Name: a video protocol of retroviral infection in primary intestinal organoid culture
Uploaded: 2014-08-11T18:30:00.000+00:00
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Description: Watch this Scientific Journal Video about A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture at JoVE.com

辜BK和Mustata RC是由亨利·戴尔先生奖学金由威康信托和安德森 - 罗尔夫A被支持的医学研究理事会（MRC）的支持。芬克J的由威康信托基金会4年博士，计划的支持。

在以下协议中使用的所有老鼠被安放在无特定病原体条件下，所有的程序都根据英国内政部的规定执行。 1，准备工作<ol><li>准备媒体1小时提前使用（ 见表1和材料清单详细介绍）和预暖在37℃水浴中至少10分钟后再使用。 </li></ol> 2，培养小肠（SI）的组织体注意:除非另有说明，所有的孵育均在加湿培养箱中于37℃，5％CO 2中进行。设备和试剂进入与活细胞接触必须是无菌的。 预暖的组织培养板中是很重要的，因为它可以防止地下室基质（基质胶或BME）的水滴从播种时撒出。另外，地下室基质应保持在冰上在任何时候。储存于-20°C和解冻在使用前冰。 <ol><li>隔离囊<ol><li>对于小肠隔离按照国家的规章制度牺牲小鼠，然后放置在其背面的动物，并用70％乙醇的腹部。从腹股沟到胸骨进行纵向正中切口。首先，切开皮肤，然后皮下组织。从盲肠到胃中取出小肠。隐窝从十二指肠，空肠和回肠中分离，可用于类器官培养。 </li><li>不含钙或镁（PBS0）洗分离小肠预冷磷酸盐缓冲盐水。 </li><li>切割组织成3-5厘米长的小段，并用剪刀剖开纵向。使用镊子传播组织。 </li><li>刮去用盖玻片绒毛。注意，过多的力会导致组织撕裂，并会减少在下面的步骤隐窝的产率。 </li><li>转移组织到50ml管含有预冷却PBS0使用设克钳。经过剧烈摇晃清洗组织碎片，改变PBS0。重复2-3倍，或直至PBS0匝数较少多云。 </li><li>孵育于50ml管装有30ml PBS0的用1mM乙二胺四乙酸（EDTA）处理30分钟，在4℃下在管式辊的组织。 </li><li>用力摇动该管和所述组织转移到另一个50ml管中含有用5mM EDTA30毫升PBS0的。在1mM EDTA的溶液（预先含有组织）将包含隐窝和绒毛的混合物。该馏分是不适合组织体的播种，因为它通常包含绒毛的高百分比。 </li><li>在4℃下孵育所述组织进一步进行1小时的管子辊。 </li><li>大力摇晃试管，并收集在干净的50ml管中的解决方案。确认隐窝的存在并估计数， 例如 ，通过用光学显微镜计数在50μl隐窝。计算需要获得50-100隐窝，并将其转移到1.5 ml吨的量UBE。 </li><li>转速为300×g离心5分钟。弃去上清液，沉淀重悬在50μl地下室矩阵，以及种子中的预温热的24孔板中。孵育板在组织培养箱5-15分钟让地下室矩阵聚合。 </li><li>覆盖用500μlENR介质（ 见表1）。无欲无求的组织体将呈现一个小的圆形囊性形状后，又经过2-3天出芽结构约24小时可见。切换到新鲜ENR媒体每3天。 </li><li>通道类器官培养，每7天。 </li></ol></li><li>传代和维护类器官<ol><li>通过刷新媒体，每3天传代1保持的组织体:3或1:5作为管腔变得充满死亡的细胞，约每7天。 </li><li>打破地下室矩阵圆顶与用1ml的Pipetman尖介质和从井将其传送到1.5ml管中。 </li><li>机械地分解，通过移液管的组织体pproximately使用50倍的罚款 ​​（ 如 ，200微升）一角。 </li><li>转速为300×g离心5分钟。 </li><li>弃上清，悬浮颗粒在150-250微升地下室矩阵。种子在预先温热的24孔板（50μl的地下室矩阵/孔）。前播种吸管地下室矩阵上下一次涂覆的尖端的壁。移液管缓慢，以避免在井中的中间，以防止基底基质扩散出来的气泡和种子。人们希望得到地下室矩阵的圆顶在井的中心。 </li><li>在孵育组织培养箱5-15分钟让地下室矩阵聚合。覆盖层中，每孔500微升ENR媒体。 </li></ol></li></ol> 3，预先感染SI类器官的治疗注意: 图1示出了转导过程。 <ol><li>外汇ENR到ENRWntNic（ 见表1）中，成长类器官在T他的媒介最少3天，直到它们都采用了胆囊形态。 Wnt3a的增加茎和帕内特细胞的数目，而烟酰胺（NIC），提高了培养效率。 </li></ol> 4，病毒制作<ol><li>铂E细胞的1个150毫米的菜是需要每个感染。种子约5×10 6个细胞用15-18毫升培养基（DMEM + 10％FBS）中嘌呤霉素（1微克/毫升）和杀稻瘟素（10微克/毫升）的存在下进行。 2-3天后转染细胞，当他们达到70-80％汇合。无嘌呤和转染前瘟改变介质的DMEM + 10％FBS中。 </li><li>加30的逆转录病毒DNA构建体的微克和240微升的聚乙烯亚胺（PEI）来分离含有1ml OPTI-MEM中的试管中。混合并在室温下孵育5分钟。 </li><li>普尔两种溶液并在室温下孵育20-30分钟。完整的混合物加入铂E细胞的培养基中，小心摇动高原E要确保DNA-PEI复合物的均匀分布。 </li><li>孵育铂E细胞的转染混合物过夜，刷新了媒体的第二天。使细胞保持在新的培养基中2天。 </li><li>只收集在50毫升猎鹰管中，用0.45微米的过滤器和离心机在8000 XG通过它在4℃下12-16小时。弃去上清，重悬在250μl的转导介质的粒料（ 见表1）。 </li></ol> 5类器官片段的制备<ol><li>一个感染的24孔板一个孔是必要的。打破地下室矩阵圆顶与用1ml的Pipetman尖中，转移到1.5 ml管。 </li><li>使用体积更细尖（ 例如 ，200微升提示），通过移液（30-50x）机械地破坏组织体。该解决方案将变得浑浊，没有整个组织体应该是可见的。 </li><li>离心机在室温下，900×g离心5分钟<html> 。 </li><li>弃上清，悬浮颗粒在500微升的细胞培养级重组蛋白酶（ 如的TrypLE）。在37℃下加热5分钟。温育后检查用光学显微镜的组织体片段的大小，计数每片段的细胞数。含5-10个细胞碎片的理想选择。如果多数片段包含多个小区的更高的温育时间可以在同一时间被增加2分钟。 </li><li>加入500微升的ENR介质终止解离过程。 </li><li>离心机在室温下，900×g离心5分钟。除去上清液，并保持颗粒冰或4℃。 </li></ol> 6逆转录病毒转导<ol><li>结合类器官片段与250μL的48孔板的一个孔中的逆转录病毒解决方案（从第4.5节）的。缓慢的移液用1ml的Pipetman尖轻轻混匀。 </li><li>封板用封口膜。 </li></ol>E"&gt; 7。Spinoculation和电镀<ol><li>离心反应板在32℃，600 XG，1小时。小心地取出封口膜孵育板在组织培养箱6小时。 </li></ol> 8，播种感染类器官片段<ol><li>从井中转移感染类器官片段和传导介质到1.5ml管中，并旋在900×g离心5分钟。 </li><li>弃去上清液，把含在冰上沉淀的管5分钟，冷却。加入100μl地下室矩阵的和缓慢上下吹打重悬沉淀。 </li><li>种子滴50微升的地下室矩阵β细胞混合"在一个新的24孔板中。孵育板在37℃下进行5-15分钟，直到基底基质固化。 </li><li>不添加聚凝胺传导介质井和孵化盘在组织培养的孵化器。更换介质每2-3天。 </li></ol> 9，选择<ol><li> SE开始2-3天后，加入嘌呤霉素（1微克/毫升）对媒体经文。 </li><li>当该片段开始形成类器官用补充有嘌呤霉素（1微克/毫升）的ENR媒体更换转导介质。 </li></ol> 10，感染后的SI类器官的治疗<ol><li>根据"传代并保持组织体"的协议（第2.2.1节）文化转类器官。 1-2周后，SI类器官将重拾萌芽结构。 </li><li>以下出芽结构的外观通过在1μM的工作浓度加入4 - 羟基他莫昔（4-OHT）诱导的miRNA或cDNA的表达。请注意，此步骤仅当使用逆转录病毒载体，从Addgene（的pMSCV-loxP的红色荧光-loxP的EGFP-普罗-WPRE（32702）的pMSCV-loxP的红色荧光-loxP的3xHA - 普罗-WPRE（32703），和的pMSCV有效-flip - 普罗 - 红色荧光-GFP-miRNA的（32704））和类器官表达Cre-ERT2。 </li></ol> 11。确认感染与expr的分裂国家/抑制利率的基因<ol><li>如果使用的逆转录病毒载体，从Addgene（32702，32703及32704）转导效率可以通过观察红色荧光表达的证实。此外，所关注的基因的表达或抑制可以通过Western印迹证实，使用GFP或3xHA表位（用于基因表达）和定量PCR（基因敲除），例举在辜等人 4。 </li></ol>

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The authors have nothing to disclose. The authors have no conflict of interest declared.

<html>以实现高转导效率的某些方面是至关重要的。一个是预处理组织体与ENRWntNic媒体的，直到它们通过一个圆形囊性形状。这增加干细胞的数量以及获得转基因的稳定整合，以及增加在SI类器官的存活率在整个转导过程，从而有机会。另一个参数是以下spinoculation孵育时间。过短或过长孵化效果不佳转导效率和组织体的存活率较差，分别为。该spinoculation步骤不是必需的，尽管它显著增加转导组织体的百分比。最后，高滴度的病毒的关键是成功的转导。这是依赖于包装细胞系和病毒的类型。铂-E细胞系和小鼠干细胞病毒（MSCV）的组合，被发现产生效价为组织体的转导不够高。 ve_content"&gt;以下是用于故障诊断可以帮助实现成功转导的提示。首先，如果在包装细胞系的转染较差，确保细胞的汇合度为70-80％之间，而的温育时间池PEI-DNA混合物是20-30分钟之间。的组织体的转导过程中的生存高度依赖于片段的大小。太长胰蛋白酶消化使多数片段为包含低于3细胞，并由此减少组织体的生存能力。另一个因素是Wnt信号的条件培养基的活性，如果活性太低升压它通过加成CHIR99021中为5μM可以增加存活的工作浓度。CHIR99021抑制GSK3，从而增加Wnt信号传导。此外，Y-27362，其防止失巢凋亡加入到转导的介质，以提高组织体可生存，由于组织体被破坏，以片段（含有1-10个细胞）之前的转导。作为60;如上所述，spinoculation后的培养时间不超过6小时。最后，如​​果转差被观察到影响的病毒滴度和插入的逆转录病毒载体的大小限制上述的考虑因素。敲除的效率高度依赖于miRNA的。由于效率与目标基因和miRNA的组合而变化，值得执行效率筛选以鉴定那些工作最好。 该技术被限制在类器官系统的上皮现象。在未来有可能，学习通过病原体或重建共培养感染或免疫介导的疾病与免疫系统衍生的组分分别。此外，逆转录病毒只能携带一个相对较小的大小的插入片段。因此，天然存在的调控区域也被排除在外，因此，转基因的表达不能模仿的的内源基因。如上面所提到的，击倒效率是依赖于靶基因和miRNA上。如果用合适的敲低效率没有的miRNA可以发现它可以限制该技术的使用为特定目的基因。 从理论上讲，类器官都与用于细胞系所有的标准化操作技术兼容。逆转录病毒转导是第一个方法来进行报告4，和最近的BAC（细菌人工染色体）-transgenesis已变得可用5。以2-3周，共生成时间，病毒质粒转染包装细胞系的转染后，它比转基因（TG）小鼠的产生显著更快。通过维持体内隐窝绒毛结构而含有干细胞以及肠上皮所有分化的细胞谱系的组织体培养体系桥接TG动物和先前使用的细胞培养物之间的间隙。 <pCLASS ="jove_content"&gt;这里所描述的协议提供了一种方法，通过GAIN-执行的内胚层上皮细胞表型分析在体外和损失-功能的研究。这使得以解决成体干细胞生物学生理学相关的问题是可行的，具有Tg小鼠的最小需求。例如，可避免通过使用来自于新生突变体与围产期致死6类器官条件性敲除小鼠的产生。此外，该技术可以应用于来自于先前建立的基因敲除小鼠进行追加击倒7,8研究旁系同源物的作用，组织体。 继成立小肠类器官，原文化协议的适配使得胰腺，肝脏，结肠和胃上皮9-11培养。此外，人体肠道类器官和肿瘤组织体已被来自正常人的活组织切片检查，主要亚丁OMA与大肠癌的活检10。病毒感染协议可以很容易地扩展到这些类型的组织体，并提供了执行功能性研究在人来源的组织的前所未有的方法。 两者合计，小肠组织体逆转录病毒转导是研究干细胞维持，分化和细胞命运的决定，以及细胞信号传导和细胞 - 细胞相互作用的宝贵资源。

需要高通量功能基因，以增加我们的生物体内的认识，以改善目前的基础科学和医学。小鼠遗传学一直是金标准调查的体内基因功能，尽管它是既费时又费钱。细胞系，作为其他共同选择，具有较高的吞吐能力，同时为更便宜。然而，它们可通过它们不能重现正确的微环境，从而生理反应见于体内的阻碍，因此，有一个明确的需要一种易于处理的模型系统，它允许成本/时间效率高通量分析而模仿在体内的转基因（TG）小鼠的实验中观察到的生理反应。 为内胚层上皮这样的一个模型系统出现在2009年1。在从LGR5阳性肠道干细胞的发现所获得的知识是大约对应于所需的干细胞维持细胞外基质和生长因子的适当位置信息。利用这些信息成为可能，建立"小胆"也被称为类器官2。最近一个共识命名体外培养，在组织体被称为"enteroids"，建议3。样细胞系，该组织体是不断扩展的和易于治疗与配体和抑制剂。的，而不是两维然而，它们是三维的自组织结构的保留隐窝 - 绒毛组织，以及干细胞和小肠（SI）的分化的细胞谱系。类器官包括环绕管腔面积的上皮细胞单层的。突出萌芽结构对应于含有干细胞室小肠隐窝。从萌芽结构的顶端开始祖细胞分化为心肌梗死，他们篦朝向上皮衬里，其中终末分化的细胞脱落到内腔。相比的细胞系，这在体外系统更加紧密地概括了正常的生理和因此对小肠上皮有前途的模型系统。 在逆转录病毒转导的这个视频协议，我们提出了一个方法，使体外基因功能研究，在这本小说化培养体系。首先，我们通过描述化培养在一步一步地，并继续通过展示逆转录病毒随后转导过程的产生。最后，还有一款用于故障排除的其他建议。这种技术的优点在于，它可以与实时成像或药物筛选结合研究稳态，细胞命运决定和细胞 - 细胞相互作用中肠上皮。由于其结构简单和快速的周转率，组织体代表一个理想的S型变体系的研究成体干细胞生物学。除逆转录病毒转导可应用于从预先建立的转基因小鼠以及人类患者样品来自组织体。如敲入和基因敲除方法不能扩展到人类，人类SI组织体构成一个有吸引力的替代方案。 综上所述，通过逆转录病毒转导基因操作允许来自小鼠或人体组织样本，从而补充小鼠遗传学和细胞系，同时开辟新的途径在人源性组织研究小肠类器官表型分析。逆转录病毒转导使GAIN-和研究缺失型的功能的类器官培养系统4要执行。这使得它对于调查基因的功能，成体干细胞生物学和疾病而按照三个R​​（减少，改进和替换）是一种有价值的资源。

<table><tbody><tr><td>Advanced DMEM/F12&amp;nbsp;</td><td>Invitrogen</td><td>12634-034</td><td></td></tr><tr><td>Glutamax 100x&amp;nbsp;</td><td>Invitrogen</td><td>35050-068</td><td></td></tr><tr><td>HEPES 1 M</td><td>Invitrogen</td><td>15630-056</td><td></td></tr><tr><td>Penicillin-streptomycin 100x</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>B27 supplement 50x</td><td>Invitrogen</td><td>17504-044</td><td></td></tr><tr><td>N2 supplement 100x&amp;nbsp;</td><td>Invitrogen</td><td>17502-048</td><td></td></tr><tr><td>n-Acetylcysteine 500 mM</td><td>Sigma-Aldrich</td><td>A9165-5G</td><td></td></tr><tr><td>Mouse EGF 500 &amp;micro;g/ml</td><td>Invitrogen Biosource</td><td>PMG8043</td><td></td></tr><tr><td>Mouse Noggin 100 &amp;micro;g/ml</td><td>Peprotech</td><td>250-38</td><td></td></tr><tr><td>R-Spondin conditioned medium</td><td></td><td></td><td>The conditioned media is generated from HEK293 cells, for details see Sato and Clevers 2013.</td></tr><tr><td>Wnt conditioned medium</td><td></td><td></td><td>The conditioned media is generated from L cells, for details see Sato and Clevers 2013.</td></tr><tr><td>Nicotinamide 1 M</td><td>Sigma</td><td>N0636</td><td></td></tr><tr><td>Y-27632 10 &amp;micro;M</td><td>Sigma</td><td>Y0503-1MG</td><td></td></tr><tr><td>Polybrene (8 &amp;micro;g/ml)</td><td>Sigma</td><td>H9268-5G</td><td></td></tr><tr><td>Standard BD Matrigel matrix</td><td>BD Biosciences</td><td>356231</td><td>Basement Matrix Extract (Cultrex PathClear BME Reduced Growth Factor Type 2, 3533-005-02)&lt;br/&gt; supplied by AMSBIO can be used as an alterntive.</td></tr><tr><td>24-well Plate</td><td>Greiner Bio One</td><td>662960</td><td></td></tr><tr><td>48-well Plate</td><td>Greiner Bio One</td><td>677980</td><td></td></tr><tr><td>CHIR99021</td><td>Sigma</td><td>A3734-1MG</td><td></td></tr><tr><td>Platinum-E cells</td><td>Cell Biolabs</td><td>RV-102</td><td></td></tr><tr><td>Puromycin</td><td>Invitrogen</td><td>A1113802</td><td></td></tr><tr><td>Blasticidin</td><td>Invitrogen</td><td>A1113902</td><td></td></tr><tr><td>Polyethyleneimine (PEI)</td><td>Polysciences</td><td>23966</td><td></td></tr><tr><td>opti-MEM</td><td>Life Technologies</td><td>51985-034</td><td></td></tr><tr><td>TrypLE</td><td>Invitrogen</td><td>12605-010</td><td></td></tr><tr><td>Parafilm</td><td>Sigma</td><td>P7793-1EA</td><td></td></tr><tr><td>4-OHT</td><td>Sigma</td><td>H7904</td><td></td></tr></tbody></table>

a video protocol of retroviral infection in primary intestinal organoid culture

LGR5-阳性干细胞可与必要的生长补充因子EGF，头蛋白，和R-脊椎蛋白，这使我们能够培养不断扩大的初级三维上皮结构体外 。这两个"小胆"，也被称为类器官的结构和生理特性，非常类似于它们在体内的同行。这使得它们对于小肠上皮有吸引力的模型系统。利用逆转录病毒转导，基因功能现在可以通过条件基因过表达或敲除执行。此视频演示类器官培养的方法，反转录病毒的产生，并组织体的逆转录病毒转导，以协助在体外小肠上皮表型分析。这种新颖的器官系统模型结合逆转录病毒介导的基因表达提供了基因功能的体外快速分析的重要工具，而不costl的需要y和费时代的转基因动物。

组织体是准备当中央管腔由于死细胞（ 图2）的存在下变暗被分裂。预处理后组织体的2-3天应采用圆形囊性形态（ 图3）。这增加干细胞的数量，提高获得稳定整合的机会。病毒颗粒的尺寸可由于细胞碎片的沉淀物的大小变化而变化的贡献后的病毒上清的离心分离，最有可能的。没有明显的相关性，以转导效率已经观察到。在选择过程非转类器官会死，那些具有稳定的整合将继续一段时间。从MSCV-eGFP的逆转录病毒的荧光蛋白可以在细胞存活的组织体，其具有一个囊状形态，在2-3天内以下转导（ 图4）源于被观察到。 <img alt<html> ="图1"FO:内容宽度="6英寸"SRC ="/文件/ ftp_upload / 51765 / 51765fig1highres.jpg"WIDTH ="600"/&gt; 图1中的逆转录病毒转导过程的示意图。之前感染组织体是经预处理的使用ENRWntNic直到他们通过囊状结构（步骤1）。铂-E细胞作为包装细胞系，并进行培养，直至达到70-80％汇合。此后它们被转用PEI的逆转录病毒构建。病毒收获在2天后（第2步）。组织体用胰蛋白酶消化，得到含有1-10细胞（步骤3）的片段，然后被感染（步骤4）。以下spinoculation增加感染效率（步骤5），在受感染的组织体碎片接种后（步骤6）和2-3天的选择为阳性的克隆与稳定整合可以进行（步骤7）。 1765fig2highres.jpg"WIDTH ="500"/&gt; 图后4-6天培养的组织体2代表图像的内腔充满了死亡的细胞，使其显得暗。组织体在这个阶段就可以传代。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51765/51765fig3highres.jpg" width="500" /> 小肠类器官3-4天ENRWntNic培养基图3。代表形象。该组织体采用胆囊形态。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51765/51765fig4highres.jpg" width="500" /> 小肠组织体图4代表性图像（一），显示病毒的转基因表达（B，绿色荧光蛋白）。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td colspan="2"> ADVANCED的DMEM / F 12 + + + </td></tr><tr><td colspan="2">储存在4℃下4周</td></tr><tr><td>先进的DMEM / F12 </td><td> 500毫升</td></tr><tr><td> Glutamax的100X </td><td> 5毫升</td></tr><tr><td> HEPES 1米</td><td> 5毫升</td></tr><tr><td>抗生素100X </td><td> 5毫升</td></tr><tr><td colspan="2"> ENRWntNic培养基（20毫升）中 </td></tr><tr><td colspan="2">储存在4℃下2周</td></tr><tr><td>先进的DMEM / F 12 + + + </td><td> 7.2毫升</td></tr><tr><td> B27添加剂（50X） </td><td> 400μL </td></tr><tr><td> N2补充（100X） </td><td> 200微升</td></tr><tr><td>乙酰半胱氨酸（500毫米） </td><td> 50微升</td></tr><tr><td>鼠表皮生长因子（500微克/毫升） </td><td> 2微升</td></tr><tr><td>鼠标头蛋白（100微克/毫升） </td><td> 20微升</td></tr><tr><td> R-脊椎康迪特提一下媒体</td><td> 2毫升</td></tr><tr><td> Wnt3a的条件培养基</td><td> 10毫升</td></tr><tr><td>烟酰胺（1μM） </td><td> 200微升</td></tr><tr><td colspan="2"> 传导介质（20毫升）中 </td></tr><tr><td colspan="2">准备好新鲜</td></tr><tr><td> ENRWntNic中</td><td> 20毫升</td></tr><tr><td> Y-27632（10微米） </td><td> 20微升</td></tr><tr><td>聚凝胺（8微克/毫升） </td><td> 20微升</td></tr><tr><td colspan="2"> ENR培养基（20毫升）中 </td></tr><tr><td colspan="2">储存在4℃下4周</td></tr><tr><td>先进的DMEM / F 12 + + + </td><td> 17.4毫升</td></tr><tr><td> B27添加剂（50X） </td><td> 400μL </td></tr><tr><td> N2补充（100X） </td><td> 200微升</td></tr><tr><td>乙酰半胱氨酸（500毫米） </td><td> 50微升</TD&gt; </tr><tr><td>鼠表皮生长因子（500微克/毫升） </td><td> 2微升</td></tr><tr><td>鼠标头蛋白（100微克/毫升） </td><td> 20微升</td></tr><tr><td> R-脊椎调理液</td><td> 2毫升</td></tr><tr><td colspan="2"> 媒体铂E细胞（500毫升） </td></tr><tr><td colspan="2">储存在4℃下进行12周</td></tr><tr><td> DMEM </td><td> 449.45毫升</td></tr><tr><td>胎牛血清（FBS） </td><td> 50毫升</td></tr><tr><td>嘌呤霉素（1微克/毫升） </td><td> 50微升</td></tr><tr><td>杀稻瘟素（10微克/毫升） </td><td> 500微升</td></tr></tbody></table> 表1媒体高级的DMEM / F12 + + +，ENRWntNic介质，转导中，ENR中程和中的铂E细胞组成。

Watch this Scientific Journal Video about A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture at JoVE.com

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

本协议解释主要LGR5-positve化培养和逆转录病毒转导的后续表现。这使Cre重组酶诱导表达递送转基因或敲除，并允许功能性研究，以进行在小说体外器官 模型系统。

逆转录病毒感染的原发性肠化培养的视频协议

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding ...

a-video-protocol-retroviral-infection-primary-intestinal-organoid

Research

JoVE Journal

Biology

29.7K 次观看.  University of Cambridge. 本协议解释主要LGR5-positve化培养和逆转录病毒转导的后续表现。这使Cre重组酶诱导表达递送转基因或敲除，并允许功能性研究，以进行在小说体外器官 模型系统。 

视频: A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

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 ...

科研

医学

Genetic Engineering of Primary Mouse Intestinal Organoids Using Magnetic Nanoparticle Transduction Viral Vectors for Frozen Sectioning

Intestinal organoid cultures provide a unique opportunity to investigate intestinal stem cell and crypt biology in vitro, although efficient approaches to manipulate gene expression in organoids have made limited progress in this arena. While CRISPR/Cas9 technology allows for precise genome editing of cells for organoid generation, this strategy requires extensive selection and screening by sequence analysis, which is both time-consuming and costly. Here, we provide a detailed protocol for efficient viral transduction of intestinal organoids. This approach is rapid and highly efficient, thus decreasing the time and expense inherent in CRISPR/Cas9 technology. We also present a protocol to generate frozen sections from intact organoid cultures for further analysis with immunohistochemical or immunofluorescent staining, which can be used to confirm gene expression or silencing. After successful transduction of viral vectors for gene expression or silencing is achieved, intestinal stem cell and crypt function can be rapidly assessed. Although most organoid studies employ in vitro assays, organoids can also be delivered to mice for in vivo functional analyses. Moreover, our approaches are advantageous for predicting therapeutic responses to drugs because currently available therapies generally function by modulating gene expression or protein function rather than altering the genome.

We describe step-by-step instructions to: 1) efficiently engineer intestinal organoids using magnetic nanoparticles for lenti- or retroviral transduction, and, 2) generate frozen sections from engineered organoids. This approach provides a powerful tool to efficiently alter gene expression in organoids for investigation of downstream effects.

使用磁纳米粒子转导病毒载体进行冷冻切片的原小鼠肠道器官的遗传工程

Intestinal organoid cultures provide a unique opportunity to investigate intestinal stem cell and crypt biology in vitro, although efficient approaches to ...

发育生物学

Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. Three cellular sources have been used: Embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) or adult stem cells. Here, culture of mouse and human gastric organoids derived from adult stem cells is described and used for infection with the gastric pathogen Helicobacter pylori. Human gastric glands are isolated from resection material, seeded in a basement matrix and embedded in medium containing growth factors epidermal growth factor (EGF), R-spondin, Noggin, Wnt, fibroblast growth factor (FGF) 10, gastrin and transforming growth factor (TGF) beta inhibitor. In these conditions, gastric glands grow into 3-dimensional organoids containing 4 lineages of the stomach. The organoids expand indefinitely and can be frozen and thawed similarly as cell lines. For infection studies, bacteria are microinjected into the lumen of the organoids. Infected organoids are processed for imaging. The described methods can be adapted to other organoids and infections with other bacteria, viruses or parasites. This allows the study of infection-induced changes in primary cells.

Stem cell derived cultures harbor tremendous potential to model infectious diseases. Here, the culture of mouse and human gastric organoids derived from adult stem cells is described. The organoids are microinjected with the gastric pathogen Helicobacter pylori.

类器官的模型传染病:人类的文化与小鼠胃组织体与幽门螺杆菌的显微注射

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. ...

免疫与感染

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 ...

Adapting Gastrointestinal Organoids for Pathogen Infection and Single Cell Sequencing under Biosafety Level 3 (BSL-3) Conditions

Human intestinal organoids constitute the best cellular model to study pathogen infections of the gastrointestinal tract. These organoids can be derived from all sections of the GI tract (gastric, jejunum, duodenum, ileum, colon, rectum) and, upon differentiation, contain most of the cell types that are naturally found in each individual section. For example, intestinal organoids contain nutrient-absorbing enterocytes, secretory cells (Goblet, Paneth, and enteroendocrine), stem cells, as well as all lineage-specific differentiation intermediates (e.g.,&#160;early or immature cell types). The greatest advantage in using gastrointestinal tract-derived organoids to study infectious diseases is the possibility of precisely identifying which cell type is targeted by the enteric pathogen and to address whether the different sections of the gastrointestinal tract and their specific cell types similarly respond to pathogen challenges. Over the past years, gastrointestinal models, as well as organoids from other tissues, have been employed to study viral tropism and the mechanisms of pathogenesis. However, utilizing all the advantages of using organoids when employing highly pathogenic viruses represents a technical challenge and requires strict biosafety considerations. Additionally, as organoids are often grown in three dimensions, the basolateral side of the cells is facing the outside of the organoid while their apical side is facing the inside (lumen) of the organoids. This organization poses a challenge for enteric pathogens as many enteric infections initiate from the apical/luminal side of the cells following ingestion. The following manuscript will provide a comprehensive protocol to prepare human intestinal organoids for infection with enteric pathogens by considering the infection side (apical vs. basolateral) to perform single-cell RNA sequencing to characterize cell-type-specific host/pathogen interactions. This method details the preparation of the organoids as well as the considerations needed to perform this work under biosafety level 3 (BSL-3) containment conditions.

Adapting Gastrointestinal Organoids for Pathogen Infection and Single Cell Sequencing under Biosafety Level 3 BSL-3 Conditions

This protocol describes how to infect human intestinal organoids from either their apical or basolateral side to characterize host/pathogen interactions at the single-cell level using single-cell RNA sequencing (scRNAseq) technology.

在生物安全3级（BSL-3）条件下使胃肠道类器官适应病原体感染和单细胞测序

Human intestinal organoids constitute the best cellular model to study pathogen infections of the gastrointestinal tract. These organoids can be derived ...

Co-Culture of Murine Small Intestine Epithelial Organoids with Innate Lymphoid Cells

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate balance of mucosal homeostasis. These 3D, multi-cellular systems offer a reductionist model for addressing multi-factorial diseases and resolving technical difficulties that arise when studying rare cell types such as tissue-resident innate lymphoid cells (ILCs). This article describes a murine system that combines small intestine organoids and small intestine lamina propria derived helper-like type-1 ILCs (ILC1s), which can be readily extended to other ILC or immune populations. ILCs are a tissue-resident population that is particularly enriched in the mucosa, where they promote homeostasis and rapidly respond to damage or infection. Organoid co-cultures with ILCs have already begun shedding light on new epithelial-immune signaling modules in the gut, revealing how different ILC subsets impact intestinal epithelial barrier integrity and regeneration. This protocol will enable further investigations into reciprocal interactions between epithelial and immune cells, which hold the potential to provide new insights into the mechanisms of mucosal homeostasis and inflammation.

This protocol offers detailed instructions for establishing murine small intestine organoids, isolating type-1 innate lymphoid cells from the murine small intestine lamina propria, and establishing 3-dimensional (3D) co-cultures between both cell types to study bi-directional interactions between intestinal epithelial cells and type-1 innate lymphoid cells.

小鼠小肠上皮类器官与先天性淋巴细胞的共培养

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate ...

病毒感染的人肠道类器官单层的钙成像

Live Calcium Imaging of Virus-Infected Human Intestinal Organoid Monolayers Using Genetically Encoded Calcium Indicators

Calcium signaling is an integral regulator of nearly every tissue. Within the intestinal epithelium, calcium is involved in the regulation of secretory activity, actin dynamics, inflammatory responses, stem cell proliferation, and many other uncharacterized cellular functions. As such, mapping calcium signaling dynamics within the intestinal epithelium can provide insight into homeostatic cellular processes and unveil unique responses to various stimuli. Human intestinal organoids (HIOs) are a high-throughput, human-derived model to study the intestinal epithelium and thus represent a useful system to investigate calcium dynamics. This paper describes a protocol to stably transduce HIOs with genetically encoded calcium indicators (GECIs), perform live fluorescence microscopy, and analyze imaging data to meaningfully characterize calcium signals. As a representative example, 3-dimensional HIOs were transduced with lentivirus to stably express GCaMP6s, a green fluorescent protein-based cytosolic GECI. The engineered HIOs were then dispersed into a single-cell suspension and seeded as monolayers. After differentiation, the HIO monolayers were infected with rotavirus and/or treated with drugs known to stimulate a calcium response. An epifluorescence microscope fitted with a temperature-controlled, humidified live-imaging chamber allowed for long-term imaging of infected or drug-treated monolayers. Following imaging, acquired images were analyzed using the freely available analysis software, ImageJ. Overall, this work establishes an adaptable pipeline for characterizing cellular signaling in HIOs.

作者聚焦：研究肠道上皮信号传导的病毒破坏 – 研究见解和未来方向 

This protocol describes an approach for performing calcium imaging in virus-infected human intestinal organoids and offers an approach to analysis.

使用基因编码的钙指示剂对病毒感染的人肠道类器官单层进行活体钙成像

Calcium signaling is an integral regulator of nearly every tissue. Within the intestinal epithelium, calcium is involved in the regulation of secretory ...

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

协议的慢病毒转导与肠组织体下游分析

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

逆转录病毒感染的原发性肠化培养的视频协议

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在体外化培养基层小鼠结肠肿瘤

使用磁纳米粒子转导病毒载体进行冷冻切片的原小鼠肠道器官的遗传工程

类器官的模型传染病:人类的文化与小鼠胃组织体与幽门螺杆菌的显微注射

该

在生物安全3级（BSL-3）条件下使胃肠道类器官适应病原体感染和单细胞测序

小鼠小肠上皮类器官与先天性淋巴细胞的共培养

使用基因编码的钙指示剂对病毒感染的人肠道类器官单层进行活体钙成像

使用基因编码的钙指示剂对病毒感染的人肠道类器官单层进行活体钙成像

使用基因编码的钙指示剂对病毒感染的人肠道类器官单层进行活体钙成像

协议的慢病毒转导与肠组织体下游分析