The authors would like to thank Dr. Sheryl Coutermarsh-Ott, Dylan McDaniel and Bettina Heid for technical discussions. The authors thank Dr. Nanda Nanthakumar for providing the Caco-2 cells. The authors also thank The Multicultural Academic Opportunities Program (MAOP) at Virginia Tech. This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Award K01DK092355 (to I.C.A.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

所有的研究获得批准，在弗吉尼亚理工大学IACUC的指导方针进行 1.准备R-Spondin1条件培养基从HEK293T-Rspo1细胞系<ol><li> HEK293T-Rspondin1细胞的产生已如前所述10。种子的HEK293T-Rspondin1分泌细胞:5-10％汇合，约8×10 5 -1.7×10 6个细胞，在为T-175烧瓶用40ml 1×Dulbecco氏改良的Eagle培养基（DMEM）+ 10％胎牛血清（ FBS）的作为生长培养基，并在37℃+ 5％CO 2孵育。 注:对于HEK293T-Rspondin1分泌细胞将作为R-spondin1条件培养基生长培养基。 R-spondin1可替代地购买作为在500纳克/毫升的终浓度中使用的重组生长因子。 </li><li>成长HEK293T-Rspondin1细胞七天或直至达到通过亮视野显微镜测定95％汇合。收获40毫升条件培养基，然后到50毫升锥形管中。丢弃HEK293T-Rspondin1分泌细胞的T形175烧瓶中。 </li><li>离心机含有的条件培养基在300×g离心10分钟，在4℃下在管，以沉淀细胞碎片。收集上清液，称为R-spondin1条件培养基，并分装成15ml试管。商店等分在-20℃下短期或-80℃长期储存。 注意:一旦解冻，则R-Spondin1条件培养基可以存储最多1周4℃。 </li></ol> 2.类器官生长培养基的制备和收获小肠隐窝试剂&#160;<ol><li>收获前一天到，放置在冰上冷冻蛋白基质中并在4℃下解冻过夜。高压釜解剖剪刀，镊子和载玻片，将用于为隐窝收获。 </li><li>第二天，开始通过制备40ml含有补充剂和生长因子类器官的生长培养基中加入库存浓度至40ml 1×DM的EM / F-12。媒体补充剂含有:10mM的2- [4-（2-羟乙基）哌嗪-1-基]乙磺酸（HEPES） - 400微升，1×补充谷氨酰胺- 400微升，1×B27无维生素A - 400微升，1×N 2 - 200微升，1毫摩尔N-乙酰半胱氨酸- 40微升，100纳克/毫升间头蛋白- 40微升，50纳克/毫升间的EGF - 4微升和发生在一个37℃的水浴中，直到使用。在水浴中温R-spondin1的15毫升等分至37℃。 </li><li>温暖3无菌24孔板至37℃至少30分钟通过在培养箱中放置。制备每只小鼠1×磷酸盐缓冲盐水（PBS）+ 2mM的乙二胺四乙酸（EDTA）和凉爽50毫升至4℃。准备每只小鼠100毫升1X含10％FBS，冷却至4℃PBS。 </li></ol> 3.收获小家鼠小肠隐窝为化培养1 <ol><li>牺牲一个雄性C57B6 / J鼠标6-12岁周龄标准的啮齿动物食物和水可用募集根据广告机构指引自由采食 。通过CO 2安乐死窒息颈椎脱位。 注意:保持胴体上的冰上，直到收获的组织，并在生物安全柜进行组织收获，以尽量减少污染的风险。 注:男性或女性鼠标可能被用来生成类器官。 </li><li> （可选）刮胡子鼠标在70％乙醇浸入之前删除的皮毛。浸没在70％乙醇鼠标2分钟，四肢脚用鼠标接触电路板的背侧钉扎板。 </li><li>使用预消毒解剖剪刀和镊子，使鼠标的腹侧部分的中线切口。使在生殖器在进行颅到颈部的底部切口。用镊子横向打开皮肤切口和针皮肤钉扎板以暴露腹膜。 </li><li>使在腹部的腹膜一个中线切口以解剖剪刀和第折Ë腹膜与横向镊子和脚钉扎板，以露出腹部器官。 注:请注意，以避免削减腹部器官。 </li><li>与通过切割连接到胃，并连接到盲肠远端结小肠的近端结解剖镊子和剪刀除去从腹腔小肠。放置小肠中含有10ml冰冷的1×PBS中的无菌培养皿。 </li><li>冲洗载小肠用冰冷1×PBS中使用1毫升吸移管的管腔内的内容。直到小肠的腔中的杂物被除去重复此步骤。 </li><li>切小肠解剖剪刀成3-4大致相等的长度条和纵向奠定一个新的无菌培养皿条。对于每个条带，插入小肠的腔内部的解剖剪刀的刀刃，使一个切口的整个长度ÒF中的条。使用镊子横向折叠打开切口，以揭露小肠腔内。 </li><li>使用无菌载玻片轻轻刮去小肠的腔表面上的绒毛。切小肠进入1-2厘米长条与解剖剪刀且这些条带转移到含有10ml冰冷的1×PBS中的50ml锥形管中。 </li><li>通过反转50ml锥形管轻轻混匀的内容，并允许组织内容沉降到50ml锥形管的底部。吸出1×PBS中并通过用10ml冰冷的1X PBS中洗涤该组织重复此三次。在最后的洗涤留在冰上锥形管含有10ml 1×PBS中，小肠组织片段10分钟。 </li><li>吸出PBS 1X，并添加25毫升1X PBS + 2毫摩尔EDTA的。放置在4℃或在冰桶45分钟在摇摆平台上。虽然组织培养，标签6的15毫升锥形管"分数＃1-6"。 </li><li&gt;温育后，允许组织内容沉降到管和抽吸1×PBS + 2mM EDTA的的底部。加入10 mL 1×PBS中+ 10％FBS的并用手剧烈摇动该管的10倍。 </li><li>允许组织内容沉降到管底部。除去并将上清液转移到标有"级分1"管15毫升锥形。重复1×PBS + 10％FBS的振荡工序（3.11）的五倍，并且为了传送上清液内容至适当标记的馏分管。 </li><li>离心在125×g离心5分钟。吸出上清，重悬粒料在5ml预热1×DMEM / F-12，用无生长因子加入。 </li><li>离心在78×g离心2分钟。抽吸将4ml上清并在1ml剩余介质的悬浮每个粒料。 </li><li>从每一级分移除20个ml和添加到载玻片。隐窝可视化和碎片光显微镜下，并确定哪个组合分数，并相应池实现的最大佩尔切隐窝碎片比ntage。 注:分数2-6产生被镀隐窝的比例最大。到池的部分是最常见的有4分和5分与6分3分。 </li><li>在125 XG待镀5分钟离心馏分。吸上清留下50-100微升其余颗粒之上。 </li><li>保持在冰上管和1ml蛋白基质添加到合并的级分。吸取上下慢慢地防止除气泡。 </li><li>从37℃培养箱除去预热24孔板，加入50μl的蛋白基质/隐窝悬浮液在每个中间良好。引晶的板转移到37℃培养箱中10分钟，以使蛋白基质滴固化。 </li><li>加入450微升事先准备好的器官样生长介质+ R-spondin1的50微升空调每孔媒体。在37℃+ 5％CO 2的过夜孵育板。 </li><li>加入50-100微升R-spondin1空调中介的每天每一个好。每个第 3天更换整个器官样生长介质与新鲜烹制的类器官的生长介质450微升/步骤2.2很好的描述。此外50-100添加微升R-spondin1条件培养基，每孔。 注:该蛋白基质降保持其完整性一个星期，但后7天进行传代类器官。 </li></ol> 4.传代每类器官第 7天<ol><li>在冰上解冻过夜蛋白基质，在4℃下传代的前一天。温暖的无菌24孔板至37℃至少30分钟。准备在步骤2.2中所述类器官的生长培养基，并保持在37℃直到使用。 </li><li>从孵化器中取出类器官板展开板传代冰。从3-4井10毫升吸管吸生长介质启动。 注意:保持在10毫升吸管的送气媒体，因为这将被用于去除在下一步骤中的蛋白基质下降。 </li><li>在如盗版阱，吸管上下以撞出从板的蛋白基质下降。用10毫升吸管的尖端小心刮擦是在撞出蛋白基质降很有帮助。传送内容到15毫升锥形管中。 </li><li>孵育15毫升在冰上锥形管10分钟，并离心在125 xg离心在4℃下5分钟。在锥形管的底部观察到白色沉淀。轻轻吸并丢弃类器官粒料离开100-200微升粒料以上上述大部分上清液/旧类器官生长培养基。 </li><li>通过使用连接到1ml注射器23号针头拆散类器官隐窝。吸并弹出10-15倍，以解离隐窝。减少空气气泡的形成由于过度移液。 </li><li>悬浮组织体于500μl蛋白基质和添加每孔50μl的在一个新的预温热24孔板中。添加预先制备的类器官的生长培养基的450微升+50微升的R-spondin1的每口井条件培养基。孵育在37℃下板过夜。 </li></ol> 5.电镀类器官在14日的模式识别受体刺激与和的PAMP 李斯特菌基因表达分析<ol><li>两天来挑战，连胜了之前L.单核细胞上的BHI琼脂平板上。 </li><li>次日挑L.单核细胞集落，并在37℃下过夜生长在10ml BHI肉汤以200rpm振荡。 </li><li>在冰上解冻过夜蛋白基质，在4℃下类器官挑战的前一天。温暖的无菌24孔板至37℃至少30分钟。准备在步骤2.2中所述类器官的生长培养基，并保持在37℃直到使用。 </li><li>从孵化器中取出化培养，并开始组织体传代。 </li><li>从3-4孔中吸出介质并保持吸气介质在10毫升pi​​pete因为这将被用于去除蛋白质矩阵降。吸取和下来以撞出从板的蛋白基质下降。用10毫升吸管的尖端小心刮擦是在撞出蛋白基质降很有帮助。传送内容到15毫升锥形管中。 </li><li>孵育15毫升在冰上锥形管10分钟。离心机在125×g离心在4℃下10分钟。在锥形管的底部观察到白色沉淀。轻轻吸出上清液留下约100微升残留上清落后。 </li><li>重悬在5ml冰冷的1×PBS中的类器官沉淀并除去一个10微升等分试样用于计数。的10微升等分试样加入到血细胞计数器的中间无玻璃盖玻片。轻轻覆盖在下降顶部的玻璃盖玻片。 注:血细胞计数将与类器官堵塞如果载玻片没有首先去除。 </li><li>通过光学显微镜计数组织体的总数在每个栅格/血细胞计数仪的整个腔室，并确定类器官浓度:NUM共组织体BER每10微升计数。 </li><li>删除15毫升锥形管，将允许组织体的足够数量的待接种用于测定和离心机该悬浮液在125×g离心10分钟，一个适当的体积。 注:每一个24孔板的40-100组织体之间的范围是足够的用于RNA分析。典型的类器官大小的范围可以从300微米至1000微米的直径。 </li><li>吸出上清液并重新悬浮在500毫升的蛋白质基质的粒料不产生气泡。加入50毫升，每孔类器官/蛋白基质悬浮液在24孔板中。 注:蛋白质基质的用于悬浮组织体将用于治疗的条件和技术重复的数目而变化的量。 </li><li>加入500μl类器官生长培养基（无N-乙酰基-半胱氨酸）到每个孔中并在37℃+ 5％CO 2孵育1小时。 </li><li>准备的PAMP的2倍浓度（ 即鞭毛，脂多糖）和生活L.菌等 。现场测量L的OD 菌和牛排在BHI板计数。以1×10 6菌落形成单位（CFU）/ ml的治疗类器官。 注:OD到CFU的决心可能会有所不同细菌的类型，应该事先进行评估，以刺激类器官。例如，0.6≈1×10 9 CFU / ml的李斯特的OD 菌，而且还应当评估独立地之前的实验。 </li><li>连胜走出L的处理股票的连续稀释菌等 ，以准确地确定治疗的CFU /毫升。作者发现，加入100μl的在BHI琼脂平板1×10 8稀释足以计数菌落以确定准确的CFU /毫升。 </li><li>准备L的高温中被杀死的解决方案通过采取活L的1毫升等分试样菌等在5000 xg离心10分钟， 单核细胞溶液和离心机。吸出上清液并洗涤将细菌沉淀用1毫升1×PBS中。 <ol><li>离心机L.在5000 xg离心10分钟， 单核细胞和在1毫升1×PBS中的悬浮颗粒。热火杀L.菌等中在80-90℃进行1小时加热1.7 15mL的聚丙烯管中。文化热的100微升等分打死L.在BHI琼脂平板上的单核细胞 ，以确认没有活的细菌依然存在。 </li><li>加入500微升适当2X PAMP和/或微生物治疗的500微升驻地媒体在由用户确定指定的孔中。孵育在37℃+ 5％CO 2的24小时。 注意:添加2倍PAMP /微生物治疗的500微升等分试样以500μl的驻地媒体会带来处理浓度以1ml的总体积为1x。 </li></ol></li><li>次日，手动计数L。单核细胞集落为准确测定治疗CFU /毫升。从处理过的组织体板吸媒体和洗井Ť重稀土次用1×PBS。 </li><li>通过酚/氯仿11或根据制造商的说明一个RNA提取试剂盒继续进行RNA提取。使用标准的定量RT-PCR 12评估基因表达。 </li></ol> 6.电镀类器官对PAMP和单增李斯特菌挑战上清液中的蛋白质分析14天<ol><li>激发前两天，启动Caco-2细胞的细胞培养物，接种密度≈1×10 6细胞在用1×DMEM + 20％FBS的T-75烧瓶中，并在37℃+ 5孵育的Caco-2细胞％的CO 2。开始L的细菌培养上BHI板菌和在细菌培养箱中于37℃孵育板。 </li><li>次日挑L.单核细胞集落，并在37℃下生长在10ml BHI肉汤过夜。在冰上解冻过夜蛋白基质，在4℃下类器官挑战的前一天。 </li><li>第二天温暖的无菌96孔黑色壁板到37℃，至少30分钟。制备类器官的生长培养基没有在步骤2.2中所述，并保持在37℃直到使用N-乙酰基半胱氨酸。从孵化器中取出化培养，并开始组织体传代。 注意:典型类器官大小可以在直径范围从300微米到1000微米。 </li><li>从3-4孔中吸出介质并保持吸气介质在10毫升pi​​pete因为这将被用于去除蛋白质矩阵降。用移液管重悬以逐出板蛋白质矩阵降。用10毫升吸管轻轻地刮取是撞出蛋白基质下降有帮助。传送内容到15毫升锥形管中。 </li><li>孵育15毫升在冰上锥形管10分钟。离心机在125×g离心在4℃下10分钟。在锥形管的底部观察到白色沉淀。轻轻吸出上清液留下100μl的剩余上清液后面。 </li><li>悬浮颗粒类器官在冰冷的5毫升1X PBS和取出10微升等分用于计数。的10微升等分试样加入到血细胞计数器的中间，并且轻轻覆盖在玻璃盖玻片。 注:血细胞计数将与类器官堵塞如果载玻片没有首先去除。 </li><li>通过光学显微镜计数组织体的总数在血细胞计数器/整个腔室的每一个网格和确定类器官浓度:每10ml计数总组织体的数量。 </li><li>删除15毫升锥形管，将允许组织体的足够数量的待接种用于测定和离心机该悬浮液在125×g离心10分钟，一个适当的体积。 注:蛋白质基质的用于悬浮组织体将用于治疗的条件和技术复制量而变化的量。作者发现，40-100类器官是足够的分泌蛋白分析。 </li><li>吸出上清液并重新悬浮于500μl蛋白基质的颗粒/孔，而不会产生气泡。 注:蛋白质基质的用于悬浮组织体将用于治疗的条件和技术复制量而变化的量。 </li><li>离开至少两列空的96孔黑壁组织培养板中，因为这些将作为井用Caco-2细胞对标准曲线的生成晶种。加入50μl蛋白基质+类器官悬浮液的每孔到预先加温96孔黑壁组织培养板中。储存在37℃的板10分钟以容许蛋白质基质固化。 </li><li>加入100微升类器官生长培养基（无N-乙酰基-半胱氨酸）到每个孔中并在37℃+ 5％CO 2孵育1小时。 </li><li>准备的PAMP的2倍浓度和生活L.菌等。加入100毫升每孔每一个给定处理条件，并培育24小时。 </li><li>翌日板的Caco-2细胞中的标准曲线孔中。通过加热无菌1X PBS，0.25％胰蛋白酶和1x DMEM + 20％FBS T开头的Ø37℃。 </li><li>除去含有Caco-2细胞在T-75烧瓶中，并吸出细胞培养基。清洗细胞，用10ml 1×PBS中。吸出1×PBS中，加2ml的0.25％胰蛋白酶和地点的T-75烧瓶中37℃孵化15分钟。 </li><li>可视化在光学显微镜下的烧瓶以确保细胞脱离然后8毫升1×DMEM + 20％FBS中的添加到分离的Caco-2细胞并转移到15毫升锥形管中。删除10微升等分试样并使用血细胞计数器通过光学显微镜计数细胞。 注:60,000个细胞/孔工作良好和稀释的上部的细胞数可以用于产生标准曲线的情况下进行向下至2,500个细胞/孔。 </li><li>悬浮Caco-2细胞中的蛋白质基质和添加每孔50微升至适当的孔中。允许蛋白质基质在37℃下固化为Caco-2细胞。 注:在Caco-2细胞的生长培养基不需要以下蛋白基质凝固无以复加。 </li><li>以下24个小时的治疗时间为类器官检测，吸并保存类器官细胞的上清液媒体和保持在冰上。用1×PBS洗涤各孔三次。加入100微升100％甲醇中以每孔包括的Caco-2标准曲线孔中，并在4℃下或在冰上孵育20-30分钟来固定类器官。 注:不要求的Caco-2标准曲线孔用1×PBS洗涤，因为它们可以有甲醇直接添加。 </li><li>以下的甲醇孵育，用冰冷的1×PBS洗涤各孔三次。储存在4℃的板长达1-2周。 </li><li>在1微克的浓度加入核染色染料/毫升每孔，用铝箔覆盖板，并在4℃孵育过夜。 </li><li>用96孔板读数器来测量核染色染料的激发/发射（在350nm / 461nm分别）和正常化每类器官以及到Caco-2细胞的标准曲线。 </li><li>进行到下游测定法，诸如酶联免疫吸附<sup&gt; 12，细胞培养物上清液正火细胞数目。 </li></ol>

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The authors declare that they have no competing financial interests.

肠组织体的培养和维护是可以通过用足够的组织培养技术的任何个人掌握的过程。有在传代相比，在更传统的单层生长的细胞时微妙之处，但这些微妙之处不难克服。该方法的关键步骤涉及能够对组织体生长到足够高的密度以获得最佳播种。作为通常可以用细胞系取得了较大的接种密度是不实际的实验，必须缩小与类器官。当有多个处理组，这就变得尤为明显。 该协议的目?...

的能力，以收集和小肠，结肠，胰腺，肝脏和脑培养初级组织体已经描述并有密切关系理解一个多种生理学代表现象为组织生物学1-5令人兴奋的进展。描述小肠类器官培养和维护的方法，首次报道了佐藤等人出来汉斯Clevers 1的实验室。在此之前的方法，收获和证明原肠上皮细胞的培养在限于并在维持上皮细胞的生长是无效的。方法包括通过用酶，例如胶原酶和分散酶，这将最终导致混合初级成纤维细胞6的生长培养组织的离解。这些条件也将时间在维持上皮细胞培养物的限制。最小的无上皮细胞小生境将形成，因为上皮细胞会进入细胞凋亡，由于缺乏适当的生长因子或接触丧失完整性，称为anokis 7。所述3D-类器官培养系统的出现提供了含有肠细胞类型的光谱中持续培养1培养原肠细胞的方法。这些上皮组织体具有以上的细胞系是它们是由多种分化的细胞的优点，并更好地模仿它们从体内 8衍生的器官。该过程最终"生长在培养皿迷你肠"已被证明是用于评估肠上皮细胞在不同刺激的反应的有价值的工具。调查主肠细胞微生物病原体相关的分子模式（PAMP）的相互作用是相关的免疫学领域，因为这些分子模式可以调节从主机和微生物9多样的响应。不仅可以调查，现在寻求与鼠标类器官这些互动，但他们可以从人类以及2中培养。该技术具有显着改变的个性化医学的潜力，很容易让人猜测的进步，这种技术将在不久的将来成为可能。 该方法的总的目标是提供一种用于培养，扩展的协议，和治疗肠组织体与各种刺激。这种刺激可以最终范围从疫苗，细菌的PAMP，活的病原体，胃肠（GI）和癌症治疗。小鼠肠道组织体的分离和培养是改编自佐藤等人虽然有从原来的方法略有偏差，最终产品是化培养按照本协议时仍然实现。此方法集中在与非同质细胞结构，其必须基于小区n导电的测定时，可以考虑到工作时，描述为合适正常化的适当技术红棕色。

<table><tbody><tr><td>Fetal Bovine Serum (FBS)</td><td>Atlanta Biologicals</td><td>S11050</td><td>(Section 1,3,6) Or equivalent brand</td></tr><tr><td>Sorvall Legend XTR Centrifuge</td><td>Thermo</td><td></td><td>(Section 1,3)</td></tr><tr><td>DMEM</td><td>GE Healthcare</td><td>Sh30243.01</td><td>(Section 1,6) For Caco-2 and HEK293 Rspondin1 cells</td></tr><tr><td>HEK293T-Rspondin1 secreting cell line</td><td></td><td></td><td>(Section 1) Described and modified from Kim, K.A. et al. Lentiviral particles contained RSPO1(NM_138683) ORF cDNA cloned into a pReceiver-Lv105 backbone custom ordered and purchased from GeneCopoeia.&amp;nbsp;</td></tr><tr><td>50 ml conical tube</td><td>Falcon</td><td>352070</td><td>(Section 1) Or equivalent brand</td></tr><tr><td>T-175 Flask</td><td>Corning</td><td>431079</td><td>(Section 1) Or equivalent brand&amp;nbsp;</td></tr><tr><td>Protein Matrix</td><td>Corning</td><td>356231</td><td>(Section 2,3,4,5,6) Matrigel Growth Factor Reduced&amp;nbsp;</td></tr><tr><td>HyClone Dulbecco&#039;s (DPBS)</td><td>GE Healthcare</td><td>SH30264.01</td><td>(Section 2,3)</td></tr><tr><td>DMEM/F12&amp;nbsp;</td><td>Life Technologies</td><td>12634-010</td><td>(Section 2,3) Advanced DMEM/F12</td></tr><tr><td>Corning 24 Well TC Plates</td><td>Corning</td><td>3524</td><td>(Section 2)</td></tr><tr><td>N2 Supplement 100x&amp;nbsp;</td><td>Life Technologies</td><td>17502-048</td><td>(Section 2)</td></tr><tr><td>B27 without vitamin A 50x&amp;nbsp;</td><td>Life Technologies</td><td>12587-010&amp;nbsp;</td><td>(Section 2)</td></tr><tr><td>Trizol</td><td>Life Technologies</td><td>15596-026</td><td>(Section 2)</td></tr><tr><td>Glutamine Supplement (Glutamax)</td><td>Life Technologies</td><td>35050-061</td><td>(Section 2) Can Combine with Advanced DMEM/ F12</td></tr><tr><td>HEPES (1 M)</td><td>Life Technologies</td><td>15630-080</td><td>(Section 2) Can Combine with Advanced DMEM/ F12&amp;nbsp;</td></tr><tr><td>10ml Serological Pipet</td><td>Falcon</td><td>357551</td><td>(Section 2) Or equivalent brand</td></tr><tr><td>Murine Noggin</td><td>Peprotech</td><td>250-38</td><td>(Section 2) Stock = 100 mg/ml</td></tr><tr><td>N-Acetyl-L-cysteine</td><td>Sigma-Aldrich</td><td>A9165</td><td>(Section 2) Stock = 1M</td></tr><tr><td>Recombinant Mouse EGF</td><td>Biolegend</td><td>585608</td><td>(Section 2) Stock = 500 mg/ml</td></tr><tr><td>Rocker Variable</td><td>Bioexpres</td><td></td><td>(Section 3)</td></tr><tr><td>dissecting scissors</td><td></td><td></td><td>(Section 3)</td></tr><tr><td>forceps</td><td></td><td></td><td>(Section 3)</td></tr><tr><td>glass slides</td><td></td><td></td><td>(Section 3)</td></tr><tr><td>dissecting tweezers</td><td></td><td></td><td>(Section 3)</td></tr><tr><td>25 ml Serological Pipet</td><td>Falcon</td><td></td><td>(Section 3)</td></tr><tr><td>EDTA&amp;nbsp;</td><td>Sigma-Aldrich</td><td>SLBB9821</td><td>(Section 3) 0.5M or alternative TC grade EDTA</td></tr><tr><td>Sterile Petri Dish 100mm x 15mm</td><td>Fisher</td><td>FB0875712</td><td>(Section 3) Or equal sized TC dish</td></tr><tr><td>1ml Syringe</td><td>Becton Dickinson</td><td>309659</td><td>(Section 4)</td></tr><tr><td>Precision Glide Needle</td><td>Becton Dickinson</td><td>305120</td><td>(Section 4) 23G x 1 1/4 (0.6mm x 30mm)</td></tr><tr><td>Flagellin from Bacillus subtilis</td><td>Invivogen</td><td>tlrl-bsfla&amp;nbsp;</td><td>(Section 5,6)</td></tr><tr><td>Listeria monocytogenes</td><td>ATCC</td><td>19115</td><td>(Section 5,6)&amp;nbsp;(Murray&amp;nbsp;et&amp;nbsp;al.)&amp;nbsp;</td></tr><tr><td>Hemocytometer</td><td>Sigma-Aldrich</td><td>Z359629-1EA</td><td>(Section 5,6)Or equivalent brand</td></tr><tr><td>BBL Brain Heart Infusion Agar</td><td>Becton Dickinson</td><td>211065</td><td>(Section 5)</td></tr><tr><td>Bacto Brain Heart Infusion</td><td>Becton Dickinson</td><td>237500</td><td>(Section 5)</td></tr><tr><td>Caco-2</td><td>ATCC</td><td>HTB-37</td><td>(Section 6)</td></tr><tr><td>Trypsin&amp;nbsp;</td><td>gibco</td><td>25200056</td><td>(section 6)</td></tr><tr><td>Methanol</td><td>Fisher</td><td>A412-4</td><td>(Section 6)</td></tr><tr><td>SpectraMax M5</td><td>Molecuar Devices</td><td></td><td>(Section 6)</td></tr><tr><td>96 Well Assay Plate</td><td>Corning</td><td>3603</td><td>(Section 6) Black Plate, Clear Bottom TC treated</td></tr><tr><td>Nuclear Staining Dye</td><td>Life Technologies</td><td>H1399</td><td>(section 6) Hoechst 33342</td></tr><tr><td>T-75 Flask</td><td>Corning</td><td>430641</td><td>(Section 6) Or equivalent brand</td></tr><tr><td>15 ml conical tube</td><td>Falcon</td><td>352096</td><td>(Section1,3) Or equivalent brand</td></tr><tr><td>1.7 ml polypropylene tube</td><td>Bioexpress</td><td>C-3262-1</td><td>Or equivalent brand</td></tr><tr><td>Quick-RNA MiniPrep</td><td>Zymo Research</td><td>R1054</td><td>Or equivalent brand</td></tr><tr><td>TNF-alpha&amp;nbsp;</td><td>Applied Biosystems</td><td>Mm 00443260_g1</td><td>Taqman gene expression assay kit</td></tr><tr><td>IL-6&amp;nbsp;</td><td>Applied Biosystems</td><td>(Mm 00446190_m1</td><td>Taqman gene expression assay kit</td></tr><tr><td>IL-1beta</td><td>Applied Biosystems</td><td>Mm 00434228_m1</td><td>Taqman gene expression assay kit</td></tr><tr><td>IL-18</td><td>Applied Biosystems</td><td>Mm 00434225_m1</td><td>Taqman gene expression assay kit</td></tr><tr><td>18s</td><td>Applied Biosystems</td><td>Hs 99999901_s1</td><td>Taqman gene expression assay kit</td></tr><tr><td>7500 Fast Real Time PCR System</td><td>Applied Biosystems</td><td></td><td></td></tr><tr><td>Nexus gradient Mastercycler</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>TaqMan Fast Universal PCR Master Mix</td><td>Life Technologies</td><td>4352042</td><td></td></tr><tr><td>High Capacity cDNA Reverse Transcription Kit</td><td>Life Technologies/Applied Biosystems</td><td>4368814</td><td></td></tr><tr><td>Fast Optical 96-Well Reaction Plate, 0.1 mL</td><td>Life Technologies/Applied Biosystems</td><td>4346907</td><td></td></tr><tr><td>Recombinant Mouse R-Spondin 1 Protein</td><td>R&amp;amp;D Systems</td><td>3474-RS-050</td><td>500 ng/ml</td></tr><tr><td>chloroform</td><td>Sigma-Aldrich</td><td>C7559</td><td></td></tr></tbody></table>

the ex vivo culture and pattern recognition receptor stimulation of mouse intestinal organoids

主肠组织体是具有显著影响粘膜免疫学领域的潜力的一个有价值的模型系统。然而，的类器官生长特性的复杂性进行了调查显著警告。具体而言，每个单独的器官样的生长模式是高度可变的，并创建在培养的上皮细胞的异质群体。有了这样的警告，共同组织培养做法不能简单应用到类器官系统，由于蜂窝结构的复杂性。计数和电镀对细胞数，这是单独分离的细胞，如细胞系共同仅基于，不是组织体的可靠方法，除非被施加一些正常化技术。正火总蛋白含量因驻地蛋白基质制成复合物。在细胞数量，形状和细胞类型而言，这些特征应在评估分泌CON时加以考虑从类器官质量帐篷。该协议已经产生勾勒出一个简单的程序，以文化和治疗小肠类器官与病原微生物和病原相关分子模式（PAMP）。它还强调当蛋白质分析被这样的挑战之后进行应该应用的标准化技术。

按照此协议，培养肠类器官时，球体特征形类器官会收获后出现。加入R-spondin1条件培养基的每日将启动类器官的生长和出芽。组织体的生长显示于图 1A - F，且代表肠组织体上天1，2，4，5，6和第14天图1F表示在第14天组织体的非均匀生长特性。 一旦组织体生长到足够数量的，?...

Watch this Scientific Journal Video about The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids at JoVE.com

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

这里，收获，维护和治疗与病原体相关的分子模式（PAMP），并李斯特菌小鼠小肠组织体的协议被描述，以及强调基因表达和蛋白质正确规范化技术。

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 ...

the-ex-vivo-culture-pattern-recognition-receptor-stimulation-mouse

Research

JoVE Journal

Immunology and Infection

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

13.5K 次观看.  Virginia Maryland College of Veterinary Medicine, Virginia Tech. 这里，收获，维护和治疗与病原体相关的分子模式（PAMP），并李斯特菌小鼠小肠组织体的协议被描述，以及强调基因表达和蛋白质正确规范化技术。 

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

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

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 Organoid Reconstitution Assay (ORA) for the Functional Analysis of Intestinal Stem and Niche Cells

The intestinal epithelium is characterized by an extremely rapid turnover rate. In mammals, the entire epithelial lining is renewed within 4 - 5 days. Adult intestinal stem cells reside at the bottom of the crypts of Lieberk&#252;hn, are earmarked by expression of the Lgr5 gene, and preserve homeostasis through their characteristic high proliferative rate1. Throughout the small intestine, Lgr5+ stem cells are intermingled with specialized secretory cells called Paneth cells. Paneth cells secrete antibacterial compounds (i.e., lysozyme and cryptdins/defensins) and exert a controlling role on the intestinal flora. More recently, a novel function has been discovered for Paneth cells, namely their capacity to provide niche support to Lgr5+ stem cells through several key ligands as Wnt3, EGF, and Dll12.
When isolated ex vivo and cultured in the presence of specific growth factors and extracellular matrix components, whole intestinal crypts give rise to long-lived and self-renewing 3D structures called organoids that highly resemble the crypt-villus epithelial architecture of the adult small intestine3. Organoid cultures, when established from whole crypts, allow the study of self-renewal and differentiation of the intestinal stem cell niche, though without addressing the contribution of its individual components, namely the Lgr5+ and Paneth cells.
Here, we describe a novel approach to the organoid assay that takes advantage of the ability of Paneth and Lgr5+ cells to associate and form organoids when co-cultured. This approach, here referred to as &#34;organoid reconstitution assay&#34; (ORA), allows the genetic and biochemical modification of Paneth or Lgr5+ stem cells, followed by reconstitution into organoids. As such, it allows the functional analysis of the two main components of the intestinal stem cell niche.

The Organoid Reconstitution Assay ORA for the Functional Analysis of Intestinal Stem and Niche Cells

Intestinal organoid cultures are established from whole crypts and do not allow the analysis of self-renewal and differentiation in a cell-specific fashion. This protocol describes reconstitution of sorted stem (Lgr5+) and niche (Paneth) cells, which give rise to organoids while enabling their prior biochemical and genetic modification and functional analysis.

化重构法 (的) 对肠道干细胞和小生境细胞功能分析的研究

The intestinal epithelium is characterized by an extremely rapid turnover rate. In mammals, the entire epithelial lining is renewed within 4 - 5 days. ...

生物工程

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

The intestine displays an architecture of repetitive crypt structures consisting of different types of epithelial cells, lamina propia containing immune cells, and stroma. All of these heterogeneous cells contribute to intestinal homeostasis and participate in antimicrobial host defense. Therefore, identifying a surrogate model for studying immune response and antimicrobial activity of the intestine in an in vitro setting is extremely challenging. In vitro studies using immortalized intestinal epithelial cell lines or even primary crypt organoid culture do not represent the exact physiology of normal intestine and its microenvironment. Here, we discuss a method of culturing mouse colon tissue in a culture dish and how this ex vivo organ culture system can be implemented in studies related to antimicrobial host defense responses. In representative experiments, we showed that colons in organ culture express antimicrobial peptides in response to exogenous IL-1&#946; and IL-18. Further, the antimicrobial effector molecules produced by the colon tissues in the organ culture efficiently kill Escherichia coli in vitro. This approach, therefore, can be utilized to dissect the role of pathogen- and danger-associated molecular patterns and their cellular receptors in regulating intestinal innate immune responses and antimicrobial host defense responses.

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 ...

免疫与感染

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 ...

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding primary 3D epithelial structures in vitro. Both the architecture and physiological properties of these &#39;mini-guts&#39;, also called organoids, closely resemble their in vivo counterparts. This makes them an attractive model system for the small intestinal epithelium. Using retroviral transduction, functional genetics can now be performed by conditional gene overexpression or knockdown. This video demonstrates the procedure of organoid culture, the generation of retroviruses, and the retroviral transduction of organoids to assist phenotypic analysis of the small intestinal epithelium in vitro. This novel organotypic model system in combination with retroviral mediated gene expression provides a valuable tool for rapid analysis of gene function in vitro without the need of costly and time-consuming generation for transgenic animals.

This protocol explains primary Lgr5-positve organoid culture and the subsequent performance of retroviral transduction. This enables Cre-inducible overexpression or knockdown of the delivered transgene and allows functional studies to be carried out in the novel in vitro organotypic model system.

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 ...

生物学

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 ...

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 ...

Culture Methods to Study Apical-Specific Interactions using Intestinal Organoid Models

The lining of the gut epithelium is made up of a simple layer of specialized epithelial cells that expose their apical side to the lumen and respond to external cues. Recent optimization of in vitro culture conditions allows for the re-creation of the intestinal stem cell niche and the development of advanced 3-dimensional (3D) culture systems that recapitulate the cell composition and the organization of the epithelium. Intestinal organoids embedded in an extracellular matrix (ECM) can be maintained for long-term and self-organize to generate a well-defined, polarized epithelium that encompasses an internal lumen and an external exposed basal side. This restrictive nature of the intestinal organoids presents challenges in accessing the apical surface of the epithelium in vitro and limits the investigation of biological mechanisms such as nutrient uptake and host-microbiota/host-pathogen interactions. Here, we describe two methods that facilitate access to the apical side of the organoid epithelium and support the differentiation of specific intestinal cell types. First, we show how ECM removal induces an inversion of the epithelial cell polarity and allows for the generation of apical-out 3D organoids. Second, we describe how to generate 2-dimensional (2D) monolayers from single cell suspensions derived from intestinal organoids, comprised of&#160;mature and differentiated cell types. These techniques provide novel tools to study apical-specific interactions of the epithelium with external cues in vitro and promote the use of organoids as a platform to facilitate precision medicine.

Here we present two protocols that allow the modeling of intestinal apical-specific interactions. Organoid-derived intestinal monolayers and Air-Liquid Interface (ALI) cultures facilitate the generation of well-differentiated epithelia accessible from both luminal and basolateral sides, whereas polarity-inverted intestinal organoids expose their apical side and are amenable to high throughput assays.

使用肠道器官模型研究猿特异性相互作用的文化方法

The lining of the gut epithelium is made up of a simple layer of specialized epithelial cells that expose their apical side to the lumen and respond to ...

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 ...

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