This work was supported, in part, by NIAID, NIH, DHHS federal research grants R01 AI036525 and U19 AI082655 (CCHI) to MBS and by NIH grant DK048373 to AF. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Allergy And Infectious Diseases or the National Institutes of Health.

伦理声明:从参与协议号HP-00040025-1志愿者收集的所有血液样本。马里兰机构审查委员会的批准大学的这个协议和授权的血液样本的收集从包括在这个手稿研究健康志愿者。这项研究的目的是解释志愿者，以及所有的志愿者给予知情，抽血前签署同意书。 注:请参阅表1为中补充的准备。 见表2为三维培养基的制备。 1.培养容器的制备<ol><li>旋50毫升容器的填充口的盖子，并用50毫升无菌培养基（ 例如 ，RPMI）填充。执行层流罩无菌条件下的所有程序。 </li><li>更换帽和擦拭溅出的培养基用70％乙醇在任何表面上。 </li><li>允许在容器到incub吃了至少15分钟，O / N在室温。 </li><li>使用填充端口丢弃培养基并加入30×3-D培养基。 </li><li>请用70％乙醇的任何表面上溅出的网上平台。 </li></ol> 2.细胞的制备<ol><li>人类上皮细胞株（HCT-8）24,40。 <ol><li>在RPMI培养细胞补充有100U / ml青霉素，100微克/ ml链霉素，50μg/ ml的庆大霉素，2mM的L-谷氨酰胺，1mM丙酮酸钠，10毫米的HEPES缓冲液和10％热灭活的小牛血清（FBS ）（ 补充1）。在70％汇合，分裂细胞以1:5的比例。 </li></ol></li><li>人类结肠成纤维细胞（CCD-18Co细胞 ）24,41。 <ol><li>培养的细胞在与补充1富集的基础的Eagle培养基在汇合，分裂细胞以1:1的比例:3。 </li></ol></li><li>人脐静脉内皮细胞（HUVEC）细胞<suP&gt; 24,42。 <ol><li>在内皮基础培养基（EBM）培养细胞培养基富含100微克/毫升肝素，3微克/毫升内皮细胞生长补充剂（ECGS），并补充1在70％汇合，分裂细胞以1:1的比例:2。 </li></ol></li><li>淋巴细胞/单核细胞<ol><li>通过使用标准技术43密度梯度离心分离从血液的外周血单核细胞（PBMC）。 <ol><li>简要地说，用10毫升移液管，小心地加入30毫升稀释血（1:1血液:磷酸盐缓冲盐水（PBS）中）到含有10ml密度离心培养基的50ml圆锥形管中。离心步骤后，淋巴细胞和单核细胞将驻留在密度离心介质和等离子体层之间的"白"层。 </li><li>收集用移液管白层。通过限制密度离心媒体的集合避免粒污染。洗完后43，U本身作为PBMC或者是在液态氮冷冻保存。 注意:以突出的PBMC大体上是由淋巴细胞和单核细胞的，树突状细胞和其他细胞类型的一小部分是很重要的。 </li></ol></li></ol></li><li>成长标准培养条件下的所有细胞在37℃下在5％CO 2的气氛中。 </li></ol> 3.胶原包埋细胞的制备<ol><li>确定插入物的数量根据实验条件的号码需要加以设定。放置插入适当数量的成6孔板的孔中。 </li><li>制成悬浮液HUVEC和成纤维细胞的: <ol><li>在PBS中洗烧瓶两次，并使用胰蛋白酶，0.25％（1×）用0.05％胰蛋白酶乙二胺四乙酸四（胰蛋白酶-EDTA）分离细胞。添加足够的胰蛋白酶溶液，以覆盖细胞培养表面面积的全部。支队通常在5发生15分钟</li><li>搜集分离的细胞在装有30ml Dulbecco改良的Eagle氏培养基（DMEM）30％FBS培养基的50毫升管。 </li><li>离心管中，在500×g离心10分钟。 </li><li>悬浮细胞在30ml的DMEM-30％FBS的培养基中。 </li><li>通过稀释20微升悬浮细胞与20微升台盼蓝染料的计数存活细胞的总数。负载细胞混合物的血细胞计数器。可行的外周血单个核细胞将白清晰;没有自生能力PBMC将收购染料变成蓝色。 <ol><li> 8×10 7个细胞毫升- -以5的浓度悬浮在DMEM-30％FBS培养基中的细胞类型1。 </li></ol></li></ol></li><li>制备含细胞胶原凝胶 注意:每个容器将需要约5毫升含细胞的胶原凝胶的制备。 <ol><li>在一个50毫升锥形管中加入1×DMEM培养基，补充有50微克/ ml庆大霉素，2mM的L-谷氨酰胺和10％热inactivat制备胶原混合物编FBS加3毫克/毫升的牛Ⅰ型胶原，10μg/ ml的层粘连蛋白，40微克/毫升IV型胶原，10μg/ ml的纤连蛋白，2微克/毫升硫酸肝素蛋白聚糖和15mM的NaOH（达到生理pH）。 </li><li>关闭该管并且颠倒几次，直至混合物完全均匀混合。避免泡沫形成。 重要提示:保持冰混合物，以防止凝胶化。 <ol><li> 关键的步骤:如果混合物开发的酸性外观（淡黄色），添加无菌的1N NaOH几滴以中和该混合物。 </li></ol></li><li>加浓的HUVEC和成纤维细胞以1.0的密度- 1.2和1.5 - 2.0×10 6个细胞/ ml，分别向富含胶原混合物（如上所述）。关闭管和颠倒几次，直至混合物完全均匀混合。避免泡沫形成。重要提示:保持冰混合物，以防止凝胶化。 </li><li>添加4 - 所述含细胞胶原5毫升凝胶每个插入，预先装入6孔板，并使其在通风橱中，很少或没有运动，以凝胶化1小时。 </li><li> 1小时后，在6孔板转移到37℃，5％CO 2培养箱1 - 2小时。 </li><li>返回6孔板的罩，并且用无菌镊子的帮助下，并从插入件分离从膜的含细胞​​凝胶手术刀无菌地切断该膜。切胶脱落成小方块（约5×5毫米）。 </li><li>添加小含细胞平方到使用填充口的50ml HARV。 </li></ol></li><li>制备悬浮液HCT-8的上皮细胞: <ol><li>在PBS中洗烧瓶两次，并使用胰蛋白酶，0.25％（1×）用胰蛋白酶-EDTA处理分离细胞。添加足够的胰蛋白酶溶液，以覆盖细胞培养表面面积的全部。脱离通常为10至15分钟内发生。 </li><li>收集分离的细胞在30毫升的DMEM-30％FBS的培养基中。 </li><li> CentrifUGE DMEM-30％FBS含管在500 XG 10分钟网上平台。 </li><li>悬浮细胞在30ml的DMEM-30％FBS的培养基中。 </li><li>如3.2.5所述计数存活细胞的总数。 </li><li>重悬在DMEM-30％FBS的培养基中的HCT-8的上皮细胞以10 7个细胞ml的浓度- 1。 </li><li>加入1毫升浓HCT-8上皮细胞进50毫升HARV使用填充口。 </li></ol></li><li>通过使用填充口，添加额外的3-D培养基直到容器几乎满（约20毫升）中。 </li><li>更换盖和擦拭用70％乙醇的填充口的唇缘。 </li><li>放置一个注射器中的RWV的每个注射器端口:地方含有3 5ml的注射器 - 在一个端口4×3-D培养基，并在另一端口5ml的空注射器。 </li><li>通过拉入空注射器而大致介质的相同体积通过其它注射器口喷射除去任何可见的气泡。 </li><li>放置VESSELS在生物反应器中，打开电源，速度设定为每分钟约13至14转。 注:关键步骤:旋转速率可能需要在实验期间进行调整几次，以保持在容器内旋转，从而避免了与该容器壁接触的细胞。 </li><li>培养细胞在37℃，微重力下，5％CO 2，由RWV生物反应器提供。 </li><li>改变培养基，每3 - 第4天用新鲜的3-D培养基更换约30毫升</li><li>经过4天（±1天），请从生物反应器的船只，并添加淋巴细胞/单核细胞向血管（2×10 7 /船）。 </li><li>放置血管回生物反应器中，在37℃，5％CO 2的额外5天（±1天）。 </li><li> （培养第9天）的另外5天后，添加淋巴细胞/单核细胞的容器（2×10 7 /容器），并继续额外长达12天的培养物。 </l我&gt; <li>改变培养基，每3 - 第4天用新鲜的3-D培养基更换约30毫升</li></ol>为组织学4.收获3-D培养<ol><li>用移液管收获的援助每个小凝胶片段，现在称为"构建体，"到含有10ml的10％福尔马林缓冲液中的六孔板中。在RT留3小时或12 - 在4℃下16小时。 </li><li>用标签和加入活检泡沫垫两块到每个磁带准备组织处理和嵌入录像带。 </li><li>沉浸在10％福尔马林缓冲液卡匣。 </li><li>用钳子的帮助放置两个泡沫垫之间的固定结构。 </li><li>沉浸在10％福尔马林缓冲液卡匣。 </li><li>与嵌入，切片，染色和44标准程序进行。 </li></ol>

The authors declare that a US Non-Provisional Patent Application has been filed in the U.S.Patent and Trademark Office (Number: 13/360,539).

在这个手稿，我们描述了人肠黏膜由倍数细胞类型，包括原代人淋巴细胞，成纤维细胞和内皮细胞，以及肠上皮细胞系24的生物工程化的模型的开发。在这个3-D模型，细胞在富含胶原蛋白的细胞外基质中的微重力条件下24培养。 如先前所描述，该模型的主要特征是:（i）以模仿上皮组织单层组织的能力，（ⅱ）上皮细胞，紧密连接，桥...

在构建三维模型首次突破据报道，在80年代初，当科学家们开始研究不同类型的支架（ 例如 ，层粘连蛋白，胶原蛋白I型，Ⅳ型胶原和纤维连接蛋白）和生长因子鸡尾酒改善细胞-细胞和的"静态"3-D模型1-7的ECM相互作用。此后，随着这些模型的主要问题一直是在营养物质的传递和氧的介质和组织构建8内的限制。在对比中，其接收从周围的血管网络的营养素的稳定流量和氧的体内环境的细胞，这些模型的静态性质阻碍他们的向细胞的有效分配。例如，在体外静态模型生成大小超过几毫米细胞聚集体将总是开发缺氧，坏死芯件9。该RWV生物反应器可以解决这个问题通过提供流体动力学，允许营养物和氧10-12的有效扩散。然而，到目前为止，使用RWV生物反应器的工作已被限制为包含一种或两种的细胞类型13-17。此外，代替类似于天然组织的空间取向，这些细胞形成细胞聚集体。这些限制的主要原因是缺乏能够把电池于一体的综合时尚支架。在RWV生物反应器迄今所使用的支架组成，除了少数例外16-18，主要用于合成微珠，管状圆筒或小片13-15,19-23的。这些是刚性的材料，其组成和灵活性不能被操纵，并且其中细胞附着到其表面上。因此，这是不可能的，这些模型将提供在其中评估系统，以综合的方式，（ 例如 ，成纤维细胞，免疫细胞和内皮细胞）的各种细胞成分如基质细胞那个SHOULD被支架内分散地模拟人体组织。 在这里，我们描述了一种肠上皮细胞系和原代人淋巴细胞，内皮细胞构成的人体肠道黏膜的多细胞三维器官模型的开发，和成纤维细胞24。这些细胞培养微重力下的生物反应器RWV提供13,25-30。在我们的3-D模型，则ECM具有许多不同的特性，例如类似培养基的重量摩尔渗透压浓度（ 例如 ，在培养过程中可以忽略的扩散限制），并掺入细胞和其它相关的细胞外基质蛋白的能力，以及在在生物反应器24中使用适当的刚度。生物系统是非常复杂的，而且在过去的几年里，一直在研究黏膜对与周围环境细胞的相互作用的考试的重点的转变，而不是隔离和研究它们ñ。特别是，细胞-细胞相互作用的影响肠细胞的存活和分化的重要性是有据可查的31-34。具体来说，上皮细胞和自己的优势之间的交流对上皮细胞扩增和分化35深远的影响。事实上，它已被广泛接受，不仅细胞 - 细胞，而且细胞 - 细胞外基质相互作用是上皮细胞中的3-D培养模型的维持和分化至关重要。以前的研究已经表明，肠ECM蛋白如胶原蛋白I 24,36,37，层粘连蛋白38和纤连蛋白39在影响肠上皮细胞获得类似于天然粘膜空间定向工具。因此，新技术的发展，象我们的3-D模型24，可以模仿所需肠道的表型多样性如果研究人员打算重新创建复杂的细胞和结构建筑和肠道微环境的功能。这些模型表示在新的口服药物和疫苗候选的开发和评估的一个重要工具。

<table><tbody><tr><td>Quad Rotator/Independent Rotating Wall Vessel (RWV) bioreactor&amp;nbsp;</td><td>Synthecon</td><td>RCCs-4DQ</td><td>For up to 4 vessels. Models with more or less vessels are also available.</td></tr><tr><td>Disposable 50 ml-vessel</td><td>Synthecon</td><td>D-405</td><td>Box with 4 vessels</td></tr><tr><td>HCT-8 epithelial cells&amp;nbsp;</td><td>ATCC</td><td>CCL-244</td><td></td></tr><tr><td>CCD-18Co Fibroblasts&amp;nbsp;</td><td>ATCC</td><td>CRL-1459</td><td></td></tr><tr><td>Human Umbilical Vein Endothelial Cells</td><td>ATCC</td><td>CRL-1730</td><td>HUVEC</td></tr><tr><td>Fibroblast Growth Factor-Basic&amp;nbsp;</td><td>Sigma</td><td>F0291</td><td>bFGF</td></tr><tr><td>Stem Cell Factor&amp;nbsp;</td><td>Sigma</td><td>S7901</td><td>SCF</td></tr><tr><td>Hepatocyte Growth Factor&amp;nbsp;</td><td>Sigma</td><td>H1404</td><td>HGF</td></tr><tr><td>Endothelin 3</td><td>Sigma</td><td>E9137</td><td></td></tr><tr><td>Laminin</td><td>Sigma</td><td>L2020</td><td>Isolated from mouse Engelbreth-Holm-Swarm tumor</td></tr><tr><td>Vascular Endothelial Growth Factor&amp;nbsp;</td><td>Sigma</td><td>V7259</td><td>VEGF</td></tr><tr><td>Leukemia Inhibitory Factor&amp;nbsp;</td><td>Santa Cruz</td><td>sc-4377</td><td>(LIF</td></tr><tr><td>Adenine</td><td>Sigma</td><td>A2786</td><td></td></tr><tr><td>Insulin</td><td>Sigma</td><td>I-6634</td><td></td></tr><tr><td>3,3&#039;,5-triiodo-L-thyronine&amp;nbsp;</td><td>Sigma</td><td>T-6397</td><td>T3</td></tr><tr><td>Cholera Toxin</td><td>Sigma</td><td>C-8052</td><td></td></tr><tr><td>Fibronectin</td><td>BD</td><td>354008</td><td>Isolated from human plasma</td></tr><tr><td>apo-Transferrin</td><td>Sigma</td><td>T-1147</td><td></td></tr><tr><td>Heparin</td><td>Sigma</td><td>H3149</td><td></td></tr><tr><td>Heparan sulfate&amp;nbsp; proteoglycan</td><td>Sigma</td><td>H4777</td><td>Isolated from basement membrane of mouse&amp;nbsp; Engelbreth-Holm-Swarm tumor</td></tr><tr><td>Collagen IV</td><td>Sigma</td><td>C5533</td><td>Isolated from human placenta</td></tr><tr><td>Heat-inactivated fetal bovine serum&amp;nbsp;</td><td>Invitrogen</td><td>10437-028</td><td></td></tr><tr><td>D-MEM, powder</td><td>Invitrogen</td><td>12800-017</td><td></td></tr><tr><td>10% formalin&amp;ndash;PBS&amp;nbsp;</td><td>Fisher Scientific</td><td>SF100-4</td><td></td></tr><tr><td>Bovine type I collagen&amp;nbsp;</td><td>Invitrogen</td><td>A1064401</td><td></td></tr><tr><td>Trypsin-EDTA&amp;nbsp;</td><td>Fisher Scientific</td><td>MT25-052-CI</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Invitrogen</td><td>11360-070</td><td></td></tr><tr><td>Gentamicin&amp;nbsp;</td><td>Invitrogen</td><td>15750-060</td><td></td></tr><tr><td>Penicillin/streptomincin&amp;nbsp;</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>L-Glutamine</td><td>Invitrogen</td><td>25030-081</td><td></td></tr><tr><td>Hepes</td><td>Invitrogen</td><td>15630-080</td><td></td></tr><tr><td>Ham&#039;s F-12</td><td>Invitrogen</td><td>11765-054</td><td></td></tr><tr><td>Basal Medium Eagle</td><td>Invitrogen</td><td>21010-046</td><td>BME</td></tr><tr><td>RPMI-1640</td><td>Invitrogen</td><td>11875-093</td><td></td></tr><tr><td>Endothelial Basal Medium</td><td>Lonza</td><td>CC-3156</td><td>EBM-2</td></tr><tr><td>Endothelial cell growth supplement</td><td>Millipore</td><td>02-102</td><td>ECGS</td></tr></tbody></table>

development of a multicellular three-dimensional organotypic model of the human intestinal mucosa grown under microgravity

因为在一个三维（3-D）的环境中生长的细胞具有弥合的2-D的环境中的细胞培养的许多间隙（ 例如 ，瓶或碗碟）的潜力。事实上，人们普遍认识到，在烧瓶或菜肴生长细胞倾向于去分化并失去其最初来源的组织的专门特征。目前，主要有两种类型的3-D培养系统的模拟天然细胞外基质（ECM），其中将细胞接种到支架:（一）静态模型和使用生物反应器（B）中的模型。第一个突破是静态的三维模型。使用生物反应器，如旋转壁上容器3-D模型（RWV）生物反应器是一个较新的发展。在RWV生物反应器的原始概念是美国宇航局约翰逊航天中心在90年代初开发的，被认为是克服静态模型的局限性，如缺氧，坏死核心的发展。该RWV生物反应器可以规避日是通过提供流体动力学，允许营养物和氧的高效的扩散问题。这些生物反应器组成，其用于支持和旋转培养容器的两个不同的格式，可以通过通气源类型而不同的转子碱:（1）缓慢车削外侧容器（STLVs）与在中心同轴氧合器，或（2通过一台，硅橡胶气体转移膜）高宽比容器（HARVs）与充氧。这些血管允许有效的气体传递，同时避免气泡形成和随之而来的湍流。这些条件导致在层流和最小的剪切力模型降低了培养容器内的重力（失重）。在这里，我们描述由RWV生物反应器提供了一种肠上皮细胞系和原代人淋巴细胞，微重​​力下培养的内皮细胞和成纤维细胞构成的人体肠道黏膜的多细胞三维器官模型的发展。&lt;/ P&gt;

<html>先前我们已经设计了人肠黏膜包括肠上皮细胞系和原代人淋巴细胞，微重力条件下24（ 图1）下培养的内皮细胞和成纤维细胞的多细胞三维器官模型。成纤维细胞和内皮细胞包埋在我矩阵附加肠基底膜蛋白45（ 即 ，层粘连蛋白，胶原蛋白IV，纤连蛋白和硫酸肝素蛋白聚糖）富含胶原，并加入到RWV生物反应器。经过10 - 15天，组织染色...

Watch this Scientific Journal Video about Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity at JoVE.com

Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity

细胞在三维（3-D）的环境中生长代表的2-D的环境中（ 例如 ，瓶或菜）在细胞培养的显着改进。这里，我们描述根据通过旋转壁上容器（RWV）生物反应器提供微重力培养人肠粘膜的多细胞三维器官模型的发展。

生长在微重力人体肠道黏膜的多细胞三维器官模型的建立

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., ...

development-multicellular-three-dimensional-organotypic-model-human

Research

JoVE Journal

Bioengineering

9.8K 次观看.  University of Maryland School of Medicine. 细胞在三维（3-D）的环境中生长代表的2-D的环境中（ 例如 ，瓶或菜）在细胞培养的显着改进。这里，我们描述根据通过旋转壁上容器（RWV）生物反应器提供微重力培养人肠粘膜的多细胞三维器官模型的发展。 

视频: Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity

Tissue Engineering of the Intestine in a Murine Model

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to preoperative weights within 40 days.1 In humans, massive small bowel resection can result in short bowel syndrome, a functional malabsorptive state that confers significant morbidity, mortality, and healthcare costs including parenteral nutrition dependence, liver failure and cirrhosis, and the need for multivisceral organ transplantation.2 In this paper, we describe and document our protocol for creating tissue-engineered intestine in a mouse model with a multicellular organoid units-on-scaffold approach. Organoid units are multicellular aggregates derived from the intestine that contain both mucosal and mesenchymal elements,3 the relationship between which preserves the intestinal stem cell niche.4 In ongoing and future research, the transition of our technique into the mouse will allow for investigation of the processes involved during TESI formation by utilizing the transgenic tools available in this species.5The availability of immunocompromised mouse strains will also permit us to apply the technique to human intestinal tissue and optimize the formation of human TESI as a mouse xenograft before its transition into humans. Our method employs good manufacturing practice (GMP) reagents and materials that have already been approved for use in human patients, and therefore offers a significant advantage over approaches that rely upon decellularized animal tissues. The ultimate goal of this method is its translation to humans as a regenerative medicine therapeutic strategy for short bowel syndrome.

This article and the accompanying video present our protocol for generating tissue-engineered intestine in the mouse, using an organoid units-on-scaffold approach.

小鼠模型肠组织工程

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to ...

科研

生物工程

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of the crypt region. IESCs include active stem cells, which self-renew and differentiate into various epithelial cell types, and quiescent stem cells, which serve as the reserve stem cells in the case of injury. Regeneration of the intestinal epithelium is controlled by the self-renewing and differentiating capabilities of these active IESCs. In addition, the balance of the crypt stem cell population and maintenance of the stem cell niche are essential for intestinal regeneration. Organoid culture is an important and attractive approach to studying proteins, signaling molecules, and environmental cues that regulate stem cell survival and functions. This model is less expensive, less time-consuming, and more manipulatable than animal models. Organoids also mimic the tissue microenvironment, providing in vivo relevance. The present protocol describes the isolation of colonic crypts, embedding these isolated crypt cells into a three-dimensional gel matrix system and culturing crypt cells to form colonic organoids capable of self-organization, proliferation, self-renewal, and differentiation. This model allows one to manipulate the environment-knocking out specific proteins such as claudin-7, activating/deactivating signaling pathways, etc.-to study how these effects influence the functioning of colonic stem cells. Specifically, the role of tight junction protein claudin-7 in colonic stem cell function was examined. Claudin-7 is vital for maintaining intestinal homeostasis and barrier function and integrity. Knockout of claudin-7 in mice induces an inflammatory bowel disease-like phenotype exhibiting intestinal inflammation, epithelial hyperplasia, weight loss, mucosal ulcerations, epithelial cell sloughing, and adenomas. Previously, it was reported that claudin-7 is required for intestinal epithelial stem cell functions in the small intestine. In this protocol, a colonic organoid culture system is established to study the role of claudin-7 in the large intestine.

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

The present protocol describes establishing a murine colonic organoid system to study the activity and functioning of colonic stem cells in a claudin-7 knockout model.

小鼠结肠隐窝三维培养离体研究肠道干细胞功能

The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of ...

癌症研究

Innervation of Human Intestinal Organoids

The complexity of intestinal cytoarchitecture and function poses significant challenges for the creation of the bioengineered small intestine. Techniques for generating human intestinal organoids (HIOs) resembling human small intestine have been previously reported. HIOs contain epithelium and mesenchyme but lack other critical components of functional intestine such as the enteric nervous system (ENS), immune cells, vasculature, and microbiome. Two independent research groups have published distinct methods to innervate HIOs with an ENS. Here we discuss a unique method of incorporating the ENS into an HIO-derived bioengineered small intestine, which utilizes components of these prior reports to optimize progenitor cell identity as well as developmental timing.
Human pluripotent stem cells (hPSCs) are differentiated to independently generate HIOs and enteric neural crest cells (ENCCs) by temporal regulation of differentiation markers over a period of several days per published protocols. Once HIOs reach the mid-hindgut spheroid stage (approximately day 8), day 15-21 ENCC spheroids are dissociated, co-cultured with HIOs, and suspended within clear three-dimensional (3D) basement membrane matrix droplets. HIO + ENCC co-cultures are maintained in vitro for 28-40 days before transplantation into &#62;9-week-old immunodeficient mice for further development and maturation. Transplanted HIOs (tHIOs) with ENS can be harvested 4-20 weeks later. This method integrates elements from two previously published techniques by utilizing ENCCs generated from hPSCs and co-culturing them with HIOs at an early stage of development to maximize exposure to early developmental cues that likely contribute to the formation of a more mature intestinal morphology.

Human intestinal organoids must be innervated to better recapitulate the structure and function of the native human intestine. Here, we present one method for incorporating an enteric nervous system into these constructs.

人肠道类器官的神经支配

The complexity of intestinal cytoarchitecture and function poses significant challenges for the creation of the bioengineered small intestine. Techniques ...

发育生物学

Combining Human Organoids and Organ-on-a-Chip Technology to Model Intestinal Region-Specific Functionality

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, and metabolism of nutrients and xenobiotics while facilitating a symbiotic relationship with microbiota and restricting the invasion of microorganisms. Functional interaction between various cell types and their physical and biochemical environment is vital to establish and maintain intestinal tissue homeostasis. Modeling these complex interactions and integrated intestinal physiology in vitro is a formidable goal with the potential to transform the way new therapeutic targets and drug candidates are discovered and developed.
Organoids and Organ-on-a-Chip technologies have recently been combined to generate human-relevant intestine chips suitable for studying the functional aspects of intestinal physiology and pathophysiology in vitro. Organoids derived from the biopsies of the small (duodenum) and large intestine are seeded into the top compartment of an organ chip and then successfully expand&#160;as monolayers while preserving the distinct cellular, molecular, and functional features of each intestinal region. Human intestine tissue-specific microvascular endothelial cells are incorporated in the bottom compartment of the organ chip to recreate the epithelial-endothelial interface. This novel platform facilitates luminal exposure to nutrients, drugs, and microorganisms, enabling studies of intestinal transport, permeability, and host-microbe interactions.
Here, a detailed protocol is provided for the establishment of intestine chips representing the human duodenum (duodenum chip) and colon (colon chip), and their subsequent culture under continuous flow and peristalsis-like deformations. We demonstrate methods for assessing drug metabolism and CYP3A4 induction in duodenum chip using prototypical inducers and substrates. Lastly, we provide a step-by-step procedure&#160;for the in vitro modeling of interferon gamma (IFN&#947;)-mediated barrier disruption (leaky gut syndrome) in a colon chip, including methods for evaluating the alteration of paracellular permeability, changes in cytokine secretion, and transcriptomic profiling of the cells within the chip.

Biopsy-derived intestinal organoids and organ-on-a-chips technologies are combined into a microphysiological platform to recapitulate region-specific intestinal functionality.

结合人体类器官和芯片器官技术，对肠道区域特异性功能进行建模

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, ...

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

老鼠胎儿整肠文化系统<em&gt;前体内</em&gt;信号通路和绒毛开发的三维实时成像的操作

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

生物学

在犬 IBD Gut-on-a-Chip 系统中建立 3D 形态发生

Three-Dimensional Morphogenesis in Canine Gut-on-a-Chip Using Intestinal Organoids Derived from Inflammatory Bowel Disease Patients

Canine intestines possess similarities in anatomy, microbiology, and physiology to those of humans, and dogs naturally develop spontaneous intestinal disorders similar to humans. Overcoming the inherent limitation of three-dimensional (3D) organoids in accessing the apical surface of the intestinal epithelium has led to the generation of two-dimensional (2D) monolayer cultures, which expose the accessible luminal surface using cells derived from the organoids. The integration of these organoids and organoid-derived monolayer cultures into a microfluidic Gut-on-a-Chip system has further evolved the technology, allowing for the development of more physiologically relevant dynamic in vitro intestinal models.
In this study, we present a protocol for generating 3D morphogenesis of canine intestinal epithelium using primary intestinal tissue samples obtained from dogs affected by inflammatory bowel disease (IBD). We also outline a protocol for generating and maintaining 2D monolayer cultures and intestine-on-a-chip systems using cells derived from the 3D intestinal organoids. The protocols presented in this study serve as a foundational framework for establishing a microfluidic Gut-on-a-Chip system specifically designed for canines. By laying the groundwork for this innovative approach, we aim to expand the application of these techniques in biomedical and translational research, aligning with the principles of the One Health Initiative. By utilizing this approach, we can develop more physiologically relevant dynamic in vitro models for studying intestinal physiology in both dogs and humans. This has significant implications for biomedical and pharmaceutical applications, as it can aid in the development of more effective treatments for intestinal diseases in both species.

作者聚焦：犬 IBD Gut-on-a-Chip 模型的开发和应用，用于 3D 肠道形态发生研究 

Integration of canine intestinal organoids and a Gut-on-a-Chip microfluidic system offers relevant translational models for human intestinal diseases. Protocols presented allow for 3D morphogenesis and dynamic in vitro modeling of the gut, aiding in the development of effective treatments for intestinal diseases in dogs and humans with One Health.

使用炎症性肠病患者肠道类器官在犬肠道芯片中的三维形态发生

Canine intestines possess similarities in anatomy, microbiology, and physiology to those of humans, and dogs naturally develop spontaneous intestinal ...

医学

3D Culturing of Organoids from the Intestinal Villi Epithelium Undergoing Dedifferentiation

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is compartmentalized into crypts and villi: the stem and proliferating cells are confined to the crypts, whereas the villi epithelium contains only differentiated cells. Hence, the normal intestinal crypts, but not the villi, can give rise to organoids in 3D cultures. The procedure described here is applicable only to villus epithelium undergoing dedifferentiation leading to stemness. The method described uses the Smad4-loss-of-function:&#946;-catenin gain-of-function (Smad4KO:&#946;-cateninGOF) conditional mutant mouse. The mutation causes the intestinal villi to dedifferentiate and generate stem cells in the villi. Intestinal villi undergoing dedifferentiation are scraped off the intestine using glass slides, placed in a 70 &#181;m strainer and washed several times to filter out any loose cells or crypts prior to plating in BME-R1 matrix to determine their organoid-forming potential. Two main criteria were used to ensure that the resulting organoids were developed from the dedifferentiating villus compartment and not from the crypts: 1) microscopically evaluating&#160;the isolated villi to ensure absence of any tethered crypts, both before and after plating in the 3D matrix, and 2) monitoring the time course of organoid development from the villi. Organoid initiation from the villi occurs only two to five days after plating and appears irregularly shaped, whereas the crypt-derived organoids from the same intestinal epithelium are apparent within sixteen hours of plating and appear spherical. The limitation of the method, however, is that the number of organoids formed, and the time required for organoid initiation from the villi vary depending on the degree of dedifferentiation. Hence, depending upon the specificity of the mutation or the insult causing the dedifferentiation, the optimal stage at which villi can be harvested to assay their organoid forming potential, must be determined empirically.

The procedure describes isolation of the villi from the mouse intestinal epithelium undergoing dedifferentiation to determine their organoid forming potential.

肠道维利表皮的器官3D培养进行分化

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is ...

小鼠肠隐窝和单干细胞类器官的 3D 培养

The 3D Culturing of Organoids from Murine Intestinal Crypts and a Single Stem Cell for Organoid Research

At present, organoid culture represents an important tool for in vitro studies of different biological aspects and diseases in different organs. Murine small intestinal crypts can form organoids that mimic the intestinal epithelium when cultured in a 3D extracellular matrix. The organoids are composed of all cell types that fulfill various intestinal homeostatic functions. These include Paneth cells, enteroendocrine cells, enterocytes, goblet cells, and tuft cells. Well-characterized molecules are added into the culture medium to enrich the intestinal stem cells (ISCs) labeled with leucine-rich repeats containing G protein-coupled receptor 5 and are used to drive differentiation down specific lineages; these molecules include epidermal growth factor, Noggin (a bone morphogenetic protein), and R-spondin 1. Additionally, a protocol to generate organoids from a single erythropoietin-producing hepatocellular receptor B2 (EphB2)-positive ISC is also detailed. In this methods article, techniques to isolate small intestinal crypts and a single ISC from tissues and ensure the efficient establishment of organoids are described.

作者聚焦：来自小鼠肠隐窝的类器官的 3D 培养和用于类器官研究的单个干细胞  

We describe a protocol to isolate murine small intestinal crypts and culture intestinal 3D organoids from the crypts. Additionally, we describe a method to generate organoids from a single intestinal stem cell in the absence of a sub-epithelial cellular niche.

小鼠肠隐窝和单个干细胞的类器官的3D培养用于类器官研究

At present, organoid culture represents an important tool for in vitro studies of different biological aspects and diseases in different organs. Murine ...

生长在微重力人体肠道黏膜的多细胞三维器官模型的建立

摘要

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此视频中的章节

小鼠模型肠组织工程

小鼠结肠隐窝三维培养离体研究肠道干细胞功能

人肠道类器官的神经支配

结合人体类器官和芯片器官技术，对肠道区域特异性功能进行建模

老鼠胎儿整肠文化系统

使用炎症性肠病患者肠道类器官在犬肠道芯片中的三维形态发生

使用炎症性肠病患者肠道类器官在犬肠道芯片中的三维形态发生

使用炎症性肠病患者肠道类器官在犬肠道芯片中的三维形态发生

肠道维利表皮的器官3D培养进行分化

小鼠肠隐窝和单个干细胞的类器官的3D培养用于类器官研究