Name: establishment and genetic manipulation of murine hepatocyte organoids
Uploaded: 2022-02-12T12:30:00
Description: Watch this Scientific Journal Video about Establishment and Genetic Manipulation of Murine Hepatocyte Organoids at JoVE.com

我们感谢Hans Clevers教授慷慨地提供了细胞系来生产重组R-spondin1。这项工作得到了中国国家重点研发计划（2019YFA0111400）、国家自然科学基金（31970779）和山东大学青年交叉学科创新研究组（2020QNQT003）对H.H.的资助。

所有小鼠实验均经山东大学基础医学院动物护理与使用委员会批准，并根据内政部指南和规定（No.ECSBMSSDU2019-2-079）。
注意：该方案主要用于从分离的原代肝细胞中培养3D类器官。
1. 建立小鼠肝细胞类器官培养
注意：细胞外基质（如基质胶或BME）用作培养类器官的基底基质。将细胞外基质储存在-20°C，并在4°C或冰上预解冻。在实验过?...

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长期培养成熟肝细胞的能力是研究肝脏基础科学，药物毒理学和嗜肝性宿主微生物学感染（如疟疾和肝炎病毒）的基础。通过明确定义的生态位，这里的方案为肝细胞建立了一个培养系统。该协议驱动成熟肝细胞在3D培养物中扩增，具有异质性，概括了细胞 - 细胞相互作用，大多数肝细胞功能和遗传修饰，允许设计基因功能研究和新的治疗途径进行再生治疗。 
高质量的肝细?...

类器官是自组织的三维（3D） 体外 簇，包括自我更新的干细胞和多系分化细胞1，2。许多器官的类器官已经通过明确定义的利基因素从多能或成体干细胞中建立，包括肠道，大脑，结肠，肾脏，肝脏，胰腺，甲状腺，胃，皮肤和肺3，4，5，6，7.类器官通过模仿发育（源自胚胎或诱导多能干细胞，PSC）或稳态/再生进展（源自成体干细胞，ASCs）来概括物理细胞功能，这为疾病研究和治疗开辟了新的途径8，9。
作为哺乳动物最大的器官，肝脏主要负责储存，代谢和解毒。两种上皮细胞类型，肝细胞和胆管细胞，构建肝小叶的基本单位。肝细胞负责70-80%的肝功能10。虽然肝脏具有显着的再生能力，但在传统的单层培养过程中，肝细胞特征的快速丧失发生在失调的细胞极化和去分化，这增加了研究人员和临床医生在培养皿中建立"间隙桥接"肝脏模型的需求。然而，直到最近，来自原代肝细胞的 离体 扩增模型尚未得到很好的建立11，12，13，14，15。肝脏类器官可以从胚胎/诱导多能干细胞、成纤维细胞转化为肝细胞样细胞和组织衍生细胞中建立。肝脏类器官的发展促进了体外模型在药物筛选和肝毒性测定中的应用16，17。
在这里，我们描述了从小鼠原发性肝细胞建立肝脏类器官的详细方案。通过使用该协议，我们建立了具有两种胶原酶灌注的肝细胞类器官的 体外 培养系统。这些类器官可以长期扩增数月。它们的生理功能与肝细胞高度一致。此外，我们还详细介绍了如何使用类器官进行遗传操作，例如慢病毒感染，siRNA转导和CRISPR-Cas9工程。肝细胞类器官的繁殖揭示了使用类器官来理解肝脏生物学并开发个性化和转化医学方法的可能性。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Perfusion Buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sangon Bitech</td>
			<td>A600077-0100</td>
			<td>3.50 g/L</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sangon Bitech</td>
			<td>A100188-0500</td>
			<td>9.00 g/L</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sangon Bitech</td>
			<td>A100395-0500</td>
			<td>4.00 g/L</td>
		</tr>
		<tr>
			<td>Na2HPO4&#183;12H2O</td>
			<td>Sangon Bitech</td>
			<td>A501727-0500</td>
			<td>1.51 g/L</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sangon Bitech</td>
			<td>A501218-0001</td>
			<td>80.0 g/L</td>
		</tr>
		<tr>
			<td>NaH2PO4&#183;2H2O</td>
			<td>Sangon Bitech</td>
			<td>A610404-0500</td>
			<td>0.78 g/L</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sangon Bitech</td>
			<td>A500873-0500</td>
			<td>3.50 g/L</td>
		</tr>
		<tr>
			<td>Phenol Red</td>
			<td>Sangon Bitech</td>
			<td>A100882-0025</td>
			<td>0.06 g/L</td>
		</tr>
		<tr>
			<td>Digestion Buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sangon Bitech</td>
			<td>A501330-0005</td>
			<td>0.50 M</td>
		</tr>
		<tr>
			<td>Collagenase IV</td>
			<td>Sigma</td>
			<td>C1639</td>
			<td>0.10 mg/mL</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sangon Bitech</td>
			<td>C621110-0010</td>
			<td>23.80 g/L</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sangon Bitech</td>
			<td>A100395-0500</td>
			<td>4.00 g/L</td>
		</tr>
		<tr>
			<td>Na2HPO4&#183;12H2O</td>
			<td>Sangon Bitech</td>
			<td>A501727-0500</td>
			<td>1.51 g/L</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sangon Bitech</td>
			<td>A501218-0001</td>
			<td>80.0 g/L</td>
		</tr>
		<tr>
			<td>NaH2PO4&#183;2H2O</td>
			<td>Sangon Bitech</td>
			<td>A610404-0500</td>
			<td>0.78 g/L</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sangon Bitech</td>
			<td>A500873-0500</td>
			<td>3.50 g/L</td>
		</tr>
		<tr>
			<td>Tip-wash Buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10091148</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>C14190500BT0</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Wash Medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Invitrogen</td>
			<td>12634-010</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>100 X</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td>100 X</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Sangon Bitech</td>
			<td>&#160;A600135-0025</td>
			<td>100 X</td>
		</tr>
		<tr>
			<td>Culture Medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>A83-01</td>
			<td>Tocris</td>
			<td>2939</td>
			<td>Stock concentration 500 &#181;M, final 
			concentration 2 &#181;M</td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Invitrogen</td>
			<td>12634-010</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>B-27 supplement 50x, minus vitamin A</td>
			<td>Gibco</td>
			<td>1704-044</td>
			<td>50 X</td>
		</tr>
		<tr>
			<td>Chir 99021</td>
			<td>Tocris</td>
			<td>4423</td>
			<td>Stock concentration 30 mM, final 
			concentration 3 &#181;M</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>11330032</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Gastrin I</td>
			<td>Tocris</td>
			<td>3006</td>
			<td>Stock concentration 100 mM, final 
			concentration 10 nM</td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>100 X</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td>100 X</td>
		</tr>
		<tr>
			<td>Matrigel martix</td>
			<td>BD</td>
			<td>356231</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>N-acetylcysteine</td>
			<td>Sigma Aldrich</td>
			<td>A9165</td>
			<td>Stock concentration 500 mM, final 
			concentration 1 mM</td>
		</tr>
		<tr>
			<td>Nictinamide</td>
			<td>Sigma Aldrich</td>
			<td>N0636</td>
			<td>Stock concentration 1 M, final concentration 3 mM</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Sangon Bitech</td>
			<td>&#160;A600135-0025</td>
			<td>100 X</td>
		</tr>
		<tr>
			<td>Recombinant human EGF</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td>Stock concentration 100 &#181;g/ml,final concentration 50 ng/ml</td>
		</tr>
		<tr>
			<td>Recombinant human FGF10</td>
			<td>Peprotech</td>
			<td>100-26</td>
			<td>Stock concentration 100 &#181;g/ml, final concentration 100 ng/ml</td>
		</tr>
		<tr>
			<td>Recombinant human HGF</td>
			<td>Peprotech</td>
			<td>100-39</td>
			<td>Stock concentration 100 &#181;g/ml, final concentration 25 ng/ml</td>
		</tr>
		<tr>
			<td>Recombinant Human TGF-&#945;</td>
			<td>Peprotech</td>
			<td>100-16A</td>
			<td>Stock concentration 100 &#181;g/ml, final concentration 100 ng/ml</td>
		</tr>
		<tr>
			<td>Recombinant Human TNF-&#945;</td>
			<td>Origene</td>
			<td>TP750007-1000</td>
			<td>Stock concentration 100 &#181;g/ml, final concentration 100 ng/ml</td>
		</tr>
		<tr>
			<td>Rho kinase inhibitor Y-27632</td>
			<td>Abmole Bioscience</td>
			<td>Y-27632 dihydrochloride</td>
			<td>Stock concentration 10 mM, final concentration 10 &#181;M</td>
		</tr>
		<tr>
			<td>Rspondin-1conditioned medium</td>
			<td></td>
			<td></td>
			<td>Stable cell line generated in the Hu Lab. Final concentration 15%.</td>
		</tr>
		<tr>
			<td>Others</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#181;m filter</td>
			<td>Millipore</td>
			<td>SCGPT01RE</td>
			<td></td>
		</tr>
		<tr>
			<td>16 # silicone tube</td>
			<td>LangerPump</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>24 well, suspension</td>
			<td>Greiner bio-one</td>
			<td>GN662102-100EA</td>
			<td></td>
		</tr>
		<tr>
			<td>37 &#176;C Water Bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;m filter</td>
			<td>BD</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>stemcell</td>
			<td>AT-104</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Anti-CTNNB1</td>
			<td>BD</td>
			<td>610154</td>
			<td>Mouse</td>
		</tr>
		<tr>
			<td>Anti-GAPDH</td>
			<td>CST</td>
			<td>5174S</td>
			<td>Rabbit</td>
		</tr>
		<tr>
			<td>Anti-HDAC7</td>
			<td>Abcam</td>
			<td>ab50212</td>
			<td>Mouse</td>
		</tr>
		<tr>
			<td>Anti-KI67</td>
			<td>Abcam</td>
			<td>ab15580</td>
			<td>Rabbit</td>
		</tr>
		<tr>
			<td>Biological safety cabinet</td>
			<td>ESCO</td>
			<td>CCL-170B-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Recovery Solution</td>
			<td>Corning</td>
			<td>354253</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Centrifuge 5430</td>
			<td>eppendorf</td>
			<td>542700097</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>ESCO</td>
			<td>AC2-4S1</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sangon Bitech</td>
			<td>&#160;A100231</td>
			<td>stored at RT</td>
		</tr>
		<tr>
			<td>EVOS FL Color Imaging Systems</td>
			<td>Invitrogen</td>
			<td>AME4300</td>
			<td></td>
		</tr>
		<tr>
			<td>Hdac3 siRNA</td>
			<td>Guangzhou RiboBio Co., Ltd.</td>
			<td>siG2003180909192555</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Hdac7 lentivirus</td>
			<td>ShanghaiGenePharmaCo.,Ltd</td>
			<td>LV2020-2364</td>
			<td>stored at -80 &#176;C</td>
		</tr>
		<tr>
			<td>Hitrans G A</td>
			<td>Shanghai Genechem Co.,Ltd.</td>
			<td>REVG004</td>
			<td>Increased virus infection efficiency</td>
		</tr>
		<tr>
			<td>Image Pro Plus software</td>
			<td>Media Cybernetics, Bethesda, MD, USA</td>
			<td>version 6.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamin RNAiMAX Transfection Reagent</td>
			<td>Invitrogen</td>
			<td>13778150</td>
			<td>Increased virus infection efficiency</td>
		</tr>
		<tr>
			<td>Live cell dye</td>
			<td>Luna</td>
			<td>F23001</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Low-speed desktop centrifuge</td>
			<td>cence</td>
			<td>TD5A-WS/TD5AWS</td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleofactor-&#945; 2b Device</td>
			<td>Lonza</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Gibco</td>
			<td>31985070</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Pasteur pipette</td>
			<td>Sangon Bitech</td>
			<td>F621006-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>LangerPump</td>
			<td>BT100-2J</td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF membrane</td>
			<td>Millipore</td>
			<td>IPVH00010</td>
			<td>Activate with methanol</td>
		</tr>
		<tr>
			<td>PX458</td>
			<td>Addgene</td>
			<td>48138</td>
			<td>stored at -80 &#176;C</td>
		</tr>
		<tr>
			<td>Refrigerated centrifuge</td>
			<td>Thermo Scientific Heraeus</td>
			<td>Labofuge 400</td>
			<td></td>
		</tr>
		<tr>
			<td>RSL3</td>
			<td>MCE</td>
			<td>HY-100218A</td>
			<td>Stock concentration 10 mM, final concentration 4 &#181;M</td>
		</tr>
		<tr>
			<td>Trypsin/EDTA</td>
			<td>Gibico</td>
			<td>25200072</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Wee1 siRNA</td>
			<td>Guangzhou RiboBio Co., Ltd.</td>
			<td>siG2003180909205971</td>
			<td>stored at -20 &#176;C</td>
		</tr>
	</tbody>
</table>

establishment and genetic manipulation of murine hepatocyte organoids

肝脏是哺乳动物最大的器官。它在葡萄糖储存，蛋白质分泌，代谢和解毒中起重要作用。作为大多数肝功能的执行者，原代肝细胞的增殖能力有限。这就需要建立 离体 肝细胞扩增模型，用于肝脏生理和病理研究。在这里，我们通过两步胶原酶灌注分离小鼠肝细胞，并建立了3D类器官培养物作为"迷你肝脏"，以概括细胞 - 细胞相互作用和物理功能。类器官由异质细胞群组成，包括祖细胞和成熟肝细胞。我们详细介绍了分离和培养小鼠肝细胞或胎儿肝细胞在2-3周内形成类器官的过程，并展示了如何通过机械上下移液来通过它们。此外，我们还将介绍如何将类器官消化成单细胞，用于shRNA/异位构建的慢病毒感染，siRNA转染和CRISPR-Cas9工程。类器官可用于药物筛选，疾病建模和基础肝脏研究，通过模拟肝脏生物学和病理生物学。

来自女性Alb-Cre的肝细胞类器官;在播种后五天观察Rosa26-GFP小鼠（8周）（图1A）。类器官随着Ki67阳性染色而增殖（图1B）。通过qRT-PCR和蛋白质印迹检查通过siRNA转染或慢病毒感染在肝细胞类器官中代表性基因的干扰表达。结果如图 2A 和 2B所示。 图2C 显示了用RSL-3和CRISPR / Cas9基因组文库筛选治疗14?...

Watch this Scientific Journal Video about Establishment and Genetic Manipulation of Murine Hepatocyte Organoids at JoVE.com

Establishment and Genetic Manipulation of Murine Hepatocyte Organoids

从小鼠肝细胞建立了长期 的体外 3D类器官培养系统。这些类器官可以通过shRNA/异位构建的慢病毒感染，siRNA转染和CRISPR-Cas9工程进行遗传和遗传操纵。

小鼠肝细胞类器官的建立与遗传操作

The liver is the largest organ in mammals. It plays an important role in glucose storage, protein secretion, metabolism and detoxification. As the ...

肝脏是哺乳动物最大的器官。它在新陈代谢和解毒中起着非常重要的作用。虽然肝脏在体外具有显着的再生能力，但从长远来看，在体外培养肝细胞仍然是一个非常大的挑战。在这里，我们从成熟的肝细胞中建立肝细胞类器官。它保留了肝细胞在形态和功能上的主要特征。在本视频中，我们将详细介绍如何培养和传代这些类器官，以及如何对这些类器官进行遗传修饰。首先，肝脏灌注和消化。准备所有区域和器械，并使其无菌。清洗小鼠并打开表皮和皮肤，然后尝试找到肝脏的袋烤箱。将针头注入其中，然后捕获它。以每分钟5毫升的速度进行灌注，直到肝脏变白。将它们换入灌注缓冲液中，小心放置，观察肝脏，直到它变软。通常需要三到五分钟，然后切开肝脏并放入盘子中，然后用剪刀将它们切成小块。经常上下打气。通常，将它们制成单个细胞需要10到15分钟。然后用10到15分钟上下搅拌，并通过钳工喂它们。将它们制成单个细胞。收集50毫升管中的所有细胞，并以速度离心它们，然后收集所有每个板。尽量把它们永远放在冰冷的冰里。制作单个细胞悬浮液并用细胞计数器计数。通常肝细胞如果快乐，它具有活泼的荧光。第二部分，我们尝试让细胞坐下并进行类器官培养。我们收集所有的细胞悬浮液，并用一定的同心融合来计数细胞，然后我们混合。Metrogels.和代码介质。通常，我们以1比2或1比3的比例取介质和metrogels。仔细混合后，我们将它们放在梯度板上。我们通常在24孔板孔处加入100微升。将它们放入37度温度的培养箱中。20分钟后，我们将它们向前反转，底部在底部，然后在介质处。在两三天的反击中，我们通常会看到类器官出来。这是我们类器官的典型形象。如果我们想进行传代或做一些遗传修饰方法，我们通常需要来自类器官的单细胞，我们用涂层洗涤缓冲液从计数器板中收集所有类器官。我们放下上清液，然后在涂层处洗涤缓冲液到板中，混合它们，所以上下打气。然后我们通常用洗涤胶带缓冲器预洗所有吸头。为了避免所有类器官都倒在尖端上。然后，在冰上或没有冰上的新一批，我们通过中心融合收集所有类器官。我们对待它们，有一个弯曲的弧度来去除所有的地铁凝胶。在冰上孵育20至30分钟后，所有metrogel都被移除。我们收集所有干净的类器官，并添加胰蛋白酶使它们变成单细胞。加入胰蛋白酶后，我们将类器官处理到培养箱中。肝脏类器官需要5到10分钟才能变成单细胞。我们添加更多的洗涤缓冲液来停止三聚糖。然后上下打动他们。对于选项过程，您可以再次清洗它们以除去所有胰蛋白酶。通过中心融合洗涤后，我们可以在细胞计数器下计数它们。然后，我们将展示如何对这些肝细胞类器官进行交易或转染。首先，在试管上贴上你将要做的事情的标签，然后我们将根据制造商的说明进行视力转染。你需要，脂酰事实驯服INI MaX的摄政王。我们添加。控制和特定的基因siRNA，并根据制造商的说明以最佳方式孵育它们。然后根据细胞浓度，我们加入RAN max并彻底混合它们。然后，我们还将RAN max与最佳值分开添加，并将它们放在冰上。5分钟后，我们分别用不同的siRNA，特异性基因RANi和II对照分别调出RAN max混合物并彻底混合。再次将它们放在冰上，然后我们收集所有的类器官，缓慢的中央融合，滴下所有的上清液，我们将RNAi max，siRNA混合物加入到这个细胞板中，并彻底地上下激发它。简而言之，将细胞和标记RNAi max和siRNA混合物在37度下臀化并孵育4至5小时。最后一部分，这些肝细胞类器官也可能被Lenti病毒感染。类似的信息作为siRNA转染进行。对照和siRNA病毒管准备充分，并根据制造商的说明添加最佳。然后用像聚绿色这样的感染区域，然后我们添加不同的Lenti病毒，根据制造商的说明宪法。将类器官培养物中的单细胞悬浮液和各种颗粒在1.5毫升的eppendorf管中混合，加入，孔，混合它们板块该混合物，并在32度下浓缩1小时。浓缩物融合将在500 G下进行，在37度下孵育4至5小时后，细胞悬浮液将被收集并置于metrogel中。然后再次将它们放入培养箱中。器官对形成效率或其他功能分析可以在7至10天后进行。以下是我们的代表性结果。在图1A中，您可以看到来自小鼠的ALB-Cre Rosa 26-GFP或RFP肝细胞类器官。在图1B中，您可以看到染色中的Ki67阳性信号，这表明我们的肝细胞类器官正在增殖。在图2A和2B中，这是我们基因干扰表达的代表性基因表达。在肝细胞类器官中进行遗传操作后，干扰效果非常有效。这是图2C，你可以看到在我们的小鼠类器官中使用卡森石技术进行基因操作后。结论，我们的研究结果表明，肝细胞类器官可以在体外扩增并进行遗传操纵。总之，在本视频中，我们展示了如何进行肝脏灌注和消化以获得肝细胞。如何捕获肝细胞并使肝细胞类器官通过。此外，我们还展示了如何进行siRNA和载体转染或Lenti病毒感染，以修饰这些类器官的遗传。此外，我们的类器官有两个短缺。首先，我们没有使用每个核心或毛皮核心来纯化肝细胞。其次，我认为应该尝试更多的生长因子和信号传导来改善类器官的生长时间。

Research

JoVE Journal

Biology

Method for Culture of Early Chick Embryos ex vivo (New Culture)

Method for Culture of Early Chick Embryos ex vivo New Culture

文化早期鸡胚体外（新文化）的方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

科研

生物学

Double Whole Mount in situ Hybridization of Early Chick Embryos

双整装原位杂交技术在早期鸡胚

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

成人的心肌细胞的分离和遗传操作共聚焦成像

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and ...

DNA Electroporation, Isolation and Imaging of Myofibers

DNA电穿孔，肌纤维的分离和成像

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently ...

An Improved Time- and Labor- Efficient Protocol for Mouse Primary Hepatocyte Isolation

一种改进的小鼠原代肝细胞分离时间和劳动效率方案

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on ...

Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe

使用活性氧敏感荧光探针分析小鼠肠道类器官中的氧化应激

Reactive oxygen species (ROS) play essential roles in intestinal homeostasis. ROS are natural by-products of cell metabolism. They are produced in ...

The Establishment of a Murine Mandibular Molar Extraction Socket Healing Model

小鼠下颌磨牙拔牙窝愈合模型的建立

This study introduces the development of a molar extraction model in the murine mandible to provide a practicable model for studying alveolar bone ...

Establishment, Maintenance, Differentiation, Genetic Manipulation, and Transplantation of Mouse and Human Lacrimal Gland Organoids

小鼠和人泪腺类器官的建立、维持、分化、遗传操作和移植

The lacrimal gland is an essential organ for ocular surface homeostasis. By producing the aqueous part of the tear film, it protects the eye from ...

Ex Vivo Expansion and Genetic Manipulation of Mouse Hematopoietic Stem Cells in Polyvinyl Alcohol-Based Cultures

Ex Vivo Expansion and Genetic Manipulation of Mouse Hematopoietic Stem Cells in Polyvinyl Alcohol-Based Cultures

离体 聚乙烯醇基培养物中小鼠造血干细胞的扩增和遗传操作

Self-renewing&#160;multipotent&#160;hematopoietic stem cells (HSCs) are an important cell type due to their abilities to support hematopoiesis throughout ...

Culture of Piglet Intestinal 3D Organoids from Cryopreserved Epithelial Crypts and Establishment of Cell Monolayers

冷冻保存的上皮隐窝仔猪肠道3D类器官的培养和细胞单层的建立

Intestinal organoids are increasingly being used to study the gut epithelium for digestive disease modeling, or to investigate interactions with drugs, ...