Name: generation and quantitative characterization of functional and polarized biliary epithelial cysts
Uploaded: 2020-05-16T15:00:00
Description: Watch this Scientific Journal Video about Generation and Quantitative Characterization of Functional and Polarized Biliary Epithelial Cysts at JoVE.com

我们感谢尼古拉斯·拉鲁索博士（美国明尼苏达州罗切斯特市梅奥诊所），他亲切地提供了NRC细胞系。
这项工作得到了 iLite RHU 计划 （授予 ANR-16-RHUS-0005） 和 Dhu Hepatinov 的财政支持。
我们感谢伊莎贝尔·加辛和丽索·德·西梅里·巴黎·萨克莱在成像方面的支持。

1. 囊肿的生成
注：如果凝胶允许嵌入细胞，则此协议可与任何类型的水凝胶一起执行。
<ol><li>水凝胶涂层 注：室滑道适当的水凝胶涂层是避免在井底形成 2D 细胞层的关键步骤，这可能会干扰后续囊肿成像并影响囊肿形成效率的计算。<ol>
<li>为确保凝胶溶液的均匀性，在4°C（O/N）下解冻水凝胶。</li>
		<li>在 -20 °C 下，在冰或 O/N 上预冷移液器尖端，在 -20°C O/N 下使用 8 井?...

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为了研究3D细胞结构的器官生成和维护，各种组织已经建模，使用不同的细胞起源，但也不同类型的细胞外基质，包括合成水凝胶8，9，10，21。然而，由于缺乏3D定量分析，使得在器官形成或功能7、8、9、10、15、18?...

近三十年来，体外研究领域向3D培养系统发展。出现了许多协议，用于在支架/矩阵存在或不存在的情况下，在一滴、搅拌、微流体装置或浮式 1 中，将 3D 中的细胞培养为球体或聚合体。3D 培养方法的使用已经证明了其比二维 （2D） 培养物的优势，尤其是上皮细胞，这些细胞在 3D 结构（称为囊肿或囊肿）中表现出自组织。在这种情况下，细胞形成一个包围流明的单层，其中细胞获得其完整的上皮表型与改善的生理特异性功能2。
许多研究有助于开发在自然基质中形成这些上皮器官的方法。这允许重新综述体内细胞和细胞-微环境相互作用，得到上皮表型3，4，5，6，7的建立和稳定性。最近，特别是为了开发可移植器官和破译微环境对上皮程序的要求，开发了合成水凝胶，以增强上皮8、9、10的形成。不幸的是，这些研究报告定性数据，或目前计算方法使用内部参考，如囊肿与非囊肿的比例在2D平面8，9，10。这排除了上皮器官的效率、稳定性或形态和生理表征方面的不同研究之间的任何比较。
使用微流体装置对珠子上皮细胞进行微封装，取得了更逼真的定量和比较结果。利用这项技术，根据不同3D细胞结构11、12的形态，形成并分化了不同细胞类型的器官。然而，这项技术不容易使用，需要使用洁净室来生产微流体设备。这项技术已为几种水凝胶建立，但需要技术适应才能应用于其他水凝胶，从而限制其多功能性。因此，大多数旨在开发上皮器官的研究都依赖于将上皮细胞嵌入到水凝胶中。在这些方法中，整个三生培养培养中凝胶结构和细胞分布的高异质性往往被忽视。因此，大多数分析都与单个 2D 图像相关，这些图像仅非常粗略地表示整个 3D 体积中各种蜂窝对象的分布。
影响胆管的疾病，如胆管癌、胆道炎、原发性鼻窦炎等，是导致死亡和发病的主要原因。除了肝移植，没有有效的治疗方法，这些情况13。努力调查胆管的形成，疾病的原因，和进展将允许开发新的疗法14。
使用正常或患者衍生、分化或祖传细胞系的囊肿、球状体或管状结构的胆管状模型已开发15、16、17、18、19、20。各种研究有重述胆血管细胞极性，胆血管细胞标记的表达，胆碱的存在，胆血管细胞分泌和再吸收能力，以及流明的形成和阻塞;所有这些都代表了胆血管细胞表型、形态学和功能15、17、19的重要特征。其他人报告说，维持患者衍生的胆道器官长时间20。最近，我们调查了生化和生物物理线索对胆囊肿器官形成21中的作用。重要的是，在胆道球类和管7，22中研究了胆道间闭塞的发病机。此外，还利用胆道球体15、20成功研究了主性胆囊炎的主要特征，如胆管细胞衰老、亲炎细胞因子的分泌以及巨噬细胞的招募。然而，在生理上调节胆血管细胞表型、生理学和微环境的可重复体外三维定量模型仍需要解决这些问题。此外，只有少数出版物报告囊肿形成效率21，23。这是一个重要点，特别是在调查器官发生，疾病原因，以及药物反应与胆血管细胞功能和极化的相关性。此外，由于从协议到协议使用的基架/矩阵存在差异，因此很难在系统之间进行比较。为了解决这些问题，我们提出了一种定量、可靠和通用的方法，用于产生一种模拟流明形成、胆血管细胞极化和胆血管细胞分泌功能的胆道囊肿。重要的是，在评估随时间、囊肿形成效率、尺寸、可行性、极化和功能时，我们通过 3D 凝胶沿 Z 轴进行系统分析。此外，我们使用天然水凝胶和普通大鼠胆结细胞（NRC）作为协议的例子，但其他天然或合成水凝胶，以及上皮细胞可用于形成三维囊结构。

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			<td dir="LTR" height="21" style="height:21px;width:408px;">10 &#181;l- Pipette Eppendorf Research Plus</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">3120000020</td>
			<td style="width:261px;"></td>
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			<td dir="LTR" height="21" style="height:21px;width:408px;">100 &#181;l - Pipette Eppendorf Research Plus</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">3120000046</td>
			<td></td>
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			<td dir="LTR" height="21" style="height:21px;width:408px;">1000 &#181;l - Pipette Eppendorf Research Plus</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">3120000062</td>
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			<td dir="LTR" height="21" style="height:21px;width:408px;">1X PBS</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">14190-094</td>
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			<td dir="LTR" height="21" style="height:21px;width:408px;">200 &#181;l - Pipette Eppendorf Research Plus</td>
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			<td dir="LTR" style="width:156px;">3120000054</td>
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			<td dir="LTR" height="21" style="height:21px;width:408px;">3,3&#8242;,5-Triiodo-L-thyronine sodium salt</td>
			<td dir="LTR" style="width:180px;">Sigma-Aldrich</td>
			<td dir="LTR" style="width:156px;">T5516</td>
			<td>NRC complete medium final concentration = 3.4 &#181;g/mL</td>
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			<td dir="LTR" height="21" style="height:21px;width:408px;">Acetic acid</td>
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			<td>0.02N final</td>
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			<td dir="LTR" height="21" style="height:21px;width:408px;">Aerosol barrier pipettes tips 10 &#181;l (Fisherbrand)</td>
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			<td dir="LTR" style="width:156px;">2707439</td>
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			<td dir="LTR" height="21" style="height:21px;width:408px;">Aerosol barrier pipettes tips 1000 &#181;l (Fisherbrand)</td>
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			<td dir="LTR" style="width:156px;">2707404</td>
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			<td dir="LTR" height="21" style="height:21px;width:408px;">Aerosol barrier pipettes tips 200 &#181;l (Fisherbrand)</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">2707430</td>
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			<td dir="LTR" height="21" style="height:21px;width:408px;">Antibiotic Antimicotic Solution (100X)</td>
			<td dir="LTR" style="width:180px;">Sigma-Aldrich</td>
			<td dir="LTR" style="width:156px;">A5955</td>
			<td>NRC complete medium final concentration = 1:100 dilution</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Bovine pituitary extract</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">13028-014</td>
			<td>NRC complete medium final concentration = 30 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Bovine serum albumin</td>
			<td dir="LTR" style="width:180px;">Sigma-Aldrich</td>
			<td dir="LTR" style="width:156px;">A2153</td>
			<td style="width:261px;">1:1000 dilution</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Chemically Defined Lipid Concentrate (100X)</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">11905-031</td>
			<td>NRC complete medium final concentration = 1:100 dilution</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Collagen high concentration, rat tail</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">354249</td>
			<td dir="LTR" style="width:261px;">50 &#181;g/mL final concentration</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Dexamethasone</td>
			<td dir="LTR" style="width:180px;">Sigma-Aldrich</td>
			<td dir="LTR" style="width:156px;">D4902</td>
			<td>NRC complete medium final concentration = 0.393 &#181;g/mL</td>
		</tr>
		<tr height="42">
			<td dir="LTR" height="42" style="height:42px;width:408px;">DMEM F12</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">21331-020</td>
			<td style="width:261px;">NRC complete medium final concentration = 1X</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">E-cadherin Rabbit anti-Human, Rat, Polyclonal</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">PA5-32178</td>
			<td dir="LTR" style="width:261px;">1:400 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:22px;width:408px;">Eclipse TE300 inverted microscope</td>
			<td style="width:180px;">Nikon</td>
			<td style="width:156px;"></td>
			<td>imaging</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Ethanolamine</td>
			<td dir="LTR" style="width:180px;">Sigma-Aldrich</td>
			<td dir="LTR" style="width:156px;">E9508</td>
			<td>NRC complete medium final concentration = 0.32 mM</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Fetal calf serum</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">10270-106</td>
			<td>NRC complete medium final concentration = 5:100 dilution</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Fluoroshield with DAPI (Mounting medium)</td>
			<td dir="LTR" style="width:180px;">Sigma-Aldrich</td>
			<td dir="LTR" style="width:156px;">F6057</td>
			<td style="width:261px;"></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Formaldehyde 16% (W/V)</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">28906</td>
			<td>4% (W/V)</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Goat serum</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">16210-064</td>
			<td style="width:261px;">1:10 dilution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:408px;">Hamamatsu camera (Digital camera C11440 ORCA - flash 4.OLT)</td>
			<td style="width:180px;">Hamamatsu</td>
			<td style="width:156px;"></td>
			<td>imaging</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Hoechst 33258</td>
			<td dir="LTR" style="width:180px;">Sigma-Aldrich</td>
			<td dir="LTR" style="width:156px;">B1155</td>
			<td style="width:261px;">5 &#181;g/mL final concentration</td>
		</tr>
		<tr height="42">
			<td dir="LTR" height="42" style="height:42px;width:408px;">IgG (H+L) Highly Cross-Adsorbed Goat anti-Rabbit, Alexa Fluor Plus 647</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">A32733</td>
			<td dir="LTR" style="width:261px;">1:500 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">ImageJ version 2.0.0-rc-69/1.52n</td>
			<td style="width:180px;"></td>
			<td style="width:156px;"></td>
			<td>Open source image processing software</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Insulin-Transferrin-Selenium (100X)</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">51300-044</td>
			<td>NRC complete medium final concentration = 1:100 dilution</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">L-Glutamine (100X)</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">25030-024</td>
			<td>NRC complete medium final concentration = 1:100 dilution</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Matrigel GFR (stock concentration 9.7 mg/mL)</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">356231</td>
			<td dir="LTR" style="width:261px;">4:10 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NIS Elements software version 4.50.00</td>
			<td style="width:180px;">Nikon</td>
			<td style="width:156px;"></td>
			<td>image acquisition and display</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Non-Essential-Amino-Acids-Solution (100X)</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">11140-035</td>
			<td>NRC complete medium final concentration = 1:100 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Objective Plan Fluor 10X/0.30 Ph1 DL (&#8734;/1.2 WD 15.2)</td>
			<td style="width:180px;">Nikon</td>
			<td style="width:156px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Prolong Gold Antifade Reagent</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">P36931</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Propidium Iodide (PI)</td>
			<td dir="LTR" style="width:180px;">Sigma-Aldrich</td>
			<td dir="LTR" style="width:156px;">P4170</td>
			<td>20 &#181;g/mL final concentration</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Rhodamine Phalloidin</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">R415</td>
			<td dir="LTR" style="width:261px;">16.2 nM final concentration</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Sir-Actin / Verapamil kit</td>
			<td dir="LTR" style="width:180px;">Spirochrome</td>
			<td dir="LTR" style="width:156px;">SC001</td>
			<td dir="LTR" style="width:261px;">10 &#181;M final concentration</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Soybean trypsin inhibitor</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">17075-029</td>
			<td>NRC complete medium final concentration = 50 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Sterile cell strainer 40 &#181;m (Fisherbrand)</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">22363547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Sterile pipettes 10 mL (Fisherbrand)</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">1367811E</td>
			<td style="width:261px;"></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Sterile pipettes 5 mL (Fisherbrand)</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">1367811D</td>
			<td style="width:261px;"></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Sterile tubes 1.5 mL (Fisherbrand)</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">11926955</td>
			<td style="width:261px;"></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Sterile tubes 15 mL (Fisherbrand)</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">7200886</td>
			<td style="width:261px;"></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Sterile tubes 50 mL (Fisherbrand)</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">553913</td>
			<td style="width:261px;"></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Sucrose</td>
			<td dir="LTR" style="width:180px;">Sigma-Aldrich</td>
			<td dir="LTR" style="width:156px;">S0389</td>
			<td>5:100 dilution</td>
		</tr>
		<tr height="24">
			<td dir="LTR" height="24" style="height:24px;width:408px;">Tissue culture treated flask 25cm2 (Falcon)</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">353108</td>
			<td style="width:261px;"></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Triton X-100</td>
			<td dir="LTR" style="width:180px;">Sigma-Aldrich</td>
			<td>T8787</td>
			<td>5:1000 dilution</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Trypsin-EDTA (0.05%) phenol red</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">25300-054</td>
			<td>1X</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Tween-20</td>
			<td dir="LTR" style="width:180px;">Sigma-Aldrich</td>
			<td dir="LTR" style="width:156px;">P1379</td>
			<td>5:10000 dilution</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Vitamin (100X)</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">11120-037</td>
			<td>NRC complete medium final concentration = 1:100 dilution</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">&#956;-Slide 8 Well ibiTreat, Ibidi</td>
			<td dir="LTR" style="width:180px;">Clinisciences</td>
			<td dir="LTR" style="width:156px;">80826</td>
			<td style="width:261px;"></td>
		</tr>
	</tbody>
</table>

generation and quantitative characterization of functional and polarized biliary epithelial cysts

胆结细胞，在肝脏中排行胆管的上皮细胞，监督胆汁的形成和修饰。近二十年来，在肝脏疾病的背景下，基于胆血管细胞的三维（3D）模型已经出现，如囊肿、球状体或管状结构，以模拟组织拓扑进行器官生成、疾病建模和药物筛选研究。这些结构主要是通过在水凝胶中嵌入胆血管细胞获得的。主要目的是通过解决上皮极性、功能性和形态特性来研究自组织性。然而，很少有研究侧重于囊肿形成效率。在这种情况下，效率通常从单个平面的图像中量化。进行功能测定和结构分析时不表示水凝胶聚合异质性和副作用引起的囊肿分布的潜在异质性。因此，定量分析一完成，不能用于比较一个条款到另一个。此外，此方法不允许比较不同矩阵和细胞类型的 3D 增长潜力。此外，没有提及免疫抑制囊肿的实验故障排除。本文提供了一种可靠而通用的方法，表明初始细胞分布与囊肿形成的异质垂直分布有关。在10天的时间过程中，沿水凝胶深度进行Z-堆栈分析，随后对嵌入在水凝胶中的胆血管细胞细胞进行跟踪分析。该方法可获得囊肿形成效率和生长的强健动力学。我们还提出了评估囊肿极性和分泌函数的方法。最后，还提供了优化免疫抑制方案的其他提示，以限制成像的囊肿崩溃。这种方法可以应用于其他三D细胞培养研究，从而打开将一个系统与另一个系统进行比较的可能性。

囊肿的形成和表征 3D细胞培养系统是研究器官生成和疾病建模的重要工具。不幸的是，这些方法大多数是定性的或使用在单个平面上执行的内部定量，通过比较囊肿和非囊肿的数量，在可变和经常未指定的体积，防止任何比较之间的各种研究7，8，9，10，15，18，23。<sup class="xref...

Watch this Scientific Journal Video about Generation and Quantitative Characterization of Functional and Polarized Biliary Epithelial Cysts at JoVE.com

Generation and Quantitative Characterization of Functional and Polarized Biliary Epithelial Cysts

三维（3D）细胞系统是研究器官生成的相关模型。提出了一种基于水凝胶的胆囊肿产生及其表征方法。该协议通过一种简单可靠的方法来评估囊肿形成效率、尺寸并测试其功能，解开了 3D 表征的障碍。

功能性与极化胆囊肿的生成与定量特征

Cholangiocytes, the epithelial cells that line up the bile ducts in the liver, oversee bile formation and modification. In the last twenty years, in the ...

这是生成胆道囊肿的第一个协议，提供系统分析，允许随着时间的推移评估囊肿的形成、效率和大小。此方法允许对上皮囊肿形成进行定量评估，并使得可以评估这一过程作为上皮细胞或水凝胶类型的函数。由于胆道疾病的治疗选择有限，该协议为标准化药物研究、确定正常的治疗靶点和了解疾病机制打开了大门。简单的方法可以解决管状结构领域生物工程中新信息机制的重要问题。在开始实验之前，在4摄氏度下解冻适当的水凝胶溶液，并在零下20摄氏度的一夜之间解冻预冷移液器尖端和8井室滑动。第二天早上，将水凝胶和八井室滑轨放在冰上，在冷普通大鼠胆囊细胞完整介质中加入适当体积的水凝胶，获得500微升40%水凝胶溶液。然后使用冷移液器尖端将 50 微升水凝胶溶液添加到腔室滑轨的每个井的中心，并使用移液器尖端将溶液尽可能均匀地铺在井底的整个表面上，而不会产生气泡。为准备实验细胞，当水凝胶聚合时，用预热的PBS清洗70%汇合正常大鼠胆血管细胞培养中的细胞，并在细胞培养培养箱中用5毫升新鲜预加热PBS孵育细胞20分钟。在孵育结束时，用一毫升预加热的 Trypsin-EDTA 更换PBS，将烧瓶返回细胞培养箱5至10分钟。当细胞分离时，用四毫升预热完全正常的大鼠胆黄细胞介质中和反应，将细胞转移到15毫升管中离心。然后将颗粒重新悬浮在五毫升预热介质中，并通过 40 微米滤株将细胞过滤到 50 毫升管中进行计数。要产生囊肿，将适当体积的水凝胶加入冷完全正常大鼠胆囊细胞介质中，在1，600微升的介质中获得1，600微升的80%水凝胶溶液，并将细胞稀释至每毫升冷完全正常大鼠胆细胞介质浓度的5倍至第5个细胞。立即混合细胞和水凝胶细胞溶液，并在水凝胶涂层室滑轨的每个井中加入400微升的细胞，注意避免气泡。为确保获得可重复和显著的结果，请务必小心处理水凝胶，并彻底混合初始细胞氢溶液，以获得均质溶液。当所有细胞都播种后，将幻灯片放在细胞培养箱中。在培养两天后，从每一个井的一角取出250微升的介质，注意不要冲洗水凝胶，并慢慢用250微升的新鲜培养基取代废弃的下一杯。对于囊肿成像，在适当的培养日，在配备图像采集软件的相位对比度显微镜上选择 10 X 目标，打开白灯并选择亮场成像选项。选择播放以打开相机并聚焦囊肿领域。设置曝光时间并打开自动捕获文件夹窗口，以允许自动保存图像。打开捕获 Z 系列窗口，重置默认位置，并使用 Z 形螺钉定义 Z 堆栈的顶部和底部平原，根据目标和分辨率级别调整 Z 步数。然后单击"立即运行"以启动采集。映像后，打开斐济的 Z 堆栈。要复制堆栈，请单击图像、重复和复制堆栈。若要从重复的堆栈创建最小强度投影，请在图像菜单下选择堆栈和 Z 项目。选择投影类型的最小强度，然后单击"确定"。要从投影中减去背景，请在过程菜单下选择减去背景并选择 500 像素的滚动球半径和浅色背景，以使囊肿比背景更对比度。要测量近似囊肿直径，请缩放目标囊肿，选择直线工具，然后按键盘上的 T 按钮。在最终投影上绘制一条穿过每个囊肿直径的线。为每个囊肿创建的下一个感兴趣区域将添加到兴趣管理器区域。计算所有囊肿后，单击 Z 堆栈窗口以选择它，然后单击"全部显示"以查看计数囊肿。沿 Z 堆栈移动光标，检查每个图像中是否计算了所有囊肿，并尽可能向兴趣管理器区域添加新囊肿。计算所有囊肿后，选择感兴趣区域集并单击更多并保存以保存数据。要确定每个囊肿的大小，在仍选择感兴趣区域时，请单击"度量"。将显示一个窗口，列出每个囊肿及其估计大小。然后以 CSV 格式保存数据。如所证明，记录囊肿的数量和它们各自的大小随着时间的推移，可以分析囊肿的形成和生长的演变。起始细胞组和10天大囊肿的可行性可以通过活体/死染色来评估。死亡细胞在第10天占细胞总数的不到3%，大多以分离细胞或小聚集物形式位于囊肿之外，尽管在一些大囊肿中观察到一些坏死细胞碎片堆积。与氟化物和霍奇斯特的孵化允许评估氟化物从基底到腹腔发光空间的形成和分泌。值得注意的是，使用多药耐药抑制剂进行预处理抑制氟雷丝素的分泌，表明流明中荧光氟素的积累是由于通过耐多药的运输器分泌的，而不是从细胞间空间泄漏的。E-cadherin表达的染色显示，正常的大鼠胆血管细胞在水凝胶中保持其上皮表型至少10天。在准备供免疫荧光评估的囊肿时，在饱和期间将牛血清白蛋白保持在0.1%或更少是保持囊肿完整性的关键，因为较高的浓度会导致囊肿缩回和流明崩溃。隔夜水凝胶、解冻、移液器尖端和腔室滑动记录、在冰上工作以及均匀地将同源细胞水凝胶混合物均匀地扩散到培养室滑轨上，都是实验成功的关键。此方法可用于产生来自其他上皮细胞或水凝胶的囊肿，从而可以比较，从而更好地了解上皮极化。这种定量方法可用于测试药物或基因突变对胆道功能和器官生成的影响。

Research

JoVE Journal

Bioengineering

Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing

通过微接触印刷的定向功能性心肌组织的产生

Advanced heart failure represents a major unmet clinical challenge,  arising from the loss of viable and/or fully functional cardiac muscle  cells. ...

科研

生物工程

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

腺苷酸环化酶的药物引起的过敏性：含量简化和小型化的小分子和siRNA筛选应用

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

Construction and Characterization of a Novel Vocal Fold Bioreactor

一种新型声带生物反应器的构建与鉴定

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To ...

Fabrication and Characterization of a Conformal Skin-like Electronic System for Quantitative, Cutaneous Wound Management

制作定量，皮肤伤口管理一个形皮肤般的电子系统与表征

Recent advances in the development of electronic technologies and biomedical devices offer opportunities for non-invasive, quantitative assessment of ...

Double Emulsion Generation Using a Polydimethylsiloxane (PDMS) Co-axial Flow Focus Device

Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

复乳代使用聚二甲基硅氧烷（PDMS）同轴流量聚焦设备

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper ...

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

提高2D和3D皮肤<em&gt;体外</em&gt;模型使用大分子拥挤

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

用脱细胞支架龙ESC衍生鼠气道上皮细胞的生成

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

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 ...

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 ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

使用高通量微流体设备生成动态环境条件

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...