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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Advanced DMEM/ F12</td>
			<td>Life Technologies</td>
			<td>12634-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffered saline, modified, w/o Calcium chloride &#38; 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&#160;</td>
			<td>BD Bioscience</td>
			<td>244620</td>
			<td></td>
		</tr>
		<tr>
			<td>BD&#160;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&#160;</td>
			<td>Bio-Rad</td>
			<td>1725125&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysing Matrix S (1/8&#34;), 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>100x15 Petri Dish</td>
			<td>Falcon</td>
			<td>5687</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate 6well 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 micron 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

13.9K 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

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

在Live主机转利什曼原虫寄生虫在体内成像

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

科研

免疫与感染

Use of the EpiAirway Model for Characterizing Long-term Host-pathogen Interactions

Nontypeable Haemophilus influenzae (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory mucosa 1; 2. To study the mechanisms by which these organisms survive on and inside respiratory tissues, a model in which successful long-term co-culture of bacteria and human cells can be performed is required. We use primary human respiratory epithelial tissues raised to the air-liquid interface, the EpiAirway model (MatTek, Ashland, MA). These are non-immortalized, well-differentiated, 3-dimensional tissues that contain tight junctions, ciliated and nonciliated cells, goblet cells that produce mucin, and retain the ability to produce cytokines in response to infection. 
This biologically relevant in vitro model of the human upper airway can be used in a number of ways; the overall goal of this method is to perform long-term co-culture of EpiAirway tissues with NTHi and quantitate cell-associated and internalized bacteria over time. As well, mucin production and the cytokine profile of the infected co-cultures can be determined. This approach improves upon existing methods in that many current protocols use submerged monolayer or Transwell cultures of human cells, which are not capable of supporting bacterial infections over extended periods3. For example, if an organism can replicate in the overlying media, this can result in unacceptable levels of cytotoxicity and loss of host cells, arresting the experiment. The EpiAirway model allows characterization of long-term host-pathogen interactions. Further, since the source for the EpiAirway is normal human tracheo-bronchial cells rather than an immortalized line, each is an excellent representation of actual human upper respiratory tract tissue, both in structure and in function4.
For this method, the EpiAirway tissues are weaned off of anti-microbial and anti-fungal compounds for 2 days prior to delivery, and all procedures are performed under antibiotic-free conditions. This necessitates special considerations, since both bacteria and primary human tissues are used in the same biosafety cabinet, and are co-cultured for extended periods.

This method allows characterization of extended bacterial co-culture with EpiAirways, primary human respiratory epithelial tissue grown at the air-liquid interface, a biologically relevant in vitro model. The approach can be used with any microbe that is amenable to long-term co-culture.

使用的EpiAirway模型，用于描述长期宿主 - 病原体相互作用

Nontypeable Haemophilus influenzae  (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory ...

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 ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

人胎盘及蜕膜器官培养研究在母胎界面感染

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

黏膜衬里流体为体内上呼吸道免疫介质水平量化的无创性取样

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

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, ...

Optimization of the Cuff Technique for Murine Heart Transplantation

Murine cardiac transplantation has been performed for more than 40 years. With advancements in microsurgery, certain new techniques have been used to improve surgical efficiency. In our lab, we have optimized the cuff technique with two major steps. First, we used the inner tube technique to insert a temporary inner tube into the external jugular vein and carotid artery blood vessel to facilitate eversion of the vessel over the cuff. Second, we performed complete heterotopic cardiac transplantation through the collaboration of two experienced surgeons. These modifications effectively reduced the operation time to 25 minutes, with a success rate of 95%. In this report, we describe these procedures in detail and provide a supplemental video. We believe that this report on the improved cuff technique will offer practical guidance for murine heterotopic heart transplantation and will enhance the utility of this mouse model for basic research.

We introduce an inner tube approach to the cuff technique for mouse cervical heterotopic heart transplantation to help evert the vessel over the cuff. We found that cooperation between two experienced surgeons markedly shortens the operation time.

穆林心脏移植袖口技术的优化

Murine cardiac transplantation has been performed for more than 40 years. With advancements in microsurgery, certain new techniques have been used to ...

Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis

When developing novel antimicrobials, the success of animal trials is dependent on accurate extrapolation of antimicrobial efficacy from in vitro tests to animal infections in vivo. The existing in vitro tests typically overestimate antimicrobial efficacy as the presence of host tissue as a diffusion barrier is not accounted for. To overcome this bottleneck, we have developed an ex vivo porcine corneal model of bacterial keratitis using Pseudomonas aeruginosa as a prototypic organism. This article describes the preparation of the porcine cornea and protocol for establishment of the infection. Bespoke glass molds enable straightforward setup of the cornea for infection studies. The model mimics in vivo infection as bacterial proliferation is dependent on the ability of the bacterium to damage corneal tissue. Establishment of infection is verified as an increase in the number of colony forming units assessed via viable plate counts. The results demonstrate that infection can be established in a highly reproducible fashion in the ex vivo corneas using the method described here. The model can be extended in the future to mimic keratitis caused by microorganisms other than P. aeruginosa. The ultimate aim of the model is to investigate the effect of antimicrobial chemotherapy on the progress of bacterial infection in a scenario more representative of in vivo infections. In so doing, the model described here will reduce the use of animals for testing, improve success rates in clinical trials and ultimately enable rapid translation of novel antimicrobials to the clinic.

This article describes a step-by-step protocol to set up an ex vivo porcine model of bacterial keratitis. Pseudomonas aeruginosa is used as a prototypic organism. This innovative model mimics in vivo infection as bacterial proliferation is dependent on the ability of the bacterium to damage corneal tissue.

建立猪外维沃角膜模型研究药物治疗细菌性角膜炎

When developing novel antimicrobials, the success of animal trials is dependent on accurate extrapolation of antimicrobial efficacy from in vitro tests to ...

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important role in the dissemination and pathogenesis of this opportunistic fungal pathogen. However, methods to visualize fungal hyphae in the GI tract in vivo are challenging which limits the understanding of environmental signals in controlling this morphogenesis process. The protocol described here demonstrates a novel ex vivo method for visualization of hyphal morphogenesis in gut homogenate extracts. Using an ex vivo assay, this study demonstrates that cecal contents from antibiotic treated mice, but not from untreated control mice, promote C. albicans hyphal morphogenesis in the gut content. Further, adding back specific groups of gut metabolites to the cecal contents from antibiotic-treated mice differentially regulates hyphal morphogenesis ex vivo. Taken together, this protocol represents a novel method to identify and investigate the environmental signals that control C. albicans hyphal morphogenesis in the GI tract.

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

The ex vivo assay described in this study using gut homogenate extracts and immunofluorescence staining represents a novel method to examine the hyphal morphogenesis of Candida albicans in the GI tract. This method can be utilized to investigate the environmental signals regulating morphogenetic transition in the gut.

前体内分析研究胃 肠道的 念珠菌血脂磷

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important ...

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a poor correlation between antibiotic susceptibility testing (AST) results using standard diagnostic methods (e.g., broth microdilution, disk diffusion, or Etest) and clinical outcomes after antibiotic treatment. Attempts to improve AST by the use of off-the-shelf biofilm growth platforms show little improvement in results. The limited ability of in vitro biofilm systems to mimic the physicochemical environment of the CF lung and, therefore bacterial physiology and biofilm architecture, also acts as a brake on the discovery of novel therapies for CF infection. Here, we present a protocol to perform AST of CF pathogens grown as mature, in vivo-like biofilms in an ex vivo CF lung model comprised of pig bronchiolar tissue and synthetic CF sputum (ex vivo&#160;pig lung, EVPL).
Several in vitro&#160;assays exist for biofilm susceptibility testing, using either standard laboratory medium or various formulations of synthetic CF sputum in microtiter plates. Both growth medium and biofilm substrate (polystyrene plate vs. bronchiolar tissue) are likely to affect biofilm antibiotic tolerance. We show enhanced tolerance of clinical Pseudomonas aeruginosa and Staphylococcus aureus isolates in the ex vivo model; the effects of antibiotic treatment of biofilms is not correlated with the minimum inhibitory concentration (MIC) in standard microdilution assays or a sensitive/resistant classification in disk diffusion assays.
The ex vivo platform could be used for bespoke biofilm AST of patient samples and as an enhanced testing platform for potential antibiofilm agents during pharmaceutical research and development. Improving the prescription or acceleration of antibiofilm drug discovery through the use of more in vivo-like testing platforms could drastically improve health outcomes for individuals with CF, as well as reduce the costs of clinical treatment and discovery research.

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

This workflow can be used to perform antibiotic susceptibility testing using an established&#160;ex vivo model of bacterial biofilm in the lungs of individuals with cystic fibrosis. Use of this model could enhance the clinical validity of MBEC (minimal biofilm eradication concentration) assays.

在囊性纤维化肺中的伪肌瘤和金黄色葡萄球菌生物膜的前体内模型中进行抗生素疗效测试

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a ...