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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1250 &#181;L Griptip - Filter</td>
			<td>Integra</td>
			<td>4445</td>
			<td></td>
		</tr>
		<tr>
			<td>300 &#181;L Griptip - Filter</td>
			<td>Integra</td>
			<td>4435</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;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&#38;D Systems (Bio-Techne)</td>
			<td>3532-010-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO), >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&#160; (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&#160; 40R&#160;</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;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 &#181;L&#160;</td>
			<td>Integra</td>
			<td>3018</td>
			<td></td>
		</tr>
		<tr>
			<td>Single Channel EVOLVE 20-200 &#181;L&#160;</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

597 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

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

利用生物发光成像之间调查的协同作用肺炎链球菌 A型流感病毒的婴儿小鼠

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

科研

免疫与感染

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

鼠标应用结扎Peyer氏修补肠袢含量评价M细胞的细菌吸收

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

建立的液体覆盖的文化偏光人类呼吸道上皮的CALU-3细胞研究呼吸系统病原宿主细胞的反应<em在体外</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

利用RNA干扰调查的先天免疫反应的小鼠巨噬细胞

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The molecular basis of this resistance, which is known to be multifactorial, remains incompletely understood. Recent studies found that the differential expression of erythrocyte microRNAs, once translocated into malaria parasites, affect both gene regulation and parasite growth. These miRNAs were later shown to inhibit mRNA translation by forming a chimeric RNA transcript via 5&#39; RNA fusion with discreet subsets of parasite mRNAs. Here, the techniques that were used to study the functional role and putative mechanism underlying erythrocyte microRNAs on the gene regulation and translational potential of P. falciparum, including the transfection of modified synthetic microRNAs into host erythrocytes, will be detailed.&#160; Finally, a polysome gradient method is used to determine the extent of translation of these transcripts. Together, these techniques allowed us to demonstrate that the dysregulated levels of erythrocyte microRNAs contribute to cell-intrinsic malaria resistance of sickle erythrocytes.

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

Human microRNAs translocate from host erythrocytes to Plasmodium falciparum parasites. Here, the techniques used to transfect synthetic microRNAs into host erythrocytes and isolate all RNAs from P. falciparum are described. In addition, this paper will detail a method of polysome isolation in P. falciparum to determine the ribosomal occupancy and translational potential of parasite transcripts.

方法调查小RNA和核糖体占用的调控作用<em&gt;恶性疟原虫</em

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

人类的过表达和纯化<em&gt;顺</emβ-肾上腺素转移酶<em&gt;大肠杆菌</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Using Phylogenetic Analysis to Investigate Eukaryotic Gene Origin

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct a tree to show the evolutionary relationship among different taxa (classification units) at the molecular level. Phylogenetic analysis can also be used to investigate domain relationships within an individual taxon, particularly for organisms that have undergone substantial change in morphology and physiology, but for which researchers lack fossil evidence due to the organisms&#39; long evolutionary history or scarcity of fossilization.
In this text, a detailed protocol is described for using the phylogenetic method, including amino acid sequence alignment using Clustal Omega, and subsequent phylogenetic tree construction using both Maximum Likelihood (ML) of Molecular Evolutionary Genetics Analysis (MEGA) and Bayesian Inference via MrBayes. To investigate the origin of eukaryotic Sugars Will Eventually be Exported Transporters (SWEET) genes, 228 SWEETs including 35 SWEET proteins from unicellular eukaryotes and 57 SemiSWEET proteins from prokaryotes were analyzed. Interestingly, SemiSWEETs were found in prokaryotes, but SWEETs were found in eukaryotes. Two phylogenetic trees constructed using theoretically distinct methods have consistently&#160;suggested that the first eukaryotic SWEET gene might stem from the fusion of a bacterial SemiSWEET gene and an archaeal SemiSWEET gene.&#160;It is worth noting that one should be cautious to draw a conclusion based only on phylogenetic analysis, although it is useful to explain the underlying relationship between different taxa, which is difficult or even impossible to discern through experimental means.

A method of constructing a phylogenetic tree based on sequence homology of SWEETs from eukaryotes and SemiSWEETs from prokaryotes is described. Phylogenetic analysis is a useful tool for explaining the evolutionary relatedness between homologous proteins or genes from different organism groups.

应用系统进化分析研究真核基因的起源

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct ...

A Co-culture Method to Investigate the Crosstalk Between X-ray Irradiated Caco-2 Cells and PBMC

The protocol adopted in this work aims at unraveling how X-rays perturb the functioning of the intestinal barrier, focusing on the interplay between colorectal tumor cells and the immune system. Colorectal carcinoma is among the most common type of cancer, typically treated by surgery, chemotherapy, and radiotherapy. Advantages of radiotherapy in targeting the tumor are well known. However, even limited exposures of healthy tissues are of great concern, particularly regarding the effects on the intestinal barrier and the immune system. The adopted setup allows to study the interplay between two cell populations in a condition more similar to the physiological one, when compared to normal cell cultures. For this purpose, we resort to different techniques and we used an in vitro co-culture model, based on Caco-2 cells differentiated as a monolayer and PBMC, sharing the same culture medium. This protocol has been developed to focus on both macroscopic effects, i.e. cell viability and Trans-Epithelial Electrical Resistance (TEER), and, through western blot, molecular alterations, i.e. the activation of inflammatory pathway in immune cells and the tight junction protein expression in Caco-2 cells. Initial evaluation of radiation effects on Caco-2 cell viability was assessed via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Trypan blue assays, while TEER was measured at fixed time intervals through an ohmmeter specifically designed for co-culture systems. In this way, the effects due to radiation, the presence of Peripheral Blood Mononuclear Cells (PBMC), and eventually their synergistic effect, can be demonstrated. Through these complementary techniques, we observed a high radio-resistance of Caco-2 within the range of 2 - 10 Gy of X-rays and an increased Caco-2 monolayer permeability when PBMCs were added. In particular, PBMC presence was found to be associated with the variation in the tight junction scaffold proteins expression.

We present a protocol to investigate the crosstalk between X-ray-irradiated Caco-2 and peripheral blood mononuclear cells (PBMC). The protocol starts with Caco-2 irradiation and set-up of the co-culture with PBMC; subsequently, trans-epithelial electrical resistance is measured regularly over 48 h and western blot performed in both Caco-2 and PBMC.

X 射线辐照 Caco-2 细胞与 PBMC 间串扰的共培养方法研究

The protocol adopted in this work aims at unraveling how X-rays perturb the functioning of the intestinal barrier, focusing on the interplay between ...

Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and migration. One critical limitation of such assays, however, is their homogenous assortment of cellular types. Repair is a complex process which demands the interaction of several cell types. Therefore, to study a culture devoid of cellular subtypes, is a concern that must be overcome if we are to understand the repair process. The gastric organoid model may alleviate this issue whereby the heterogeneous collection of cell types closely reflects that of the gastric epithelium or other native tissues in vivo. Demonstrated here is a novel, in vitro scratch-wound assay derived from human or mouse 3-dimensional organoids which can then be transferred to a gastric epithelial monolayer as either intact organoids or as a single cell suspension of dissociated organoids. The goal of the protocol is to establish organoid-derived gastric epithelial monolayers that can be used in a novel scratch-wound assay system to study gastric regeneration.

Here we describe a protocol for establishing and culturing human- and mouse-derived 3-dimensional (3D) gastric organoids, and the method for the transfer of 3D organoids to a 2-dimensional monolayer. The use of the gastric epithelial monolayer as a novel scratch-wound assay for regeneration studies is also described.

小鼠和人源性胃上皮单层培养对再生的研究

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

原发性鸡初囊-细胞培养模型研究传染性囊内病病毒发病机制

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...

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

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