Name: generation of organoids from mouse extrahepatic bile ducts
Uploaded: 2019-04-23T15:00:00
Description: Watch this Scientific Journal Video about Generation of Organoids from Mouse Extrahepatic Bile Ducts at JoVE.com

这项工作得到了美国肝病研究协会 (致 n. r.) 和国家卫生研究院、国家糖尿病和消化系统和肾脏疾病研究所 (p30 DK34933 颁发给 n. r.、P01 DK062041 至 j. l. m.) 的支持。我们感谢 Ramon Oacadiz-ruiz 博士 (密歇根大学) 协助制定这一方法。

这里描述的所有方法都已得到密歇根大学动物护理和使用机构委员会 (IACUC) 的批准。
1. 小鼠 EHBD 分离设备和材料的制备
<ol><li>在50毫升锥形管中准备播种介质和洗涤缓冲液 (材料表), 并将其保持在4°c 或冰上, 直至使用。</li>
	<li>设置手术表 (图 1 b)。准备消毒的手术器械 (图 1c)。</li>
	<li>在37°c 组织培养孵化器中放置一...

<ol>
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这项工作描述了小鼠 EHBD 胆管细胞的组织型三维模型的产生。EHBDO 培养过程中的重要步骤包括: 细致的 EHBD 解剖, 以避免胰腺细胞污染, 维护无菌条件, 以防止细菌和真菌污染, 以及离心后的精心操作, 以避免细胞材料的丢失。需要密切坚持所描述的温度条件。该技术存在一些限制。成年小鼠的乙二苯醚较小 (直径约1毫米;图 1e), 需要很巧妙的隔离。解剖显微镜可以用来协助解剖...

胆管病变是一种不治之症, 影响位于肝内和肝外胆管 (Ehbd)的胆道细胞1。一些胆管疾病, 如原发性硬化性胆管炎、胆管癌、胆道闭锁和胆总管囊肿, 主要影响乙二苯醚。由于临床前模型有限, 胆管病治疗的发展受到限制。此外, 以前的研究侧重于分类的胆管疾病: 肝脏、肝内和乙二苯醚。然而, 内和乙二苯醚具有明显的胚胎起源, 因此, 应被视为不同的分子病理。肝内胆管从肝内导管板和肝室的颅骨部分发展, 整个 EHBDs 从肝室2的尾部发育.他们还依靠不同的祖细胞隔间进行成人稳态, 包括 ehbd2,3中肝内胆管和胆道周围的海灵管。使用动物模型进行临床前研究受到费用的限制, 出于伦理原因, 应尽量减少。因此, 在体外模型中, 还原性、重现性、时间和成本效益是非常可取的。
以前对胆管病变的大多数研究都使用了正常的小鼠或大鼠癌症模型, 或来自乙二苯并 4、5、6、7的人胆管癌细胞系.然而, 这些都是转化细胞的模型, 并不重述正常的胆管细胞生物学在稳态或处于健康状态。最近在发展的有机型培养模型的进展, 使三维结构的发展, 从不同的组织类型, 包括肝胆组织, 虽然不是正常的小鼠 ehbd8,9, 10个。这些 "器官样" 结构旨在模仿原代组织, 生长在一个人工生态位, 支持器官特异性干细胞的自我更新, 11。
"有机体" 是一个宽泛的术语, 通常描述从干细胞中获得的三维组织模型。有机体可以从胚胎干细胞和诱导多能干细胞所代表的重新编程的多能干细胞中产生。它们也可以从器官特异性的成人干细胞12中产生。在以往的研究中, 已经提出了一些胆管细胞有机体模型。因此, 从人类多能干细胞中提取的有机生物已被报道为7、9、13, 并提供了一个宝贵的、时间高效的工具, 可以同时生成不同的细胞类型。然而, 这些多能干细胞衍生的有机体并不能完全反映原发性成人 EHBD 胆管细胞的结构和功能。
还提出了从人9和猫10肝的成虫干细胞中提取的有机体。猫科车型并不普遍, 用于研究目的的工具设备有限。此外, 这些肝源性成人干细胞衍生的有机体不模拟肝外胆管细胞, 而是肝内胆管细胞。
人类正常 Ehbd14和小鼠 ehbd 胆管癌15报告了 EHBD 有机体的产生。然而, 获得人类 EHBD 组织的机会极为有限, 从胆管癌的遗传小鼠模型15中获得的有机物质并不代表体内的健康胆管细胞生物学, 而是来自基因修饰细胞。
为了解决多能干细胞和肝脏衍生的胆管细胞组织模型的局限性以及临床前模型所需的对人体组织的访问有限的问题, 我们开发了一个小鼠 EHBD 有机体模型 (图 1a)。这份手稿描述了一种从成人组织中提取的小鼠 ehbd 衍生的有机体技术的发展。这些名为 Ehbdo 的 EHBD 有机体将是研究 Ehbd 胆管局稳态和疾病过程 (如胆管病变) 机制的重要体外工具。

<table border="0" cellpadding="0" cellspacing="0" style="width:799px;" width="798">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">L-WRN cell culture medium</td>
			<td style="width:104px;"></td>
			<td style="width:170px;"></td>
			<td style="width:118px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Advanced DMEM/F12</td>
			<td style="width:104px;"></td>
			<td>Life Technologies</td>
			<td style="width:118px;">12634-010</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:104px;">1%</td>
			<td>Life Technologies</td>
			<td style="width:118px;">10437-028</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Penicillin-Streptomycin</td>
			<td style="width:104px;">100 U/mL</td>
			<td>Life Technologies</td>
			<td style="width:118px;">15140-122</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Washing buffer</td>
			<td style="width:104px;"></td>
			<td></td>
			<td style="width:118px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Phosphate Buffered Saline (PBS)</td>
			<td style="width:104px;">50 mL</td>
			<td>Life Technologies</td>
			<td style="width:118px;">10010-023</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Penicillin-Streptomycin</td>
			<td style="width:104px;">125 U/mL</td>
			<td>Life Technologies</td>
			<td style="width:118px;">15140-122</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Amphotericin B&#160;</td>
			<td style="width:104px;">6.25 &#181;g/mL</td>
			<td>Life Technologies</td>
			<td style="width:118px;">15290-018</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Organoid culture medium</td>
			<td style="width:104px;"></td>
			<td></td>
			<td style="width:118px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">L-WRN Conditioned medium&#160;</td>
			<td style="width:104px;">1:1</td>
			<td style="width:170px;">ATCC</td>
			<td style="width:118px;">CRL-3276</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Advanced DMEM/F12</td>
			<td style="width:104px;">1:1</td>
			<td>Life Technologies</td>
			<td style="width:118px;">12634-010</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Penicillin-Streptomycin</td>
			<td style="width:104px;">100 U/mL</td>
			<td>Life Technologies</td>
			<td style="width:118px;">15140-122</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">N-Glutamine</td>
			<td style="width:104px;">10 &#181;l/mL</td>
			<td>Life Technologies</td>
			<td style="width:118px;">35050-061</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid, HEPES</td>
			<td style="width:104px;">10 mM</td>
			<td>Life Technologies</td>
			<td style="width:118px;">15630-080</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">B27</td>
			<td style="width:104px;">10 &#181;l/mL</td>
			<td style="width:170px;">Gibco</td>
			<td style="width:118px;">17504-044</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">N2</td>
			<td style="width:104px;">10 &#181;l/mL</td>
			<td style="width:170px;">Gibco</td>
			<td style="width:118px;">17502-048</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Organoid seeding medium</td>
			<td style="width:104px;"></td>
			<td style="width:170px;"></td>
			<td style="width:118px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Organoid culture medium&#160;</td>
			<td style="width:104px;"></td>
			<td style="width:170px;"></td>
			<td style="width:118px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Epidermal growth factor (EGF)</td>
			<td style="width:104px;">50 ng/mL</td>
			<td style="width:170px;">Invitrogen</td>
			<td style="width:118px;">PMG8041</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Fibroblast growth factor-10 (FGF10)</td>
			<td style="width:104px;">100 ng/mL</td>
			<td style="width:170px;">PeproTech</td>
			<td style="width:118px;">100-26</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Primary antibodies</td>
			<td style="width:104px;"></td>
			<td style="width:170px;"></td>
			<td style="width:118px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Anti-Cytokeratin 19 (CK19) antibody, Rabbit</td>
			<td style="width:104px;">1:250</td>
			<td style="width:170px;">Abcam</td>
			<td style="width:118px;">ab53119</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Sex-Determining Region Y-Box 9 (SOX9) antibody, Rabbit</td>
			<td style="width:104px;">1:200</td>
			<td style="width:170px;">Santa Cruz</td>
			<td style="width:118px;">sc-20095</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Pancreatic Duodenal Homeobox 1 (PDX1) antibody, Rabbit</td>
			<td style="width:104px;">1:2000</td>
			<td style="width:170px;">DSRB</td>
			<td style="width:118px;">F109-D12</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">E-cadherin antibody, Goat</td>
			<td style="width:104px;">1:500</td>
			<td style="width:170px;">Santa Cruz</td>
			<td style="width:118px;">sc-31020</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Acetylated &#945;-tubulin antibody, Mouse</td>
			<td style="width:104px;">1:500</td>
			<td style="width:170px;">Sigma-Aldrich</td>
			<td style="width:118px;">T6793</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Secondary antibodies</td>
			<td style="width:104px;"></td>
			<td style="width:170px;"></td>
			<td style="width:118px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">488 labeled anti-rabbit, Donkey IgG</td>
			<td style="width:104px;">1:1000</td>
			<td style="width:170px;">Invitrogen</td>
			<td style="width:118px;">A-21206</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">594 labeled anti-goat, Donkey IgG</td>
			<td style="width:104px;">1:1000</td>
			<td style="width:170px;">Invitrogen</td>
			<td style="width:118px;">A-11058</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">568 labeled anti-mouse, Goat IgG2b</td>
			<td style="width:104px;">1:500</td>
			<td style="width:170px;">Invitrogen</td>
			<td style="width:118px;">A-21144</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">TopFlash Wnt reporter assay</td>
			<td style="width:104px;"></td>
			<td style="width:170px;"></td>
			<td style="width:118px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">TopFlash HEK293 cell line</td>
			<td style="width:104px;"></td>
			<td style="width:170px;">ATCC</td>
			<td style="width:118px;">CRL-1573</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Luciferase Assay Kit</td>
			<td style="width:104px;"></td>
			<td style="width:170px;">Biotium</td>
			<td style="width:118px;">30003-2</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:408px;">0.05% Trypsin-EDTA</td>
			<td style="width:104px;"></td>
			<td style="width:170px;">Life Technologies</td>
			<td style="width:118px;">25300054</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:408px;">0.4% Trypan Blue Solution</td>
			<td style="width:104px;"></td>
			<td style="width:170px;">Life Technologies</td>
			<td style="width:118px;">15250061</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Additional materials and reagents</td>
			<td style="width:104px;"></td>
			<td style="width:170px;"></td>
			<td style="width:118px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Basement matrix, phenol free Matrigel</td>
			<td style="width:104px;"></td>
			<td style="width:170px;">CORNING</td>
			<td style="width:118px;">356237</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:408px;">Dissociation buffer, Accutase</td>
			<td style="width:104px;"></td>
			<td style="width:170px;">Gibco</td>
			<td style="width:118px;">A1110501</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:408px;">Cell culture freezing medium, Recovery</td>
			<td style="width:104px;"></td>
			<td style="width:170px;">Life Technologies</td>
			<td style="width:118px;">12648010</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:408px;">Cell strainer (70 &#181;m, steriled)</td>
			<td style="width:104px;"></td>
			<td style="width:170px;">Fisherbrand</td>
			<td style="width:118px;">22363548</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:408px;">Guanidinium thiocyanate-phenol RNA extraction, TRIzol</td>
			<td style="width:104px;"></td>
			<td style="width:170px;">Invitrogen</td>
			<td style="width:118px;">15596026</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:408px;">Specimen processing gel, HistoGel</td>
			<td style="width:104px;"></td>
			<td style="width:170px;">Thermo Fisher Scientific</td>
			<td style="width:118px;">HG-4000-012</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Universal mycoplasma detection kit</td>
			<td style="width:104px;"></td>
			<td style="width:170px;">ATCC</td>
			<td style="width:118px;">30-1012K</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:408px;">1.5 mL microcentrifuge tube</td>
			<td style="width:104px;"></td>
			<td style="width:170px;">Fisherbrand</td>
			<td style="width:118px;">05-408-129</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:408px;">24 well plate</td>
			<td style="width:104px;"></td>
			<td style="width:170px;">USA Scientific</td>
			<td style="width:118px;">CC7682-7524</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:408px;">50 mL conical centrifuge tube</td>
			<td style="width:104px;"></td>
			<td style="width:170px;">Fisher scientific</td>
			<td style="width:118px;">14-432-22</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:408px;">Fluorescence microscope</td>
			<td style="width:104px;"></td>
			<td style="width:170px;">Nikon</td>
			<td style="width:118px;">Eclipse E800</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:408px;">Inverted microscope</td>
			<td style="width:104px;"></td>
			<td style="width:170px;">Biotium</td>
			<td style="width:118px;">30003-2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Necropsy tray</td>
			<td style="width:104px;"></td>
			<td style="width:170px;">Fisherbrand</td>
			<td style="width:118px;">13-814-61</td>
		</tr>
	</tbody>
</table>

generation of organoids from mouse extrahepatic bile ducts

胆管病变, 影响肝外胆管 (Ehbd), 包括胆道闭锁, 原发性硬化性胆管炎, 和胆管癌。他们没有有效的治疗选择。研究 EHBD 的工具非常有限。我们的目的是开发一种特定于器官、多才多艺的成人干细胞衍生的、临床前的胆管细胞模型, 该模型可从野生小鼠和基因工程小鼠中轻松生成。因此, 我们报告了从成年小鼠 Ehbd 中开发 EHBD 有机体 (EHBDO) 培养系统的新技术。该模型具有成本效益, 能够轻松进行分析, 并具有多个下游应用。具体而言, 我们描述了小鼠 EHBD 分离和单细胞离解、有机体培养的启动、繁殖以及长期维护和储存的方法。本文还介绍了 EHBDO 的免疫组织化学、荧光显微镜和 mRNA 丰度定量的实时定量逆转录聚合酶链反应 (qRT-PCR)。该协议除了生产 ehbd 特异性有机体外, 还具有显著的优势。使用来自 L-WRN 细胞的条件介质可显著降低该模型的成本。与人体组织不同, 小鼠 Ehbd 的使用为培养生成提供了几乎无限的组织。生成的小鼠 Ehbdo 含有纯群体的上皮细胞, 具有内皮祖细胞和分化胆道细胞的标记。培养的有机生物通过多个通道保持均匀形态, 并可在液氮长期储存后恢复。该模型允许研究胆道祖细胞增殖, 可以在药理上操纵, 也可以从基因工程小鼠中产生。需要进行进一步的研究, 以优化培养条件, 以提高电镀效率, 评估功能细胞成熟度, 并直接分化细胞。开发共培养模型和更中性的细胞外基质也是可取的。

我们的协议描述了小鼠 EHBD 有机体的产生, 这些有机体是组织特异性和成体干细胞衍生的。在培养有机体后, 早在 EHBD 分离后1天就可以观察到囊性结构的形成。在培养过程中, 通常不会观察到成纤维细胞的污染。从新生儿或成人 (2个月以上) 小鼠中分离, EHBDO 的电镀效率约为 2% (图 2B)。从成年小鼠身上提取的 EHBD 有机体的电镀效率在第2段增加到 11%, 并?...

Watch this Scientific Journal Video about Generation of Organoids from Mouse Extrahepatic Bile Ducts at JoVE.com

Generation of Organoids from Mouse Extrahepatic Bile Ducts

该方案描述了小鼠肝外胆管三维器官系统的产生。这些胆道有机物质可以维持在培养中, 以研究胆管细胞生物学。胆道有机体表达的标志物的祖先和胆道细胞, 是由偏振上皮细胞。

小鼠肝外胆管有机体的产生

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have ...

这种粘膜外肝胆管器官系统可以解决临床前胆管病变模型的有限获取，以及多能干细胞和肝衍生胆细胞器官模型的局限性。我们的模型是成人组织特异性，还原性，可重复性，时间和成本效益。它将对无法获得人体组织的实验室特别有利。我们的技术允许培养几乎无限数量的肝胆管细胞，以研究组织再生和细胞对细胞的相互作用。演示程序将是 JunyaShiota， 一个博士后从我的实验室。对于肝内胆管隔离，将成年小鼠放在一个上部位置，然后沿中线打开腹腔。缩回肝脏，在隔膜上休息，并使用半叶板轻轻拉近体十二指肠，以揭示肝脏下方常见的胆管。使用手术刀将外肝胆管与周围组织分离。用钳子握住普通胆管的近端，在从肝脏解剖导管的近端之前，先用十二指肠在它的交汇处上方解剖导管。立即将分离的肝胆管放入玻璃板中，在冰上含有冷洗缓冲液，然后将胆管从周围组织和肉末清洁为0.5毫米部分。当所有组织都被切碎后，将部分转移到一个管子中，里面装着500微升分离缓冲液，在37摄氏度下孵育20分钟。在分离结束时，用500微升的冰冷细胞培养剂中和缓冲液，通过18量针将组织悬浮液三联，然后通过20个量程针进行20次三联。然后通过70微米细胞滤株将产生的细胞悬浮液过滤到50毫升管中。建立胆道器官培养，通过离心收集细胞，并小心去除上经。将颗粒重新在一毫升冰冷的无菌PBS中，将细胞转移到1.5毫升管中进行第二次离心。将洗净的颗粒重新放入120微升的液化、冰冷的基底基质和板40微升的细胞中，放入三口37摄氏度加热、24孔的孔的中心，然后将板放入37摄氏度的组织培养培养箱中约15分钟。当基底基质凝固后，在将板返回孵化器之前，将 600 微升 37 摄氏度的播种介质添加到每个井中。三天以后，每三天用600微升的新鲜有机体培养培养剂代替播种介质，用倒置显微镜定期监测器官生长。要通过肝外胆管培养物，在每井将井内水管转移到单独的1.5毫升管之前，用每井400微升的冰冷PBS移液管。通过每个混合物通过25规格的针头四次分离器官，并通过离心收集细胞。然后以适当的比率重新镀金在地下室矩阵中重新暂停细胞。对于长期的肝胆管有机物储存，用室温PBS清洗每井的有机体，而不干扰地下室基质滴，并在每井中加入500微升的冰冷冷冻介质。轻轻重新暂停器官，将混合物转移到单个低温小瓶中，然后将小瓶在零下80摄氏度下放置48小时，然后将器官转移到氮气罐中，在蒸汽相中长期储存。为了准备石蜡嵌入的器官，用500微升4摄氏度PBS代替介质，并将悬浮液从每口井转移到含有液化基质的1.5毫升管中。通过离心收集器官，并使用经过修改的 P1000 移液器尖端小心去除超钠，而不会干扰颗粒。然后加入一毫升的冰冷，4%的副甲醛到器官中，在4摄氏度下过夜孵育。第二天早上，用一毫升室温PBS代替固定剂，在室温下孵育5分钟，用离心洗三次。最后一次洗涤后，将有机物重新用一毫升30%乙醇在室温下孵育5分钟，随后在室温下将一毫升70%乙醇的5分钟孵育。在70%乙醇孵育结束时，通过离心收集器官，并在室温下将有机体重新在100%乙醇的一毫升中再加5分钟。在100%乙醇孵育结束时，在微波炉中加热样品加工凝胶20秒或直到液化，并将50微升的液化凝胶加入每管有机体。将管子放在冰上，直到试样加工凝胶凝固，并将有机物的滴件从每个管转移到盒式纸盒中的蓝色海绵垫之间，在石蜡嵌入器中进一步加工。将盒式磁带放入每步编程15分钟的组织处理器中，然后将样品加工凝胶中的石蜡嵌入器官切成每节四微米，用于免疫组织化学染色和标准方案分析。与新生儿或成年小鼠分离时，肝胆管风管器官电镀效率约为2%。第二次通过后，从成年小鼠中提取的肝胆管器官的电镀效率提高至11%，并保持稳定。大多数器官都表现出囊状形态，贯穿所有具有罕见不规则器官的通道。器官在五到七天内达到生长高峰，之后它们开始积累宫内碎片并恶化。因此，为了维持器官培养，有机体应每7至10天分裂一次。当用免疫荧光分析时，肝外胆管器官由以E-cadherin标记的上皮细胞的纯种群组成。有机细胞也表现出胆道祖细胞的标记以及胆汁分化的标记。重要的是，高比例的器官细胞拥有以乙酰化α图布林为标志的原发细胞，这是正常胆血管细胞的特征，并建议适当的器官细胞极化。需要密切遵守所述温度条件。细致的胆管解剖可防止胰腺细胞污染。离心后通过仔细操作，可以避免细胞材料的流失。这些器官可以用作临床前模型，基因和药理处理，或用于测试药物和传染性物质的影响。这种方法可用于希望利用转基因小鼠模型进一步研究胆血管细胞生物学的机制的实验室。

Research

JoVE Journal

Developmental Biology

Organ Culture and Whole Mount Immunofluorescence Staining of Mouse Wolffian Ducts

器官培养和鼠标沃尔弗导管的全山免疫染色

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an ...

科研

发育生物学

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

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 ...

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

人诱导多能干细胞的前脑型脑 Organoids 标准化和可再生性的生成

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

无支架三维胰岛素表达 Pancreatoids 的实验研究体外培养的小鼠胰祖细胞

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Determining Bile Duct Density in the Mouse Liver

确定小鼠肝脏中的胆汁密度

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques ...

Generation of Multicellular Human Primary Endometrial Organoids

多细胞人类原发性子宫内膜器官的生成

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can ...

Generation of Mosaic Mammary Organoids by Differential Trypsinization

通过微分试穿素生成马赛克乳腺有机体

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

Extra Cellular Matrix-Based and Extra Cellular Matrix-Free Generation of Murine Testicular Organoids

基于细胞基和细胞外矩阵的Murine睾丸器官生成

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed ...

Generation and Culture of Lingual Organoids Derived from Adult Mouse Taste Stem Cells

从成年小鼠味干细胞中衍生的语言器官的生成与培养

The sense of taste is mediated by taste buds on the tongue, which are composed of rapidly renewing taste receptor cells (TRCs). This continual turnover is ...

Generation of 3D Whole Lung Organoids from Induced Pluripotent Stem Cells for Modeling Lung Developmental Biology and Disease

从诱导多能干细胞生成3D全肺类器官，用于模拟肺发育生物学和疾病

Human lung development and disease has been difficult to study due to the lack of biologically relevant in vitro model systems. Human induced pluripotent ...