Name: the ex vivo colon organ culture and its use in antimicrobial host defense studies
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癌症预防和得克萨斯州的研究所（CPRIT; RP160169）和德克萨斯大学西南医学中心给MHZ，这项工作是由来自克隆氏病和美国结肠炎基金会（CCFA 3711）资金支持

此处描述的所有实验使用维持在无特定病原体（SPF）在动物资源中心（ARC），德克萨斯大学西南医学中心设施6-8周龄雄性野生型（C57BL6 / J）的小鼠中进行。所有研究机构动物护理和使用委员会（IACUC）批准，并按照IACUC的指导方针和健康指南实验动物的护理和使用国家研究院进行的。 1.收集和科隆的制备<ol><li>安乐死用CO 2窒息颈椎脱位的小鼠。 </li><li>喷雾用70％乙醇和小鼠四肢钉到钉板保持鼠标背面向下在黑板上。 </li><li>使用预先灭菌的解剖剪刀和镊子，使得一个中线切口在腹部的腹膜内。通过折叠钳腹膜打开腹部。 </li><li>从腹腔无线除去肠日解剖镊子和剪刀。通过在盲肠的底部，并在直肠的另一端切割分离从小肠结肠。 </li><li>放置在含有冰冷的PBS的无菌培养皿结肠。冲洗用冰 - 冷PBS结肠的腔的用20毫升的注射器保持20G的针或口服灌胃针直至结肠管腔内的大便被完全去除的内容。 </li><li>用剪刀剪纵向结肠。通过在无菌培养皿中冰冷的PBS剧烈摇动洗结肠。 </li><li>切结肠组织成片长大约1厘米长用无菌手术刀或剪刀。 </li><li>记录结肠件的重量。 注:在第1节的所有步骤都可以内部或生物安全柜外进行。一旦冒号准备好培养，所有的步骤必须在生物安全柜内进行，以保持无菌。 </li></ol> 2.结肠组织一个文化<ol><li>放置细胞过滤网（100微米）上的6孔细胞培养板。转让所有从一个鼠标收集到的细胞过滤结肠件。 </li><li>添加含5％FBS，青霉素 - 链霉素（1×），和庆大霉素（20微克/毫升）5毫升DMEM / F12培养基。完全覆盖结肠件与媒体。 </li><li>在5％的CO 2和95％空气的培养箱孵育2小时，在37℃。 </li><li>提起细胞过滤器和吸媒体。 </li><li>放置在细胞过滤回井，加入5毫升的新鲜媒体无任何抗生素。提起细胞过滤器和吸媒体。 </li><li>重复上述洗涤步骤（步骤2.5）两次（总共3次）以除去所有的残留抗生素。 </li><li>传送从单细胞过滤结肠件到无菌的12孔细胞培养板的单个孔。 </li><li>添加含5％FBS（没有任何抗生素）的DMEM / F12培养基。调整培养基的体积与weig结肠件HT， 例如 ，100 mg组织1毫升网上平台。在注射5％CO 2和95％空气的培养箱孵育12小时，在37℃。 </li><li>收集培养物上清液在一个无菌1.5毫升管中。 </li><li>离心机在4℃下12000×g离心5分钟。分离上清液到新的1.5毫升试管中的细菌使用杀伤实验和/或其它免疫测定法。上清液可以储存在-80℃直到测定。 </li><li>收集在用于RNA分离的管结肠件。 </li></ol> 3. 大肠杆菌的杀灭试验<ol><li>接种大肠杆菌在5mL的Luria-BERTANI（LB）肉汤中15毫升管中并在37℃以200rpm振荡过夜孵育。保持文化管帽略显宽松。 </li><li>离心细菌培养管中，在1200 xg离心在4℃下10分钟。除去上清液和重悬细菌沉淀到5毫升冰冷的PBS。 </li><li>转移1毫升BACT的erial悬挂成600nm的比色皿和测量OD。使用PBS作为空白。 </li><li>计算使用预定标准曲线的菌落形成单位（CFU）。这里，假设1 OD = 2×10 9 CFU /毫升（约）。 </li><li>稀释细菌悬浮液，使纸浆悬浮液1×10 5个/毫升。 </li><li>转移结肠器官培养上清液（500微升/孔）收集在第2.10节到24孔细胞培养板的两个孔。 </li><li>添加10μL的大肠杆菌培养物（1,000 CFU）成一个孔含有500微升结肠器官培养上清液。离开其它公没有任何大肠杆菌接种，以确认该结肠器官培养上清液中不包含任何污染。 </li><li>孵育培养基（DMEM / F12加上5％FBS）中的相同数量的细菌（1,000 CFU）而没有任何抗生素作为对照。 </li><li>孵育在37℃下1小时。 </li><li>将每个样本下拉式无线的50微升SE在麦康凯琼脂平板上。在37℃下孵育MacConkey琼脂平板过夜。 </li><li>计数菌落数并计算CFU /毫升。 </li></ol> 4.外在和内在因素对结肠抗菌宿主防御反应的影响注:抗微生物杀灭试验如这里所描述，可以通过检查的病原相关分子模式（PAMP）和细胞因子对器官培养物上清液的杀菌活性的影响。用IL-1β和IL-18这样的实验的一个例子如下所述。 <ol><li>如第1节收集小鼠冒号。 </li><li>放置在无菌纸巾结肠。纵向切开结肠成使用剪刀（ 图2）三个部分。 </li><li>如在第1节中所述洗冰冷PBS结肠的每个部分。 </li><li>切大肠切成小块的每一个部分和无菌权衡。传输各部分的片的结肠成放置在三口井6孔细胞培养板（ 图2）三个独立的细胞过滤器（100微米）。 </li><li>加入2毫升含5％FBS，青霉素 - 链霉素（1×），和庆大霉素（20微克/毫升）的DMEM / F12培养基。 </li><li>在注射5％CO 2和95％空气的培养箱孵育2小时，在37℃。 </li><li>如在2.4-2.6描述洗结肠件。传输单个细胞过滤的结肠件到无菌的12孔细胞培养板的单个孔。含有从单个小鼠的结肠的三个部分的三个孔应被指定为未处理，IL-1β和IL-18（ 图2）。 </li><li>添加含5％FBS（没有任何抗生素）的DMEM / F12培养基。调整培养基的体积与重量的结肠件， 例如 ，用于100 mg组织的1毫升介质。 </li><li>刺激用IL-1β（20毫微克/毫升）或IL-18（20毫微克/毫升）的结肠器官培养12小时。未处理的冒号<html>器官培养作为对照。 </li><li>在注射5％CO 2和95％空气培养箱中在37℃12小时培养后，收集培养物上清液中的无菌1.5mL管中。 </li><li>转移500μL培养上清于24孔板的一式两份孔中。 </li><li>接种大肠杆菌在结肠癌器官培养物上清液的大肠杆菌 （1,000 CFU）如第3.对于每个条件描述的，接种大肠杆菌在单个井。无大肠杆菌另一孔含有相同的器官培养物上清液将作为细菌污染的控制。 </li><li>孵化培养基中的细菌相同量没有任何抗生素作为对照。 </li><li>孵育在37℃下1小时。 </li><li>将每个样品滴在麦康凯琼脂平板50微升。孵育MacConkey琼脂平板过夜，在37℃。 </li><li>计数菌落数并计算CFU /毫升（ 图4）。 </li></ol>e_title"&gt; 5。抗菌基因表达的测量<ol><li>结肠器官培养的过夜培养（12小时）后，如在第2和4所描述，用2ml冰冷的PBS（2×）洗结肠件。 </li><li>收集结肠片成2毫升RNA酶/ DNA酶无螺丝帽筒。放置管在冰上。 </li><li>加入1毫升的商业Trizol试剂和裂解矩阵珠入管。 </li><li>裂解用自动组织匀浆的组织。 </li><li>收集组织溶解液到一个新的离心管中。 </li><li>使用标准协议分离RNA。 </li><li>测量RNA浓度。 </li><li>用去离子水适当稀释的RNA，并使用500ng的RNA合成cDNA。 </li><li>使用有针对性的抗菌基因，细胞因子，趋化因子和其他感兴趣的基因的实时RT-PCR分析的基因。 </li></ol>

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The authors have no competing financial interests.

肠上皮细胞在它们的生长方面的要求非常敏感，因此，难以培养。用EDTA处理分离的上皮细胞不以常规的细胞培养基如DMEM 8生存。因此，使用隔离隐窝或主要上皮细胞宿主 - 病原体相互作用的研究是非常具有挑战性的。最近，Sato等人。描述的地穴化培养系统，这是非常有前途的和有用的相关肠道病理生理学研究13。而组织体代表肠隐窝的许多特征，有人担心作为...

肠道代表充当共生微生物屏障的动态系统，地对抗入侵的病原体，并调节微生物组成1。肠上皮细胞，包括肠，杯状细胞，帕内特细胞和肠内分泌细胞，是主要的细胞群提供对抗肠道微生物的宿主防御反应。杯状细胞产生上创建上皮层2的顶部上的非军事区的粘蛋白。的帕内特细胞和肠上皮产生的抗微生物肽，细胞因子，和构成抗微生物宿主防御反应和有助于形成肠道微生物组合物3,4活性氧和氮物种。除了上皮细胞，免疫细胞，包括巨噬细胞，树突细胞，嗜中性粒细胞，自然杀伤细胞，淋巴细胞和茵<html> 7 -固有层和粘膜下层德淋巴样细胞通过产生细胞因子，趋化因子和其他介质5发挥肠道抗菌宿主防御反应的关键作用。为了了解粘膜免疫系统如何监管和微生物提供了对微生物感染的保护，应考虑肠道的异质细胞群的复杂的相互作用是非常重要的。但是，包括所有的肠的功能的体外模型是不可用的。因此，在肠道内的宿主 - 病原体相互作用的分子研究是极具挑战性的。 在过去的几年中，模仿肠粘膜的各方面的几个模型系统已被开发用于研究参与炎症性肠疾病（IBD）和其它胃肠道病症8的病理生理过程-="外部参照"&gt; 14。永生化肠上皮细胞系通常用于研究上皮细胞特异性应答。然而，由于差异基因表达和永生化细胞的功能，从利用这些细胞不经常匹配与那些在体内研究观察到的获得的数据。肠隐窝化培养最近出现作为评估的肠上皮细胞对不同刺激13的反应的潜在工具。在这个系统中，隐窝干细胞被允许生长和发展三维类器官结构。而类器官培养系统是用于研究肠上皮的许多方面非常有用的，它不模仿免疫细胞，上皮细胞和微生物产品的复杂的相互作用。肠组织的体外培养提供体内宿主防御反应的一个更好的代表性。在该方法中，小肠的部分是在一个细胞培养板机智培养ħ适当的媒体，允许不同类型的肠细胞群是代谢活性为至少48小时。因此，器官的离体培养，可以用于测量抗微生物的基因的表达和肠道的特定刺激宿主防御反应。 调查人员一直在使用体外器官培养系统来研究肠道对15微生物感染的宿主防御反应- 21。我们最近通过了器官培养系统研究抗菌宿主防御反应的炎性体在小鼠结肠22的作用。炎性为胱天蛋白酶-1的激活，这是需要用于生产成熟的IL-1β和IL-18的分子的平台。我们发现，IL-1β和IL-18诱导的抗微生物肽，有效地杀死共生pathobionts诸如大肠杆菌 </em&gt;。此观察与炎性缺陷小鼠结肠22增加大肠杆菌的负担是一致的。该系统因此可以被用来研究如炎性肠病（IBD）和结肠直肠癌（CRC）的模式识别受体（的PRRs）等天然免疫分子在肠道抗菌宿主防御反应肠道疾病中的作用，以及发病。有超过200的IBD易感性基因，突变在许多这些基因与在肠道中改变的微生物组合物相关联。这是重要的临床意义，以确定通过其IBD易感基因调节肠道菌群的确切机制。该方法的总体目标是引进体外结肠器官培养的基本协议并展示如何此培养方法可用于研究肠的抗微生物宿主防御反应。

<table><tbody><tr><td>Advanced DMEM/ F12</td><td>Life Technologies</td><td>12634-010</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate buffered saline, modified, w/o Calcium chloride &amp;amp; Magnesium chloride</td><td>Sigma</td><td>5634</td><td></td></tr><tr><td>FBS, heat inactivated</td><td>Sigma</td><td>F4135</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Life Technologies</td><td>15070063</td><td></td></tr><tr><td>Gentamicin solution</td><td>Sigma</td><td>G1272</td><td></td></tr><tr><td>Mouse IL-1b recombinan</td><td>Reprokine</td><td>RKP10749</td><td></td></tr><tr><td>Mouse IL-18 recombinant</td><td>Reprokine</td><td>RKP70380</td><td></td></tr><tr><td>TRIzol Reagent</td><td>Thermo Fisher Scientific</td><td>15596018</td><td></td></tr><tr><td>Difco Luria-Bertani Broth&amp;nbsp;</td><td>BD Bioscience</td><td>244620</td><td></td></tr><tr><td>BD&amp;nbsp;Difco Dehydrated Culture Media: MacConkey Agar</td><td>Fisher Scientific</td><td>DF0075-17-1</td><td></td></tr><tr><td>NanoDrop 1000 Spectrophotometer</td><td>Thermo Scientific</td><td></td><td>Uded to measure RNA concentration</td></tr><tr><td>UV/Vis Spectrophotometer</td><td>BECKMAN</td><td>DU 530</td><td>Used to determine E. coli count</td></tr><tr><td>iScript RT Supermix, 100 rxns</td><td>Bio-Rad</td><td>1708841</td><td></td></tr><tr><td>iTaq Univer SYBR Green Supermix&amp;nbsp;</td><td>Bio-Rad</td><td>1725125&amp;nbsp;</td><td></td></tr><tr><td>Lysing Matrix S (1/8&quot;), 2 ml Tube</td><td>MP Biomedicals</td><td>116925500</td><td>Used to homgenize colon organ for RNA isolation</td></tr><tr><td>FastPrep-24 5G System</td><td>Bio-Rad</td><td>116005500</td><td></td></tr><tr><td>100 mm x 15 mm Petri Dish</td><td>Falcon</td><td>5687</td><td></td></tr><tr><td>Plate 6 well ps TC CS100, Cellstar, 6w, tc, F-bottom (Flat), w/lid, sterile</td><td>Cellstar</td><td>5085</td><td></td></tr><tr><td>100 &amp;mu;m cell strainer</td><td>Falcon</td><td>5698</td><td></td></tr><tr><td>Sorvall Legend Micro 21R Centrifuge</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr><tr><td>Sorvall ST40R Centrifuge</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr><tr><td>Forma Scientific orbital shaker</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr></tbody></table>

the ex vivo colon organ culture and its use in antimicrobial host defense studies

肠道显示由不同类型的上皮细胞，含有免疫细胞薄层propia，和基质的重复隐窝结构的架构。所有这些异构细胞有助于肠道内环境稳定，参与抗菌宿主防御。因此，确定了在体外环境研究免疫反应和肠的抗微生物活性的替代模型是极具挑战性。 体外研究使用永生化肠上皮细胞系或甚至小学隐窝化培养并不代表正常的肠道和微环境的确切生理学。在这里，我们在培养皿以及如何体外器官培养系统可以在与抗微生物宿主防御反应的研究来实现讨论培养小鼠结肠组织的方法。在代表的实验中，我们发现，在器官培养冒号表达RESP抗菌肽ONSE外源性的IL-1β和IL-18。此外，通过在器官培养的结肠组织中产生抗菌效应分子有效地杀灭体外大肠杆菌 。这种方法，因此，可以利用解剖在调节肠道先天免疫反应和抗微生物宿主防御反应的病原体和危险相关的分子模式和它们的细胞受体的作用。

在器官培养冒号的代表性图片示于图1。结肠件在培养保持代谢和生理活性。它们有效地应对加入到培养基外源刺激。结肠组织进行体外培养，并用外源性刺激的刺激， 例如 ，IL-1β和IL-18的制备的示意工作流程，示于图2。在图3中显示，在器官培养冒号表达的抗微生物肽，例如代表数据β防御2（BD2），Reg3γ，S100A8 S100A9，?...

Watch this Scientific Journal Video about The Ex Vivo Colon Organ Culture and Its Use in Antimicrobial Host Defense Studies at JoVE.com

The Ex Vivo Colon Organ Culture and Its Use in Antimicrobial Host Defense Studies

The ex vivo organ culture allows investigation of biological processes in the context of the intact tissue architecture. Here, we introduce a method of ex vivo culture of the mouse colon, which can be used to study innate immunity and antimicrobial host defense in the intestine.

该<em&gt;离体</em&gt;结肠器官培养及使用抗菌宿主防御研究

The intestine displays an architecture of repetitive crypt structures consisting of different types of epithelial cells, lamina propia containing immune ...

the-ex-vivo-colon-organ-culture-its-use-antimicrobial-host-defense

Research

JoVE Journal

Immunology and Infection

The Ex Vivo Colon Organ Culture and Its Use in Antimicrobial Host Defense Studies

14.0K 次观看.  UT Southwestern Medical Center. The ex vivo organ culture allows investigation of biological processes in the context of the intact tissue architecture. Here, we introduce a method of ex vivo culture of the mouse colon, which can be used to study innate immunity and antimicrobial host defense in the intestine. 

视频: The Ex Vivo Colon Organ Culture and Its Use in Antimicrobial Host Defense Studies

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the gastrointestinal tract, as resident microbiota occurs and prevents colonisation by exogenous bacteria. Indeed, more than 600 species of bacteria are found in the oral cavity, and a single individual may carry around 100 different at any time. Oral bacteria possess the ability to adhere to the various niches in the oral ecosystem, thus becoming integrated within the resident microbial communities, and favouring growth and survival. However, the flow of bacteria into the gut during swallowing has been proposed to disturb the balance of the gut microbiota. In fact, oral administration of P. gingivalis shifted bacterial composition in the ileal microflora. We used a synthetic community as a simplified representation of the natural oral ecosystem, to elucidate the survival and viability of oral bacteria subjected to simulated gastrointestinal transit conditions. Fourteen species were selected, subjected to in vitro salivary, gastric, and intestinal digestion processes, and presented to a multicompartment cell model containing Caco-2 and HT29-MTX cells to simulate the gut mucosal epithelium. This model served to unravel the impact of swallowed bacteria on cells involved in the enterohepatic circulation. Using synthetic communities allows for controllability and reproducibility. Thus, this methodology can be adapted to assess pathogen viability and subsequent inflammation-associated changes, colonization capacity of probiotic mixtures, and ultimately, potential bacterial impact on the presystemic circulation.

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

Gut host-microbe interactions were assessed using a novel approach combining a synthetic oral community, in vitro gastrointestinal digestion, and a model of the small intestine epithelium. We present a method that can be adapted to evaluate cell invasion of pathogens and multi-species biofilms, or even to test probiotic formulations&#39; survivability.

体外肠道宿主-微生物界面合成细菌联合的可行性评价

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the ...

科研

生物化学

In vitro Organoid Culture of Primary Mouse Colon Tumors

Several human and murine colon cancer cell lines have been established, physiologic integrity of colon tumors such as multiple cell layers, basal-apical polarity, ability to differentiate, and anoikis are not maintained in colon cancer derived cell lines. The present study demonstrates a method for culturing primary mouse colon tumor organoids adapted from Sato T et al. 1, which retains important physiologic features of colon tumors. This method consists of mouse colon tumor tissue collection, adjacent normal colon epithelium dissociation, colon tumor cells digestion into single cells, embedding colon tumor cells into matrigel, and selective culture based on the principle that tumor cells maintain growth on limiting nutrient conditions compared to normal epithelial cells.
The primary tumor organoids if isolated from genetically modified mice provide a very useful system to assess tumor autonomous function of specific genes. Moreover, the tumor organoids are amenable to genetic manipulation by virus meditated gene delivery; therefore signaling pathways involved in the colon tumorigenesis could also be extensively investigated by overexpression or knockdown. Primary tumor organoids culture provides a physiologic relevant and feasible means to study the mechanisms and therapeutic modalities for colon tumorigenesis.

In vitro Organoid Culture of Primary Mouse Colon Tumors

A simple method to establish primary murine colon tumor organoid is described. This method utilizes the feature that colon tumor cells survive and grow into organoids in media containing limited growth factors, whereas normal colon epithelial do not.

在体外化培养基层小鼠结肠肿瘤

Several  human and murine colon cancer cell lines have been established, physiologic  integrity of colon tumors such as multiple cell layers, basal-apical ...

医学

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

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Here, a protocol to harvest, maintain, and treat mouse small intestinal organoids with pathogen associated molecular patterns (PAMPs) and Listeria monocytogenes is described, as well as emphasis on gene expression and proper normalization techniques for protein.

该<em&gt;离体</em&gt;文化与模式识别小鼠肠道组织体受体刺激

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the ...

免疫与感染

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 ...

A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper homeostasis directly depends on these interactions, as they shape an entire immunological response inducing tolerance against food antigens while at the same time mounting effective immune responses against pathogenic microbes accidentally ingested with food.
Intestinal homeostasis is preserved also through various complex interactions between the microbiota (including food-associated beneficial bacterial strains) and the host, that regulate the attachment/degradation of mucus, the production of antimicrobial peptides by the epithelial barrier, and the "education" of epithelial cells' that controls the tolerogenic or immunogenic phenotype of unique, gut-resident lymphoid cells' populations. These interactions have been so far very difficult to reproduce with in vitro assays using either cultured cell lines or peripheral blood mononuclear cells. In addition, mouse models differ substantially in components of the intestinal mucosa (mucus layer organization, commensal bacteria community) with respect to the human gut. Thus, studies of a variety of treatments to be brought in the clinics for important stress-related or pathological conditions such as irritable bowel syndrome, inflammatory bowel disease or colorectal cancer have been difficult to carry out.
To address these issues, we developed a novel system that enables us to stimulate explants of human intestinal mucosa that retain their in situ conditioning by the host microbiota and immune response, in a polarized fashion. Polarized apical stimulation is of great importance for the outcome of the elicited immune response. It has been repeatedly shown that the same stimuli can produce completely different responses when they bypass the apical face of the intestinal epithelium, stimulating epithelial cells basolaterally or coming into direct contact with lamina propria components, switching the phenotype from tolerogenic to immunogenic and causing unnecessary and excessive inflammation in the area.
We achieved polarized stimulation by gluing a cave cylinder which delimited the area of stimulation on the apical face of the mucosa as will be described in the protocol. We used this model to examine, among others, differential effects of three different Lactobacilli strains. We show that this model system is very powerful to assess the immunomodulatory properties of probiotics in healthy and disease conditions.

We introduce a novel method for the maintenance of human intestinal mucosa in culture and monitoring of the response to various types of stimuli over at least 24 hrs. With our method, the polarity of the tissue is maintained, allowing for a physiological stimulation via the apical route.

文化与偏振刺激人体肠道黏膜植的新方法

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper ...

生物学

An Intestinal Gut Organ Culture System for Analyzing Host-Microbiota Interactions

The structure of the gut tissue facilitates close and mutualistic interactions between the host and the gut microbiota. These cross-talks are crucial for maintaining local and systemic homeostasis; changes to gut microbiota composition (dysbiosis) associate with a wide array of human diseases. Methods for dissecting host-microbiota interactions encompass an inherent tradeoff among preservation of physiological tissue structure (when using in vivo animal models) and the level of control over the experiment factors (as in simple in vitro cell culture systems). To address this tradeoff, Yissachar et al. recently developed an intestinal organ culture system. The system preserves a naive colon tissue construction and cellular mechanisms and it also permits tight experimental control, facilitating experimentations that cannot be readily performed in vivo. It is optimal for dissecting short-term responses of various gut components (such as epithelial, immunological and neuronal elements) to luminal perturbations (including anaerobic or aerobic microbes, whole microbiota samples from mice or humans, drugs and metabolites). Here, we present a detailed description of an optimized protocol for organ culture of multiple gut fragments using a custom-made gut culture device. Host responses to luminal perturbations can be visualized by immunofluorescence staining of tissue sections or whole-mount tissue fragments, fluorescence in-situ hybridization (FISH), or time-lapse imaging. This system supports a wide array of readouts, including next-generation sequencing, flow cytometry, and various cellular and biochemical assays. Overall, this three-dimensional organ culture system supports the culture of large, intact intestinal tissues and has broad applications for high-resolution analysis and visualization of host-microbiota interactions in the local gut environment.

This article presents a unique method for analyzing host-microbiome interactions using a novel gut organ culture system for ex vivo experiments.

分析宿主-微生物群相互作用的肠道肠道器官培养系统

The structure of the gut tissue facilitates close and mutualistic interactions between the host and the gut microbiota. These cross-talks are crucial for ...

肠道炎症对已建立的小鼠结肠样的上皮影响

Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids

The intestinal epithelium plays an essential role in human health, providing a barrier between the host and the external environment. This highly dynamic cell layer provides the first line of defense between microbial and immune populations and helps to modulate the intestinal immune response. Disruption of the epithelial barrier is a hallmark of inflammatory bowel disease (IBD) and is of interest for therapeutic targeting. The 3-dimensional colonoid culture system is an extremely useful in vitro model for studying intestinal stem cell dynamics and epithelial cell physiology in IBD pathogenesis. Ideally, establishing colonoids from the inflamed epithelial tissue of animals would be most beneficial in assessing the genetic and molecular influences on disease. However, we have shown that in vivo epithelial changes are not necessarily retained in colonoids established from mice with acute inflammation. To address this limitation, we have developed a protocol to treat colonoids with a cocktail of inflammatory mediators that are typically elevated during IBD. While this system can be applied ubiquitously to various culture conditions, this protocol emphasizes treatment on both differentiated colonoids and 2-dimensional monolayers derived from established colonoids. In a traditional culture setting, colonoids are enriched with intestinal stem cells, providing an ideal environment to study the stem cell niche. However, this system does not allow for an analysis of the features of intestinal physiology, such as barrier function. Further, traditional colonoids do not offer the opportunity to study the cellular response of terminally differentiated epithelial cells to proinflammatory stimuli. The methods presented here provide an alternative experimental framework to address these limitations. The 2-dimensional monolayer culture system also offers an opportunity for therapeutic drug screening ex vivo. This polarized layer of cells can be treated with inflammatory mediators on the basal side of the cell and concomitantly with putative therapeutics apically to determine their utility in IBD treatment.

作者聚焦:研究体外肠道炎症对已建立的小鼠结肠样蛋白的上皮效应  

We describe a protocol detailing the isolation of murine colonic crypts for the development of 3-dimensional colonoids. The established colonoids can then be terminally differentiated to reflect the cellular composition of the host epithelium prior to receiving an inflammatory challenge or being directed to establish an epithelial monolayer.

研究体 外 肠道炎症对已建立的小鼠结肠样的上皮影响

The intestinal epithelium plays an essential role in human health, providing a barrier between the host and the external environment. This highly dynamic ...

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摘要

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此视频中的章节

体外肠道宿主-微生物界面合成细菌联合的可行性评价

在体外化培养基层小鼠结肠肿瘤

小鼠结肠隐窝三维培养离体研究肠道干细胞功能

该

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

文化与偏振刺激人体肠道黏膜植的新方法

分析宿主-微生物群相互作用的肠道肠道器官培养系统

研究体外肠道炎症对已建立的小鼠结肠样的上皮影响

研究体外肠道炎症对已建立的小鼠结肠样的上皮影响

研究体外肠道炎症对已建立的小鼠结肠样的上皮影响

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摘要

探索更多视频

此视频中的章节

体外肠道宿主-微生物界面合成细菌联合的可行性评价

在体外化培养基层小鼠结肠肿瘤

小鼠结肠隐窝三维培养离体研究肠道干细胞功能

该

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