Name: growth and characterization of irradiated organoids from mammary glands
Uploaded: 2019-05-03T14:00:00
Description: Watch this Scientific Journal Video about Growth and Characterization of Irradiated Organoids from Mammary Glands at JoVE.com

我们感谢劳拉博士 l. Bronsart 提供了 GFP 和 dtomato 标记的 RAW 264.7 巨噬细胞。这项研究得到了国家卫生研究院 #R00CA201304 资助的财政支持。

动物研究是根据范德比尔特大学动物护理和使用机构委员会批准的机构准则和议定书进行的。
1. 小鼠的制备和细胞采集 (改编自 Nguyen-ngkoc 等人)
<ol><li>牺牲胸腺 Nu/Nu 小鼠 (8-10 大) 使用 co2 窒息, 然后颈椎脱位。使用70% 的乙醇清洁皮肤。</li>
	<li>使用预先消毒的剪刀和钳子切除小鼠的腹部和腹股沟乳腺。切除前切除淋巴结。在无菌1x 磷酸盐缓冲盐水中...

<ol>
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在该协议中, 我们开发了一种用于辐照乳腺有机体的可重复生长和表征的方法 (图 1)。应用20戈的辐照剂量来反映以前肿瘤细胞招募5的体内模型。在有机体形成之前的体内乳腺照射允许分离辐射损伤效应, 而不会相应的免疫细胞浸润。体外辐照正常组织模型的开发使人们能够实时观察可能导致辐射诱导的 ctc 招募11,12</su...

大约60% 的三重阴性乳腺癌 (TNBC) 患者选择保乳疗法 (BCT) 作为一种治疗形式 1。在这种治疗方式中, 含有部分乳房组织的肿瘤被切除, 周围的正常组织暴露在电离辐射下, 杀死任何残留的肿瘤细胞。治疗可减少大部分乳腺癌患者的复发;然而, 大约13.5% 的 TNBC 患者经历局部复发 2.因此, 研究辐射如何招募循环肿瘤细胞 (ctc) 将导致对局部复发3,4的重要洞察。
先前的研究表明, 正常组织的辐射增加了不同类型细胞的招募5。在 TNBC 的临床前模型中, 正常组织的照射增加了巨噬细胞, 随后肿瘤细胞被招募到正常组织5。免疫状态影响肿瘤细胞招募到辐照部位, 在免疫功能低下的受试者中观察到肿瘤细胞迁移。利用乳腺产生的有机体对这些相互作用进行复制, 可以通过显微镜和活细胞成像实时观察细胞迁移和细胞间质相互作用, 以确定辐射损伤在改变中的作用肿瘤细胞的行为。
小鼠乳腺组织已帮助阐明在乳腺发育的关键步骤。乳腺有机体是一种多细胞的三维结构, 是一种孤立的乳腺上皮, 大于 50μm6,7,8, 9,10.使用原发上皮类固醇, Simian 等人评估了乳腺分枝的必要因素 7.Shamir 等人发现, 传播可以发生没有上皮间充质过渡, 提供了洞察转移级联8。从乳腺组织中生成和表征有机体的方法已确定 6,11,12,13。然而, 据我们所知, 从乳腺中生长辐照有机体的方法还没有报道。一项生长和描述辐照有机体的协议将是重述辐射诱导的免疫和肿瘤细胞招募的关键一步。
本文报道了一种在低粘附微板中生长和表征辐照乳腺上皮细胞的方法, 这些微板上涂有一种支持球体形成的亲水性聚合物。这些有机体与巨噬细胞共同培养, 以检查免疫细胞浸润动力学。这项工作可以扩展到包括共培养有机体与脂肪细胞, 以重述乳房特征, 乳腺癌细胞可视化肿瘤细胞的招募, CD8 + T 细胞, 以研究肿瘤免疫细胞的相互作用。以前建立的协议可用于评估辐照有机体。早期的模型共同培养乳腺有机体和免疫细胞已经揭示了转移和传播的机制。德纳多等人发现, CD4 + T 细胞对肿瘤相关巨噬细胞的调节增强了乳腺腺癌的转移表型14。共同文化模型也被用来阐明生物发展的机制。Plaks 等人阐明了 CD4 + T 细胞作为乳腺器官发生的下调节剂的作用15。然而, 我们的小组是第一个建立一个程序, 可视化正常的组织照射如何影响免疫细胞的行为。由于正常组织照射已被证明可以增强肿瘤细胞的吸收5, 这一方案可以进一步发展, 以分析肿瘤细胞行为如何被正常组织和细胞的照射改变, 从而使人们对肿瘤细胞的行为有了更大的了解。癌症复发。

<table>
	<tbody>
		<tr>
			<td>10% Neutral Buffered Formalin</td>
			<td>VWR</td>
			<td>16004-128</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-cytokeratin 14</td>
			<td>abcam</td>
			<td>ab181595</td>
			<td>Lot: GR3200524-3</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A1933-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen Type I</td>
			<td>Corning</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase from Clostridium Histolyticum, Type VIII</td>
			<td>Sigma</td>
			<td>C2139</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase I</td>
			<td>Gibco</td>
			<td>17018029</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Thermofisher</td>
			<td>11320-033</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse</td>
			<td>Roche</td>
			<td>10104159001</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Fisher</td>
			<td>14190250</td>
			<td></td>
		</tr>
		<tr>
			<td>E-Cadherin</td>
			<td>Cell Signaling</td>
			<td>24E10</td>
			<td>Lot: 13</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Sigma</td>
			<td>F0926</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Gibco</td>
			<td>15750</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit secondary</td>
			<td>abcam</td>
			<td>ab150077</td>
			<td>green 
			Lot: GR3203000-1</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit secondary</td>
			<td>abcam</td>
			<td>ab150080</td>
			<td>red 
			Lot: GR3192711-1</td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Fisher</td>
			<td>62249</td>
			<td>Lot: TG2611041</td>
		</tr>
		<tr>
			<td>Insulin (10 mg/mL)</td>
			<td>Sigma</td>
			<td>I9278</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium, 100x</td>
			<td>Gibco</td>
			<td>51500-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Basement Membrane (basement membrane extracted from Engelbreth-Holm-Swarm mouse sarcoma)</td>
			<td>Corning</td>
			<td>356237</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>Vector Laboratories</td>
			<td>S-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclon Sphera 96 well plates</td>
			<td>Thermo</td>
			<td>174927</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>VWR</td>
			<td>10128-856</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen/strep</td>
			<td>Fisher</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin</td>
			<td>abcam</td>
			<td>ab176757</td>
			<td>Lot: GR3214582-16</td>
		</tr>
		<tr>
			<td>Tight Junction Protein 1</td>
			<td>Novus</td>
			<td>NBP1-85047</td>
			<td>Lot: C115428</td>
		</tr>
		<tr>
			<td>Triton X-100 (4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol)</td>
			<td>Sigma</td>
			<td>X100-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco</td>
			<td>27250-018</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20 (Polyethylene glycol sorbitan monolaurate)</td>
			<td>Sigma</td>
			<td>P1379-100ML</td>
			<td></td>
		</tr>
	</tbody>
</table>

growth and characterization of irradiated organoids from mammary glands

从消化组织中提取的有机体是多细胞三维 (3D) 结构, 比细胞单层在体内条件下更有能力进行重述。虽然它们不能在体内完全模拟复杂性, 但它们保留了原始器官的一些功能。在癌症模型中, 有机体常用于研究肿瘤细胞的侵袭。该方案旨在开发和表征正常和辐照小鼠乳腺组织中的有机体, 以评估正常组织中的辐射反应。这些有机体可用于未来的体外癌症研究, 以评估肿瘤细胞与辐照有机体的相互作用。乳腺被切除, 照射到20戈, 并在胶原酶 VIII 溶液中消化。通过离心分化分离上皮性有机硅, 并在96好低粘附微板中制备三维有机生物。有机体表达了细胞角蛋白14的特征上皮标记。在共培养实验中观察到巨噬细胞与有机体的相互作用。该模型可用于研究肿瘤间质相互作用、免疫细胞浸润和在辐照微环境中巨噬细胞极化的研究。

从小鼠乳腺成功地获得辐照上皮乳腺有机体, 并在低粘附板上进行处理和培养 (图 1)。在不同生长环境下通过播种测试了有机产量 (图 2A-G)。将细胞直接播种到经过10厘米的细胞板上, 导致成纤维细胞过度生长。在与有机体相同的病灶平面内或附近的相对照显微镜下, 对成纤维细胞进行了鉴定, 并在几天内迅速从镀金有机体中生长出来。在基底膜和胶原蛋白基质中播...

Watch this Scientific Journal Video about Growth and Characterization of Irradiated Organoids from Mammary Glands at JoVE.com

Growth and Characterization of Irradiated Organoids from Mammary Glands

对小鼠乳腺发育的有机体进行辐照, 并对其进行评估, 以评估上皮特征和与免疫细胞的相互作用。辐照有机体可用于更好地评估细胞相互作用, 这可能导致肿瘤细胞在辐照正常组织中的招募。

乳腺辐照有机体的生长与表征

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell ...

该协议可用于进一步发展，以分析电离辐射如何影响肿瘤细胞的招募，从而更好地了解癌症复发。乳腺有机体是强大的模型。它们模仿基本的体内特征，但它们允许容易分离生物变量。使用低粘附板可抵消与蛋白质基质相关的工作流程挑战。三阴性乳腺癌患者在治疗后以较高的发病率经历局部区域复发。研究辐射如何影响肿瘤和免疫细胞行为将导致对复发机制的重要见解。展示有机体共体显微镜的是哈维尔·戈麦斯，一个在西尔维娜·巴蒂斯塔实验室的研究生。从切除牺牲的小鼠的腹部和腹腺开始这个程序，详见文本协议。如文本协议所述，在照射样品后 45 分钟，将乳腺放在 35 毫米无菌细胞板中，用手术刀切碎。在大约 40 次冲程下，直到获得不超过约 1 平方毫米的组织放松和片段。现在，在50毫升离心管中将组织转移到胶原酶溶液中。每只小鼠使用10毫升胶原酶溶液。将样品管在37摄氏度下放在水浴中30至60分钟，每10分钟旋转5秒钟。当胶原酶溶液浑浊时，消化是完整的。在室温下，以 450 次-G 旋转消化溶液 10 分钟。现在观察到三层。上清液由脂肪组成，中间层为水溶液，底部为颗粒。颗粒显示为红色，因为它是上皮细胞、单个频闪细胞和红血球的混合物。在添加和去除 BSA 溶液之前，使用 BSA 溶液预涂所有移液器、移液器尖端和离心管。BSA 溶液可以重复使用，尽管每次实验前都应进行无菌过滤。要进行额外的恢复，请将上一液转移到新的 BSA 涂层 15 毫升管中。向上和向下大力移液以分散脂肪层。在室温下以450倍-G离心样品10分钟。吸上一提液，在管中留下少量介质，以避免吸气细胞颗粒。现在用原颗粒从管子中吸出水层。将 10 毫升 DMEM/F12 加入到管中，用原始颗粒将转移到第二管中。移液器大力组合和重新暂停两个颗粒。在室温下以 450 次-G 离心 10 分钟后，吸升液，并将 4 毫升 DMEM/F12 添加到管子中。在悬浮液中加入40微升脱氧核素酶，在室温下轻轻握手2至5分钟。加入六毫升的DMEM/F12和移液器彻底。在室温下以 450 次-G 离心管 10 分钟后，将上经器吸到 0.5 毫升标记。将颗粒重新在 10 毫升 DMEM/F12 和移液器中彻底。现在，通过脉冲到450次G和停止4秒后达到该速度的管离心。重复洗涤步骤三次，通过离心分化净化器官。颗粒现在应该是一种非白色的颜色，只由上皮器官组成。将颗粒在 10 毫升 DMEM/F12 中重新暂停。彻底移液，创造同质溶液。将 50 微升转移到 30 毫米培养皿中，并在 20 倍的相对比显微镜下查看。使用计数计数器计算器官的数量。将50微升的有机体放入低粘附板的每个井中。添加150微升的有机介质，使总工作量达到200微升。每两天小心更换一次介质。在DMEM介质中保持GSP或D-番茄标记的生264.7巨噬细胞，并辅以10%的FPS和1%青霉素链霉素。以所需的密度将细胞播种到器官介质中。使用活细胞相对比和荧光成像来监测巨噬细胞的渗透。小心吸气，从井中去除有机介质。在室温下用10%中性缓冲的甲醛固定样品15分钟。在1X PBS中清洗固定器官三次，5分钟。如果需要，固定样品可以在四摄氏度下储存一个星期，以进行进一步染色。洗涤后，将固定器官渗透五分钟。在室温下用5%正常山羊血清阻断固定器官，在室温下一小时，然后用PBS洗涤三次，用PBS洗5分钟。用抗细胞素-14、E-卡德林或紧密结蛋白1，在PBST的1%NGS中孵育固定器官，在室温下孵育一小时。然后在 PBST 中洗三次，洗五分钟。现在用山羊抗兔二次抗体孵育器官，在室温下用1%NGS PBST孵育一小时。用铝箔盖住，避免光线照射。洗完后，使用核染料染色核约半小时。在PBS中清洗染色的有机体五分钟。如果使用腔室滑轨，则使用盖滑点安装。将包裹在四摄氏度的铝箔中的有机体储存长达两周。辐照的有机体可以在低粘附板或地下室膜内培养，但生长速度最快的发生在低粘附板中。器官重述乳腺特征。观察了在形态上与管道和叶相似的构造。生长趋势表明，当器官在消化和分拣后立即播种时，非辐照器官的生长速度比辐照的器官快，很可能是由于DNA损伤修复机制导致细胞生长的阻止。组织体表示上皮特征，通过免疫荧光染色进行评估。辐照的器官表示上皮标记。细胞素-14，一种明性氦的标记，在辐照的有机体表面强烈地表达。此外，E-卡德林和紧密结蛋白一在器官的细胞结内表达。这些蛋白质对于适当的细胞粘附是必不可少的。辐照后，器官继续保持其上皮特征。使用荧光显微镜，可以在低粘附板中显示器官的荧光染色。然而，最清晰的可视化是通过共和显微镜获得的。通过减去背景和按器官面积进行标准化来计算校正的总荧光强度。96孔低粘附板中生长的器官也简化了共培养实验。当以乳腺中典型的浓度播种时，巨噬细胞结肠化随着辐照的器官增加而增加。执行此程序时，要记住的最重要的事情是不要过度消化有机体，并适当地净化离心分化。按照此过程，可以执行其他共培养实验。有机物可以与肿瘤细胞、其他免疫细胞和频闪细胞共同培养，以重现与复发相关的辐照微环境。这种方法对于评估正常组织损伤如何具体影响免疫细胞动力学非常重要，最终将用于了解肿瘤细胞的招募机制。乳腺器官已阐明乳腺发育的机制，并在体外有力地重述乳腺癌。我们希望我们的模型提供有关辐射诱导肿瘤细胞招募的见解。

Research

JoVE Journal

Bioengineering

Axon Stretch Growth: The Mechanotransduction of Neuronal Growth

轴突伸展生长：神经生长Mechanotransduction

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata ...

科研

生物工程

Generation and Recovery of &beta;-cell Spheroids From Step-growth PEG-peptide Hydrogels

Generation and Recovery of ÃƒÅ½Ã‚Â²-cell Spheroids From Step-growth PEG-peptide Hydrogels

产生和回收的β-细胞的细胞球体从逐步增长的PEG-肽水凝胶

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional  microenvironment with tissue-like elasticity and high permeability for  ...

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

人类乳腺上皮细胞的激素反应3D文化模式

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is ...

Intraductal Delivery to the Rabbit Mammary Gland

导管内送货到兔乳腺

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and ...

Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation

用于神经生长因子包封的新型HDL模拟纳米颗粒的制备和表征

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

微流控生物工程血管化组织和细胞器解体

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

含有宏观结构的光学组织幻影的制备与表征

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

用利穆鲁布莱巴细胞裂解液 (lal) 法检测纳米配方中的内毒素

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Focused Ion Beam Lithography to Etch Nano-architectures into Microelectrodes

聚焦 Ion 光束平版印刷到蚀刻纳米架构到微电极

With advances in electronics and fabrication technology, intracortical microelectrodes have undergone substantial improvements enabling the production of ...

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

人类多能干细胞和免疫损伤的成熟小脑有机体可伸缩生成

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...