SNSF 向 J.H.N. 提供的 310030_219210 拨款支持了此手稿的出版，没有限制。 图 1 是在 BioRender.com 的帮助下创建的。

这些程序得到了瑞士西北部和中部伦理委员会（EKNZ;项目ID 2019-00273）。所有患者在内窥镜检查前都提供了实验性使用活检的书面知情同意书。研究中使用的试剂和设备列在 材料表中。
1. 患者来源的食管类器官的细胞分离
注: 表1中提供了用于培养人食管类器官的培养基成分列表。
<ol><li>进行活检。 注:在本研究中，在食管胃十二指肠镜检查期间，使用具有 2.8 毫米工作通道的胃镜使用活检钳从一个食管段获得两个活检。</li>
	<li>将活检转移到市售的角质形成细胞无血清培养基（KSFM;钙2+ 0.09 mM，1 ng/mL EGF，50 μg/mL BPE）。 注意:活检可以在冰上储存几个小时直至使用。</li>
	<li>用 1 mL 分散酶 I （10 U/mL） 替换 KSFM 培养基，并在室温下孵育活检 10 分钟。</li>
	<li>将活检物在室温下以300× g 离心2分钟。</li>
	<li>使用 1000 μL 移液管吸出分散酶，而不接触活检和细胞碎片沉淀。</li>
	<li>用 1 mL Dulbecco 磷酸盐缓冲盐水 （DPBS） 冲洗活检。</li>
	<li>将活检物在室温下以300× g 离心2分钟。</li>
	<li>使用 1000 μL 移液管吸出上清液。</li>
	<li>将活检与500μL胰蛋白酶-EDTA（0.05%）在37°C下孵育10分钟，同时以800rpm振荡。</li>
	<li>通过重复上下移液进行机械破坏，直到获得单细胞悬浮液。</li>
	<li>使用结核菌素注射器的橡胶柱塞头通过70μm细胞过滤器过滤细胞。</li>
	<li>用 2-4 mL 大豆胰蛋白酶抑制剂 （250 μg/mL） 清洗过滤器。</li>
	<li>通过 35 μm 细胞过滤器过滤细胞，该过滤器带有 5 mL 圆底聚苯乙烯管上的卡扣帽。</li>
	<li>将单细胞溶液转移到 15 mL 锥形管中。</li>
	<li>在4°C下以300× g 离心5分钟。</li>
	<li>使用 1000 μL 移液管吸出上清液。</li>
	<li>将细胞重悬于 100 μL KSFM 培养基中。</li>
	<li>将 10 μL 台盼蓝与 10 μL 细胞悬液混合。</li>
	<li>使用自动细胞计数仪对细胞进行计数。</li>
</ol>2. 患者来源的类器官培养
<ol><li>计数后向细胞悬液中加入 1-2 mL KSFM。</li>
	<li>在4°C下以300× g 离心5分钟。</li>
	<li>用 1000 μL 移液管吸出上清液，而不干扰细胞沉淀。</li>
	<li>将细胞沉淀重悬于基底膜提取物 （BME） 水凝胶基质（每 20,000 个细胞 40 μL BME）中。 注意:加入 BME 后，将细胞保持在冰上，以防止 BME 过早凝固。</li>
	<li>切断 200 μL 移液器吸头以吸出粘稠的 BME 细胞悬浮液混合物。</li>
	<li>在预热（37°C）悬浮细胞培养板中形成40μL液滴。</li>
	<li>将板在没有培养基的情况下在37°C下孵育20-30分钟，以确保BME液滴的凝固。</li>
	<li>加入预热的 KSFM-C 培养基，补充有 10 μM Y27632（ROCK 抑制剂），用于培养的前两天。</li>
	<li>每隔一天用新的 KSFM-C 培养基（不含 Y27632 和目标细胞因子）替换培养基。</li>
	<li>吸出培养基并用移液管吸头刮去液滴，同时连续向孔中加入 1 mL 分散酶 II （1.5 U/mL）。</li>
	<li>将BME-分散酶混合物转移到15mL离心管中，并在37°C的振荡水浴中孵育20分钟以消化BME。</li>
	<li>在4°C下以250× g 离心3分钟，并吸出分散酶II。</li>
	<li>根据目标读数的方案（例如，RNA 分离、蛋白质分离或用 4% PFA 固定用于组织学）进行。</li>
</ol>3. 用于患者来源的气液界面 （ALI） 培养物的细胞分离
<ol><li>进行活检。在使用具有 2.8 mm 工作通道的胃镜进行食管胃十二指肠镜检查时，使用活检钳从一个食管段进行两次活检。</li>
	<li>将活检物放入市售的角质形成细胞无血清培养基（KSFM;Ca2+ 0.09 mM，1 ng/mL EGF，50 μg/mL BPE），并在冰上储存数小时直至使用。</li>
	<li>用 1 mL 分散酶 （10 U/mL） 交换 KSFM。之后，将活检细胞在室温下孵育10分钟。</li>
	<li>将活检物在室温下以300×g离心2分钟。</li>
	<li>使用 1000 μL 移液管取出分散酶，而不接触活检和细胞碎片沉淀。</li>
	<li>用 1 mL DPBS 洗涤活检。</li>
	<li>在室温下，将活检以300× g 旋转2分钟。</li>
	<li>用 1000 μL 移液管吸出上清液。</li>
	<li>将活检物在37°C的500μL胰蛋白酶-EDTA（0.05%）中孵育10分钟，并在孵育过程中以800rpm连续混合。</li>
	<li>通过重复的上下移液机械地破坏活检，直到获得单细胞悬浮液。</li>
	<li>将解离的细胞通过带有来自结核菌素注射器的橡胶柱塞头的 70 μm 细胞过滤器，并将细胞收集在 50 mL 锥形管中。</li>
	<li>用 2-4 mL 大豆胰蛋白酶抑制剂 （250 μg/mL） 清洗过滤器，以去除过滤器中剩余的细胞。</li>
	<li>用 35 μm 细胞过滤器卡帽将细胞过滤到 5 mL 圆底聚苯乙烯管中。</li>
	<li>将细胞转移到 15 mL 锥形管中。</li>
	<li>在4°C下以300× g 离心5分钟。</li>
	<li>用 1,000 μL 移液管取出上清液。</li>
	<li>将细胞沉淀重悬于 4 mL KSFM 培养基 （Ca2+ 0.09 mM） 中，包括 10 μM Y27632。</li>
	<li>将细胞转移到 T25 细胞培养瓶中。</li>
	<li>为了扩增原代角质形成细胞，将细胞培养至60%-80%汇合度约1周。 注:传代 （P）0 形成胰岛状细胞团，不是单层。原代角质形成细胞从 P1 开始形成单层。</li>
	<li>以 60%-80% 汇合度传代，并在 2-3 个 T25 或 1-2 个 T75 细胞培养瓶中重新接种 P1。</li>
	<li>当角质形成细胞形成单层，汇合度为 60%-80% 时，再次传代 P1。</li>
</ol>4.患者来源的气液界面（ALI）培养
<ol><li>将 P2 原代角质形成细胞接种到用于 ALI 培养的 transwell 插入物上。 注:将 400,000 个细胞接种在 12 个孔插入物（每 0.6 cm 200,000 个角质形成细胞）中，放入 500 μL KSFM（Ca2+ 0.09 mM，1 ng/mL EGF，50 μg/mL BPE）中。<ol><li>将 150,000 个细胞接种到 100 μL KSFM（Ca2+ 0.09 mM，1 ng/mL EGF，50 μg/mL BPE）的 24 个孔插入物（每 0.5 cm2 中 155,000 个角质形成细胞）。</li>
		<li>或者，在-80°C的KSFM培养基（Ca2 + 0.09mM + 10%DMSO）中冷冻24小时，然后将冷冻细胞转移到液氮中以备后用。 注意:使用冷冻小瓶时，在使用细胞进行 ALI 培养之前，请在解冻后再次传代一次。</li>
	</ol></li>
	<li>将培养基添加到transwell培养板插入物下方的下孔中。 注:对于 12 孔板:1.5 mL KSFM（Ca2+ 0.09 mM，1 ng/mL EGF，50 μg/mL BPE）。对于 24 孔板:600 μL KSFM（Ca2+ 0.09 mM，1 ng/mL EGF，50 μg/mL BPE）。</li>
	<li>2 天后更换中高钙 KSFM（Ca2+ 1.8 mM，1 ng/mL EGF，50 μg/mL BPE）。</li>
	<li>每隔一天更换一次高钙 KSFM 培养基，直到第 7 天。</li>
	<li>在第 7 天进行气运，从上室吸出培养基并用含有 10 ng/mL KGF （=FGF7）、75 μg/mL 抗坏血酸 （AA） 的高钙 KSFM（Ca2+ 1.8 mM，1 ng/mL EGF，50 μg/mL BPE）替换下室中的培养基。<ol><li>可选:将所需浓度的目标细胞因子添加到培养基中。</li>
	</ol></li>
	<li>每隔一天更换培养基，直到第 14 天。</li>
</ol>5. 跨上皮电阻 （TEER） 测量
<ol><li>将TEER仪的电极浸入装有5%次氯酸钠的24孔板的孔中10-15分钟，对TEER仪的电极进行灭菌。</li>
	<li>将电极浸入 4 个连续的孔中，用无菌 ddH2O 洗去 5% 次氯酸钠，然后让电极风干。</li>
	<li>通过将一个电极放入孔中，将第二个电极放入transwell插入物中，用PBS填充，然后测量TEER，从而设置空白。 注:TEER 测量的推荐体积:用于 12 孔板（孔中 1900 μL，插入体中 900 μL）。适用于 24 孔板（孔中 750-1000 μL，插入物中 250 μL）。</li>
	<li>用室温无菌PBS替换ALI培养物的培养基。 注意:从下孔中取出培养基，在第二步中，从Transwell插入物中取出培养基。同样，首先将 PBS 添加到插入物中，然后添加到下孔中，以防止 ALI 培养物从跨孔膜上分离。</li>
	<li>将TEER计电极置于含有ALI培养物的实验孔中，并进行TEER测量1。 注意: 在更换介质之前，每隔一天进行一次 TEER 测量。</li>
</ol>6. 大分子通量
<ol><li>将 FITC-葡聚糖 （3-5 kDa） 储备溶液稀释至工作浓度 1 mg/mL。 注意:始终保护 FITC-葡聚糖免受光照。</li>
	<li>准备一个稀释行，将FITC浓度降低（1000μg/ mL至0.25μg/ mL）作为读数的标准品。</li>
	<li>将 500 μL FITC-葡聚糖溶液 （1 mg/mL） 加入 transwell 的上室，将 1.5 mL 培养基（+/- 目标细胞因子）加入下室，并将板置于培养箱中。</li>
	<li>在相应的时间点（例如，0 分钟、15 分钟、30 分钟、60 分钟、90 分钟、120 分钟、150 分钟和 180 分钟）从下室收集 120 μL 培养基。</li>
	<li>移液器将每个时间点的复制品（50 μL/孔）放入黑色 96 孔透明平底板中。</li>
	<li>激发 490 nm 的 FITC-葡聚糖，并使用酶标仪读取 520 nm 波长的发射。</li>
	<li>按标准计算大分子通量。</li>
</ol>

人食管类器官和气液界面培养方案

作者没有什么可透露的。

所提供的程序允许培养患者来源的类器官和 ALI 培养物，成功的可能性很高。类器官方案改编自第一个发表的报告人类食管类器官产生的方案26 和最近发表的方案32。Sherill 及其同事描述了 ALI 模型22。类器官和 ALI 培养模型在研究食管疾病（如 EoE 5,26）中细胞因子和其他介质对食管上皮屏障的影响方?...

食管炎症会损害上皮屏障的完整性 1,2,3,4,5，如嗜酸性粒细胞性食管炎 （EoE） 所观察到的那样，这是一种以 Th2 为主的食管慢性炎症性疾病 6。EoE 于 1990 年代首次描述 7,8，主要由食物抗原 9,10,11,12,13 诱导。成人中最常见的 EoE 症状是吞咽困难和食物嵌塞14。在儿童中，EoE 通常表现为生长迟缓、食物拒绝、呕吐和腹痛15。全基因组关联研究 （GWAS） 已确定与上皮屏障完整性相关的 EoE 风险基因，使上皮成为 EoE 研究的重点 16,17,18。EoE 转录组学进一步揭示，分化过程受损和反应性基底区增生导致食管上皮屏障功能受损 3,5,19,20,21,22。对 EoE 是 Th2 介导的疾病6 的早期理解导致发现 IL-13 通过干扰上皮完整性作为驱动介质 3,4,21,23。实验系统允许解剖细胞因子介导的对上皮完整性的影响，从内在屏障损伤到遗传易感性，为研究免疫细胞与EoE中上皮细胞之间的复杂相互作用提供了可能性。人食管类器官和气液界面 （ALI） 培养物已被提议作为分析细胞因子刺激对上皮完整性后果的宝贵工具5。
在2009年首次发表的肠道类器官报告使用肠道Lgr5 + ASCs概括小肠上皮室24之后，建立了第一个产生成体组织特异性干细胞（ASC）衍生食管类器官的方案。DeWard等人率先从小鼠食管上皮细胞中产生类器官25。2018 年，Kasagi 等人从永生化的人食管鳞状上皮细胞系 EPC2-hTERT 和原代患者来源的细胞中生成了人食管类器官26。同年，Zhang等人成功制备了诱导多能干细胞（iPSC）衍生的食管类器官。他们阐述了 TGFβ 和骨形态发生蛋白 （BMP） 抑制对食管祖细胞 （EPC） 发育的意义，以及 Notch 信号转导在分层鳞状上皮分化中的关键作用26,27。Trisno及其同事通过将Sox2确定为Wnt抑制剂来补充这些发现，该抑制剂将发育命运指向食管分化28。随后对方案、培养基组成和培养条件的改进提高了类器官形成率，并使得冷冻保存后传代培养和回收类器官成为可能26,29,30,31,32。尽管这些类器官是研究细胞因子刺激后组织结构和潜在靶基因表达的强大工具，但食管类器官无法提供测量跨上皮抵抗 （TEER） 或大分子通量作为屏障完整性的直接测量方法。正如 Sherrill 及其同事22 之前所描述的，模拟上皮分化的 ALI 培养物4 允许直接评估上皮完整性。结合患者来源的类器官和 ALI 培养物是研究 EoE 中组织结构和上皮屏障完整性的有力工具。
以下是从食管活检中分离活细胞和建立食管类器官和 ALI 培养物的程序，这些程序可用于进一步研究细胞因子对屏障完整性的影响。

<table><tbody><tr><td>1250 &amp;micro;L Griptip - Filter</td><td>Integra</td><td>4445</td><td></td></tr><tr><td>300 &amp;micro;L Griptip - Filter</td><td>Integra</td><td>4435</td><td></td></tr><tr><td>70 &amp;micro;M cell strainer</td><td>Sarstedt</td><td>83.3945.070</td><td></td></tr><tr><td>Ascorbic Acid</td><td>Sigma-Aldrich (Merck)</td><td>A4544</td><td></td></tr><tr><td>Bovine pituitary extract</td><td>Gibco (Thermo Fischer Scientific)</td><td>3700015</td><td></td></tr><tr><td>Calcium chloride</td><td>Sigma-Aldrich (Merck)</td><td>21115</td><td></td></tr><tr><td>Cell Culture Multiwell Plates CELLSTAR for suspension cultures</td><td>Greiner Bio-One</td><td>7.657 185</td><td></td></tr><tr><td>Cultrex Basement Membrane Extract (BME), Type 2, Pathclear</td><td>R&amp;amp;D Systems (Bio-Techne)</td><td>3532-010-02</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO), &gt;99,5% BioScience Grade</td><td>Carl Roth</td><td>A994</td><td></td></tr><tr><td>Dispase I</td><td>Corning</td><td>354235</td><td></td></tr><tr><td>Dispase II</td><td>Sigma-Aldrich (Merck)</td><td>D4693</td><td></td></tr><tr><td>Dulbeccos Phosphate Buffered Saline&amp;nbsp; (DPBS)</td><td>Sigma-Aldrich (Merck)</td><td>D8537</td><td></td></tr><tr><td>EVE Automated Cell Counter</td><td>NanoEntek</td><td>EVE-MC</td><td></td></tr><tr><td>EVE Cell counting slide</td><td>NanoEntek</td><td>EVS-050</td><td></td></tr><tr><td>Falcon 5 mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap</td><td>Falcon</td><td>352235</td><td></td></tr><tr><td>Fluorescin isothiocyanate (FITC)-dextran</td><td>Sigma-Aldrich (Merck)</td><td>FD4</td><td>average mol wt 3000-5000</td></tr><tr><td>Heraeus - Megafuge&amp;nbsp; 40R&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>75004518</td><td></td></tr><tr><td>Human recombinant epidermal growth factor</td><td>Gibco (Thermo Fischer Scientific)</td><td>3700015</td><td></td></tr><tr><td>Keratinocyte-SFM</td><td>Gibco (Thermo Fischer Scientific)</td><td>17005042</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco (Thermo Fischer Scientific)</td><td>15140122</td><td></td></tr><tr><td>Recombinant Human KGF/FGF-7 Protein</td><td>R&amp;amp;D Systems (Bio-Techne)</td><td>251-KG-010/CF</td><td></td></tr><tr><td>Screw cap tube, 15 mL</td><td>Sarstedt</td><td>62.554.502</td><td></td></tr><tr><td>Single Channel EVOLVE 100-1000 &amp;micro;L&amp;nbsp;</td><td>Integra</td><td>3018</td><td></td></tr><tr><td>Single Channel EVOLVE 20-200 &amp;micro;L&amp;nbsp;</td><td>Integra</td><td>3016</td><td></td></tr><tr><td>Syringe 1 mL</td><td></td><td>1134950</td><td></td></tr><tr><td>ThermoMixer C</td><td>Eppendorf</td><td>5382000015</td><td></td></tr><tr><td>Trypsin inhibitor from Glycine max (soybean)</td><td>Sigma-Aldrich (Merck)</td><td>T9128</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>SAFC Biosciences (Merck)</td><td>59418C</td><td></td></tr><tr><td>Y27632 dihydrochloride</td><td>Tocris (Bio-Techne)</td><td>1254</td><td></td></tr></tbody></table>

three-dimensional cell culture models to investigate the epithelial barrier in eosinophilic esophagitis

食管鳞状上皮直接暴露在环境中，不断面对外来抗原，包括食物抗原和微生物。保持上皮屏障的完整性对于预防感染和避免由无害的食物源性抗原引起的炎症至关重要。本文提供了从患者活检生成人食管类器官和气液界面培养物的简化方案，以在组织稳态和疾病的背景下研究食管上皮室。这些方案是过去十年中重要的科学里程碑，描述了来自患者来源的原代细胞、类器官和气液界面培养物的三维器官样结构。它们提供了在三维框架内研究食管上皮中特定细胞因子、生长因子和信号通路功能的可能性，同时保持供体的表型和遗传特性。类器官通过评估细胞因子刺激后的转录组和蛋白质组来提供有关组织微结构的信息。相比之下，气液界面培养允许通过跨上皮阻力 （TEER） 或大分子通量测量来评估上皮屏障的完整性。将这些类器官和气液界面培养物相结合是推进食管上皮屏障受损条件研究的有力工具。

食管类器官将根据所提供的方案的指示从患者活检中提取的原代细胞生长，如倒置明场显微镜所记录的那样（图1）。上皮 ASC 在将分离的细胞接种到基底膜提取物中后，在培养的前两天开始以自组织方式形成细胞簇，充当支架。细胞簇的大小和数量在第一周内持续增加，用倒置明场显微镜观察（图2）。然而，在这一点上，细胞簇缺乏 ASC 衍生的食管类?...

Read this Scientific Article Protocol about Author Spotlight: Investigating the Pathophysiology of Eosinophilic Esophagitis at JoVE.com

作者聚焦:研究嗜酸性粒细胞性食管炎的病理生理学

在这里，提供了人食管类器官培养和气液界面培养的方案。食管类器官的气液界面培养可用于研究细胞因子对食管上皮屏障的影响。

三维细胞培养模型研究嗜酸性粒细胞性食管炎中上皮屏障 

The squamous epithelium of the esophagus is directly exposed to the environment, continuously facing foreign antigens, including food antigens and ...

我们正在研究嗜酸性粒细胞性食管炎的病理生理学，并试图揭示诱导和促进EOE慢性炎症过程的炎症级联反应。鉴定有助于疾病发病机制的新免疫介质的确定，其中心是上皮屏障的损伤和针对所述免疫介质的治疗性抗体的批准。我们已经确定 IL-20 亚家族细胞因子是炎症介质，可损害上皮分化和角化，从而通过 MAP 激酶途径导致上皮屏障损伤。

three-dimensional-cell-culture-models-to-investigate-epithelial

Research

Education

JoVE Journal

JoVE Core

Anatomy and Physiology

Immunology and Infection

Three-Dimensional Cell Culture Models to Investigate the Epithelial Barrier in Eosinophilic Esophagitis

Unit 1

Digestive System

SCIENTISTS IN ACTION

613 次观看.  University of Basel. 我们正在研究嗜酸性粒细胞性食管炎的病理生理学，并试图揭示诱导和促进EOE慢性炎症过程的炎症级联反应。鉴定有助于疾病发病机制的新免疫介质的确定，其中心是上皮屏障的损伤和针对所述免疫介质的治疗性抗体的批准。我们已经确定 IL-20 亚家族细胞因子是炎症介质，可损害上皮分化和角化，从而通过 MAP 激酶途径导致上皮屏障损伤。

视频: 作者聚焦:研究嗜酸性粒细胞性食管炎的病理生理学

Conditional Reprogramming of Pediatric Human Esophageal Epithelial Cells for Use in Tissue Engineering and Disease Investigation

Identifying and expanding patient-specific cells in culture for use in tissue engineering and disease investigation can be very challenging. Utilizing various types of stem cells to derive cell types of interest is often costly, time consuming and highly inefficient. Furthermore, undesired cell types must be removed prior to using this cell source, which requires another step in the process. In order to obtain enough esophageal epithelial cells to engineer the lumen of an esophageal construct or to screen therapeutic approaches for treating esophageal disease, native esophageal epithelial cells must be expanded without altering their gene expression or phenotype. Conditional reprogramming of esophageal epithelial tissue offers a promising approach to expanding patient-specific esophageal epithelial cells. Furthermore, these cells do not need to be sorted or purified and will return to a mature epithelial state after removing them from conditional reprogramming culture. This technique has been described in many cancer screening studies and allows for indefinite expansion of these cells over multiple passages. The ability to perform esophageal screening assays would help revolutionize the treatment of pediatric esophageal diseases like eosinophilic esophagitis by identifying the trigger mechanism causing the patient's symptoms. For those patients who suffer from congenital defect, disease or injury of the esophagus, this cell source could be used as a means to seed a synthetic construct for implantation to repair or replace the affected region.

Expansion of human pediatric esophageal epithelial cells utilizing conditional reprogramming provides investigators with a patient-specific population of cells that can be utilized for engineering esophageal constructs for autologous implantation to treat defects or injury and serve as a reservoir for therapeutic screening assays.

小儿食管上皮细胞重编程的条件在组织工程和疾病使用调查

Identifying and expanding patient-specific cells in culture for use in tissue engineering and disease investigation can be very challenging. Utilizing ...

科研

发育生物学

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

三维组织工程人食管黏膜型号生产，表征和潜在用途

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

生物工程

Modeling Oral-Esophageal Squamous Cell Carcinoma in 3D Organoids

Esophageal squamous cell carcinoma (ESCC) is prevalent worldwide, accounting for 90% of all esophageal cancer cases each year, and is the deadliest of all human squamous cell carcinomas. Despite recent progress in defining the molecular changes accompanying ESCC initiation and development, patient prognosis remains poor. The functional annotation of these molecular changes is the necessary next step and requires models that both capture the molecular features of ESCC and can be readily and inexpensively manipulated for functional annotation. Mice treated with the tobacco smoke mimetic 4-nitroquinoline 1-oxide (4NQO) predictably form ESCC and esophageal preneoplasia. Of note, 4NQO lesions also arise in the oral cavity, most commonly in the tongue, as well as the forestomach, which all share the stratified squamous epithelium. However, these mice cannot be simply manipulated for functional hypothesis testing, as generating isogenic mouse models is time- and resource-intensive. Herein, we overcome this limitation by generating single cell-derived three-dimensional (3D) organoids from mice treated with 4NQO to characterize murine ESCC or preneoplastic cells ex vivo. These organoids capture the salient features of ESCC and esophageal preneoplasia, can be cheaply and quickly leveraged to form isogenic models, and can be utilized for syngeneic transplantation experiments. We demonstrate how to generate 3D organoids from normal, preneoplastic, and SCC murine esophageal tissue and maintain and cryopreserve these organoids. The applications of these versatile organoids are broad and include the utilization of genetically engineered mice and further characterization by flow cytometry or immunohistochemistry, the generation of isogeneic organoid lines using CRISPR technologies, and drug screening or syngeneic transplantation. We believe that the widespread adoption of the techniques demonstrated in this protocol will accelerate progress in this field to combat the severe burden of ESCC.

This protocol describes the key steps to generate and characterize murine oral-esophageal 3D organoids that represent normal, preneoplastic, and squamous cell carcinoma lesions induced via chemical carcinogenesis.

在 3D 类器官中模拟口腔-食管鳞状细胞癌

Esophageal squamous cell carcinoma (ESCC) is prevalent worldwide, accounting for 90% of all esophageal cancer cases each year, and is the deadliest of all ...

癌症研究

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

工程三维上皮组织细胞外基质中嵌入

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

<meta charset="utf8"></head><body>用于研究肺屏障完整性、疾病和药物发现的气道模型

iPSC-Derived Epithelial, Mesenchymal, Endothelial, and Immune Cell Co-Culture to Model Airway Barrier Integrity in Lung Health and Disease

Human lung tissue is composed of an interconnected network of epithelium, mesenchyme, endothelium, and immune cells from the upper airway of the nasopharynx to the smallest alveolar sac. Interactions between these cells are crucial in lung development and disease, acting as a barrier against harmful chemicals and pathogens. Current in vitro co-culture models utilize immortalized cell lines with different biological backgrounds, which may not accurately represent the cellular milieu or interactions of the lung. We differentiated human iPSCs into 3D lung organoids (containing both epithelium and mesenchyme), endothelial cells, and macrophages. These were co-cultured in an air-liquid interface (ALI) format to form an epithelial/mesenchymal apical barrier invested with macrophages and a basolateral endothelial barrier (iAirway). iPSC-derived iAirways showed a reduction in barrier integrity in response to infection with respiratory viruses and cigarette toxins. This multi-lineage lung co-culture system provides a platform for studying cellular interactions, signaling pathways, and molecular mechanisms underlying lung development, homeostasis, and disease progression. iAirways closely mimic human physiology and cellular interactions, can be generated from patient-derived iPSC&#39;s, and can be customized to include different cell types of the airway. Overall, iPSC-derived iAirway models offer a versatile and powerful tool for studying barrier integrity to better understand genetic drivers for disease, pathogen response, immune regulation, and drug discovery or repurposing in vitro, with the potential to advance our understanding and treatment of airway diseases.

<meta charset="utf8"></head><body>作者聚焦:使用同基因上皮细胞、内皮细胞和免疫细胞对人类气道重塑和病毒反应进行建模

This article describes the generation of a complex, multi-cellular airway barrier model composed of induced pluripotent stem cell (iPSC)-derived lung epithelium, mesenchyme, endothelial cells, and macrophages in an air-liquid interface culture.

iPSC 衍生的上皮细胞、间充质细胞、内皮细胞和免疫细胞共培养，以模拟肺部健康和疾病中的气道屏障完整性

Human lung tissue is composed of an interconnected network of epithelium, mesenchyme, endothelium, and immune cells from the upper airway of the ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

人类舱内单聚物研究病原体、共脉和宿主肠道上皮细胞之间的相互作用

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...

免疫与感染

Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing a barrier to foreign particles. Impaired epithelial transport (ion, nutrient, or drugs) has been associated with many diseases and can have consequences that extend beyond the normal physiological functions of the transporters, such as by influencing epithelial integrity and the gut microbiome. Understanding the function and regulation of transport proteins is critical for the development of improved therapeutic interventions. The biggest challenge in the study of epithelial transport is developing a suitable model system that recapitulates important features of the native intestinal epithelial cells. Several in vitro cell culture models, such as Caco-2, T-84, and HT-29-Cl.19A cells are typically used in epithelial transport research. These cell lines represent a reductionist approach to modeling the epithelium and have been used in many mechanistic studies, including their examination of epithelial-microbial interactions. However, cell monolayers do not accurately reflect cell-cell interactions and the in vivo microenvironment. Cells grown in 3D have shown to be promising models for drug permeability studies. We show that Caco-2 cells in 3D can be used to study epithelial transporters. It is also important that studies in Caco-2 cells are complemented with other models to rule out cell specific effects and to take into account the complexity of the native intestine. Several methods have been previously used to assess the functionality of transporters, such as everted sac and uptake in isolated epithelial cells or in isolated plasma membrane vesicles. Taking into consideration the challenges in the field with respect to models and the measurement of transport function, we demonstrate here a protocol to grow Caco-2 cells in 3D and describe the use of an Ussing chamber as an effective approach to measure serotonin transport, such as in intact polarized intestinal epithelia.

We describe simple methods to study the regulation of intestinal serotonin transporter (SERT) function and expression using an in vitro cell culture model of Caco-2 cells grown in 3D&#160;and an ex vivo model of mouse intestines. These methods are applicable to the study of other epithelial transporters.

方法来研究上皮转运蛋白功能和表达在纯肠和Caco-2细胞生长在3D

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing ...

生物学

Organoid-Derived Epithelial Monolayer: A Clinically Relevant In Vitro Model for Intestinal Barrier Function

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations as the former do not faithfully represent original tissue physiology, and the availability of the latter is limited. Hence, their application hampers fundamental and drug development research. Adult stem-cell-based organoids (henceforth referred to as organoids) are miniatures of normal or diseased epithelial tissue from which they are derived. They can be established very efficiently from different gastrointestinal (GI) tract regions, have long-term expandability, and simulate tissue- and patient-specific responses to treatments in vitro. Here, the establishment of intestinal organoid-derived epithelial monolayers has been demonstrated along with methods to measure epithelial barrier integrity, permeability and transport,&#160;antimicrobial protein secretion, as well as histology. Moreover, intestinal organoid-derived monolayers can be enriched with proliferating stem and transit-amplifying cells as well as with key differentiated epithelial cells. Therefore, they represent a model system that can be tailored to study the effects of compounds on target cells and their mode of action. Although organoid cultures are technically more demanding than cell lines, once established, they can reduce failures in the later stages of drug development as they truly represent in vivo epithelium complexity and interpatient heterogeneity.

Here, we describe the preparation of human organoid-derived intestinal epithelial monolayers for studying intestinal barrier function, permeability, and transport. As organoids represent original epithelial tissue response to external stimuli, these models combine the advantages of expandability of cell lines and the relevance and complexity of primary tissue.

类器官来源的上皮单层:临床相关的肠道屏障功能体外模型

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations ...

三维细胞培养模型研究嗜酸性粒细胞性食管炎中上皮屏障

副本

摘要

探索更多视频

此视频中的章节

小儿食管上皮细胞重编程的条件在组织工程和疾病使用调查

三维组织工程人食管黏膜型号生产，表征和潜在用途

在 3D 类器官中模拟口腔-食管鳞状细胞癌

工程三维上皮组织细胞外基质中嵌入

iPSC 衍生的上皮细胞、间充质细胞、内皮细胞和免疫细胞共培养，以模拟肺部健康和疾病中的气道屏障完整性

iPSC 衍生的上皮细胞、间充质细胞、内皮细胞和免疫细胞共培养，以模拟肺部健康和疾病中的气道屏障完整性

iPSC 衍生的上皮细胞、间充质细胞、内皮细胞和免疫细胞共培养，以模拟肺部健康和疾病中的气道屏障完整性

人类舱内单聚物研究病原体、共脉和宿主肠道上皮细胞之间的相互作用

方法来研究上皮转运蛋白功能和表达在纯肠和Caco-2细胞生长在3D

类器官来源的上皮单层:临床相关的肠道屏障功能体外模型