-1GAL</td>
			<td>Xylene</td>
		</tr>
	</tbody>
</table>

maintaining human glioblastoma cellular diversity ex vivo using three-dimensional organoid culture

胶质母细胞瘤（GBM）是最常见的原发性恶性脑癌，预后极差。肿瘤内细胞和分子多样性，以及肿瘤微环境之间的复杂相互作用，可能使寻找有效的治疗方法成为一项挑战。传统的贴壁或球体培养方法可以掩盖这种复杂性，而三维类器官培养可以概括区域微环境梯度。类器官是一种三维GBM培养方法，可更好地模拟患者肿瘤结构，包含表型多样化的细胞群，可用于中等通量实验。虽然与传统培养相比，三维类器官培养更加费力和耗时，但它提供了独特的好处，可以弥合当前 体外 和 体内 系统之间的差距。类器官已成为癌症生物学家武器库中的宝贵工具，可以更好地了解肿瘤行为和耐药机制，并且其应用只会继续增长。这里详细介绍了生成和维护GBM类器官的方法。还介绍了如何使用冷冻和石蜡包埋技术进行类器官样品包埋和切片的说明，以及类器官切片的免疫组织化学和免疫荧光方案的建议，以及总类器官细胞活力的测量。

图1 显示了在10倍放大镜下通过光学显微镜观察到的早期类器官生长。 图1A 显示了中央视图中通过lrECM的单个细胞的迁移和侵袭。细胞将继续扩张并“定植”lrECM，并且通过目视检查，它们将显得更致密并最终不透明。 图1B 显示了几个成熟的类器官（在7周时），相对于一角硬币的大小没有放大倍数。
图2 显示了GBM类器官的磷酸化组蛋白H3的免疫组化染色，磷酸化组蛋白H3是活性增殖的标志物。与类器官核心相比，大多数高度增殖的细胞出现在类器官周边。阳性染色将具有棕色/铜色外观。
图3 描述了使用3D特异性发光细胞活力测定法匀浆和测量GBM类器官中总细胞数的过程。由于GBM类器官中存在大量细胞，较大的类器官结构最初通过在发光测定试剂中研磨来均质化。然后将总类器官裂解物的馏分加载到单个孔中，并用额外的发光测定试剂稀释，然后在适当的多孔板读数器上孵育和读数。
图4 表示类器官的DMSO（普通载体）控制数据。绘制的数据显示了类器官内和类器官间的一致性。在生成实验数据时，发光活性数据通常会归一化为每个样品的对照。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63745/63745fig01.jpg"> 图 1：通过光学显微镜观察 （10x）。 （A）早期类器官生长表明GBM细胞在整个富含层粘连蛋白的细胞外基质中的迁移/侵袭。（B）相对于一角硬币大小的成熟类器官。<a href="https://www.jove.com/files/ftp_upload/63745/63745fig01large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63745/63745fig02.jpg"> 图2：GBM类器官的免疫组织化学 。 （A， B）GBM类器官显示磷酸组蛋白H3染色细胞用于活性增殖。A 和 B 的比例尺分别为 600 μm 和 300 μm。 <a href="https://www.jove.com/files/ftp_upload/63745/63745fig02large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63745/63745fig03.jpg"> 图3：类器官的发光细胞活力方案 。 （A）将单个类器官移动到小离心管中并去除多余的培养基。（B） 向每个试管中加入 500 μL 1：1 PBS 和发光细胞活力测定混合物，并积极移液以分解类器官。（C） 向 96 孔板的每个孔中加入 25 μL 该类器官混合物和 75 μL 相同的 1：1 PBS 和发光细胞活力测定混合物。（D）放在摇床上2分钟，然后在室温下孵育20分钟，然后在读板器上读取发光。使用 BioRender.com 制作的图。 <a href="https://www.jove.com/files/ftp_upload/63745/63745fig03large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63745/63745fig04.jpg"> 图4：细胞活力。 0.1%二甲基亚砜（DMSO）中类器官的细胞活力数据，用于四个患者标本的四个类器官的六个技术重复。 <a href="https://www.jove.com/files/ftp_upload/63745/63745fig04large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>元件</td>
			<td>数量</td>
		</tr><tr><td>神经基础培养基减去酚红</td>
			<td>500毫升</td>
		</tr><tr><td>B-27补充剂减去维生素A（50倍）</td>
			<td>10毫升</td>
		</tr><tr><td>抗生素-抗真菌药（100x）</td>
			<td>5毫升</td>
		</tr><tr><td>丙酮酸钠（100 mM）</td>
			<td>5毫升</td>
		</tr><tr><td>0.85%氯化钠（200毫米）中的谷氨酰胺</td>
			<td>5毫升</td>
		</tr><tr><td>重组人 FGF 碱性 （250 μg/mL）</td>
			<td>20 微升</td>
		</tr><tr><td>重组人 EFG 蛋白 （250 μg/mL）</td>
			<td>20 微升</td>
		</tr><tr><td>酚红</td>
			<td>500 微升</td>
		</tr></tbody></table>表1：神经基础培养基完全（NBMc）制剂
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>试剂</td>
			<td>时间</td>
		</tr><tr><td>50%乙醇</td>
			<td>3 分钟</td>
		</tr><tr><td>75%乙醇</td>
			<td>3 分钟</td>
		</tr><tr><td>95%乙醇</td>
			<td>3 分钟</td>
		</tr><tr><td>95%乙醇</td>
			<td>4 分钟</td>
		</tr><tr><td>100%乙醇</td>
			<td>2 分钟</td>
		</tr><tr><td>100%乙醇</td>
			<td>3 分钟</td>
		</tr><tr><td>100%乙醇</td>
			<td>4 分钟</td>
		</tr><tr><td>二甲苯替代品</td>
			<td>2 分钟</td>
		</tr><tr><td>二甲苯替代品</td>
			<td>3 分钟</td>
		</tr><tr><td>二甲苯替代品</td>
			<td>4 分钟</td>
		</tr><tr><td>石蜡</td>
			<td>15 分</td>
		</tr><tr><td>石蜡</td>
			<td>15 分</td>
		</tr></tbody></table>表 2：小类器官（直径小于 3 mm）的加工计划
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>试剂</td>
			<td>时间</td>
		</tr><tr><td>50%乙醇</td>
			<td>6 分钟</td>
		</tr><tr><td>75%乙醇</td>
			<td>6 分钟</td>
		</tr><tr><td>95%乙醇</td>
			<td>5 分钟</td>
		</tr><tr><td>95%乙醇</td>
			<td>8 分钟</td>
		</tr><tr><td>100%乙醇</td>
			<td>5 分钟</td>
		</tr><tr><td>100%乙醇</td>
			<td>5 分钟</td>
		</tr><tr><td>100%乙醇</td>
			<td>8 分钟</td>
		</tr><tr><td>二甲苯替代品</td>
			<td>5 分钟</td>
		</tr><tr><td>二甲苯替代品</td>
			<td>5 分钟</td>
		</tr><tr><td>二甲苯替代品</td>
			<td>8 分钟</td>
		</tr><tr><td>石蜡</td>
			<td>30 分</td>
		</tr><tr><td>石蜡</td>
			<td>30 分</td>
		</tr></tbody></table>表 3：大型类器官（直径超过 3 mm）的处理计划
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>试剂</td>
			<td>时间</td>
		</tr><tr><td>苏木精</td>
			<td>2 分钟</td>
		</tr><tr><td>运行diH 2O</td>
			<td>2 分钟</td>
		</tr><tr><td>核苏木精澄清试剂</td>
			<td>1 分钟</td>
		</tr><tr><td>运行diH 2O</td>
			<td>1 分钟</td>
		</tr><tr><td>发蓝试剂</td>
			<td>1 分钟</td>
		</tr><tr><td>运行diH 2O</td>
			<td>2 分钟</td>
		</tr><tr><td>70%乙醇</td>
			<td>1 分钟</td>
		</tr><tr><td>100%乙醇</td>
			<td>1 分钟</td>
		</tr><tr><td>100%乙醇</td>
			<td>1 分钟</td>
		</tr><tr><td>二甲苯替代品</td>
			<td>2 分钟</td>
		</tr><tr><td>二甲苯替代品</td>
			<td>2 分钟</td>
		</tr></tbody></table>表4：苏木精复染剂

Watch this Scientific Journal Video about Maintaining Human Glioblastoma Cellular Diversity Ex vivo using Three-Dimensional Organoid Culture at JoVE.com

Maintaining Human Glioblastoma Cellular Diversity Ex vivo using Three-Dimensional Organoid Culture

在这里，我们描述了一种从主要患者标本或患者来源的细胞培养物中生成胶质母细胞瘤（GBM）类器官并将其维持至成熟的方法。这些GBM类器官含有表型不同的细胞群，并在 体外重建肿瘤微环境。

使用三维类器官培养物离 体 维持人胶质母细胞瘤细胞多样性

Glioblastoma (GBM) is the most commonly occurring primary malignant brain cancer with an extremely poor prognosis. Intra-tumoral cellular and molecular ...

寻找胶质母细胞瘤的治疗方法具有挑战性，因为肿瘤中存在的细胞多样性以及不同微环境之间的相互作用。类器官可以帮助重建一些空间多样性，我们可以用它来更好地了解肿瘤的生物学。虽然传统的细胞培养方法倾向于产生均质的肿瘤细胞群，但类器官可以更好地模拟胶质母细胞瘤的分子和细胞多样性，并更好地模拟肿瘤内相互作用。类器官可用于研究药物疗效和耐药机制。对临床治疗的耐药性可能只出现在类器官中，而不出现在传统的球体培养中，反映了这种疾病的临床现实。类器官可以帮助证明化学疗法的区域效应。它们可用于发现新的利基特定疗法，也可以作为未来个性化医疗的预测工具。一些用于制造类器官的材料对温度敏感，忽视将这些材料保持在适当的温度会导致难以创建或维护类器官。通过将蜡纸从封口膜片上取下并将其放置在两个无菌 96 孔聚合酶链反应或 PCR 板之间，开始在培养罩下制备类器官模具。确保封口膜内部清洁。对顶板施加均匀的压力，在封口膜上形成两毫米深的小酒窝，而不会产生孔洞。将盘子分开。凹陷的封口膜会粘在顶板上。然后将顶板放在干冰上 30 秒。使用无菌镊子快速将封口膜从顶板上拉下。冷冻封口膜更容易去除。将完成的封口膜模具放入有盖的无菌 10 厘米细胞培养皿中。为了从胶质母细胞瘤或GBM贴壁培养物中制备单细胞悬液，请从平板上除去用过的培养基。然后在室温下向平板中加入两毫升细胞分离溶液，并将板在37度下孵育3分钟。在显微镜下确认细胞从平板上脱离后，加入8毫升完全神经基础培养基或NBMc培养基以中和细胞分离溶液。通过单个70微米细胞过滤器过滤悬浮液。以 120 倍 G 的速度旋转细胞五分钟。然后除去上清液并将细胞重悬于一毫升NBMc中。 使用细胞非渗透染色剂计数单细胞悬液中的细胞。为了从单细胞悬浮液制备类器官，将适量的富含层粘连蛋白的细胞外基质或lrECM添加到冰桶或冷块中的小离心管中。制备每个类器官含有20， 000个细胞的细胞混合物。彻底混合lrECM细胞悬液混合物，以避免细胞沉降，从而导致类器官形成不均匀。接下来，小心地将 20 微升混合物移液到封口膜模具上，形成珍珠状液滴。确保每两到三个类器官冷却移液器吸头，以防止lrECM聚合。将所需数量的类器官移液到10厘米培养板中的封口膜模具上后，将类器官在37摄氏度下在细胞培养箱中孵育一至两个小时。类器官固化后，使用P1000尖端用NBMc培养基轻轻冲洗类器官从封口膜模具上，并将它们收集到含有20毫升NBMc的新10厘米培养板中。 将培养板放入培养箱中，不摇晃四天。四天后，更换板中的培养基。为此，在细胞培养板下方放置一张深色纸。倾斜盘子并等待 20 秒。当类器官完全沉降在培养皿底部时，用大开口玻璃移液管慢慢取出培养基。添加新培养基后，将板放入培养箱中，以80 RPM的轨道振荡器，此后每两到三天更换一次培养基。使用光学显微镜观察类器官的生长。在中心视图中，早期类器官生长显示通过lrECM迁移和单细胞侵袭。七周时的成熟类器官相对于一角硬币的大小。用磷酸化组蛋白H3对GBM类器官进行免疫组织化学染色，显示与核心相比，类器官周边具有棕色或紫色外观的高度增殖细胞。0.1%DMSO中类器官的细胞活力数据表明类器官内和类器官间的一致性。注意类器官生物量和培养基酸化。如果类器官通过培养基的速度太快，请务必更频繁地更换培养基或在附加板之间拆分类器官。成熟的类器官可用于治疗实验，或包埋和切片以用于免疫组织化学或免疫荧光。解离的类器官可用于FACS分析。这些类器官最近被用于三维功能筛选，以发现空间上不同的癌症干细胞生态位中必不可少的靶标。我们还使用这些模型在体内测试之前优先考虑不同的药物和疗法。

Research

JoVE Journal

Cancer Research

Maintaining Human Glioblastoma Cellular Diversity Ex vivo using Three-Dimensional Organoid Culture

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

Ex 活体人肺肿瘤切片中居民 CD8 T 淋巴细胞的共聚焦显微成像

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the ...

科研

癌症研究

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

胶质瘤移植中体内成像的荧光分子层析成像研究

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting ...

Longitudinal Morphological and Physiological Monitoring of Three-dimensional Tumor Spheroids Using Optical Coherence Tomography

三维肿瘤球体的纵向形态和生理监测

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, ...

Fully Human Tumor-based Matrix in Three-dimensional Spheroid Invasion Assay

三维流状入侵检测中的全人肿瘤基基质

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor ...

Evaluating the Differentiation Capacity of Mouse Prostate Epithelial Cells Using Organoid Culture

利用有机体培养评价小鼠前列腺皮细胞的分化能力

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation ...

Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions

胶质母细胞瘤干细胞在模式神经元上共同培养，以研究迁移和细胞相互作用

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite ...

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

使用胶原基支架的神经母细胞瘤三维 体外 仿生模型

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a ...

Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells

Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells

离体 小鼠尿路上皮细胞腺病毒-Cre介导的基因缺失的类器官模型

Bladder cancer is an understudied area, particularly in genetically engineered mouse models (GEMMs). Inbred GEMMs with tissue-specific Cre and loxP sites ...

Phospholipid Mediator Induced Transformation in Three-Dimensional Cultures

磷脂介质诱导的三维培养物转化

Several models have been developed to study cancer, such as rodent models and established cell lines. Valuable insights into carcinogenesis have been ...

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

小鼠结肠隐窝三维培养离体研究肠道干细胞功能

The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of ...

使用三维类器官培养物离 体 维持人胶质母细胞瘤细胞多样性

使用三维类器官培养物离体维持人胶质母细胞瘤细胞多样性