这项研究是由荷兰癌症协会 (凯) 资助的。EMCR 2012-5473) 和世界癌症研究基金国际 (WCRF; 2014-1181 号项目)

所有的程序都是根据当地的动物福利法和指导方针进行的。
1. 文书、文化媒体和菜肴的准备
<ol><li>高压釜1套肠剪刀、普通剪刀和无菌容器中的镊子。</li>
	<li>在37° c 的恒温箱中放置一个96井 (平底) 盘。</li>
	<li>用材料表中所列试剂制备10毫升的完整培养基。</li>
	<li>在37° c 的水浴中孵育整个培养基。</li>
	<li>把地下室的膜放在冰桶里融化。重组后的基底膜将在4° c 时变为液体。</li>
	<li>用冷磷酸盐缓冲盐水或简称 PBS (4 ° c) 填充4培养皿。</li>
</ol>2. 小肠棺的分离
<ol><li>牺牲 CO2吸入Lgr5-eGFP-IRES-CreERt2鼠标在 C57BL6/J 背景上。用剪刀对腹膜进行纵向解剖。</li>
	<li>用钳子抓住胃, 把它切成横。</li>
	<li>使用肠剪刀从现在起, 拉出肠道和放置在一个培养皿含有 PBS。 注: 肠剪刀有尖锐和钝尖。这些剪刀的钝尖是为了不破坏地穴绒毛建筑, 同时打开肠道。</li>
	<li>先把钝尖放进胃里, 然后轻轻地把它穿过幽门。继续用剪刀剪, 用钳子拉。</li>
	<li>一旦整个小肠被纵向打开, 在冷 pbs 中用镊子把它拿出来, 用 U 形的动作轻轻地冲洗它在 pbs 溶液中。</li>
	<li>一旦所有的粪便残渣被清除, 继续在砧板上平坦的肠道, 腔一侧向上。腔侧是容易辨认的由血管的缺席和它的苍白出现与外在部分比较。</li>
	<li>用玻璃滑动, 通过刮平的肚腑轻轻地去除绒毛。沿着整个组织的长度执行这一步骤两次。</li>
	<li>将不育的手术刀片切成 2-5 毫米的小肠。</li>
	<li>将小肠碎片放在50毫升的管中, 其中含有10毫升的冰冷 PBS。</li>
	<li>通过在 PBS 中上下移, 清除组织碎片, 去除残留的杂质。丢弃上清, 重复此步骤, 直到 PBS 完全清楚。</li>
	<li>加入15毫升冷 pbs, 使 pbs 的最终总容量为25毫升。在4° c 的滚筒上加入2毫米乙酸酸 (EDTA), 孵育45分钟。</li>
	<li>丢弃 PBS/EDTA。</li>
	<li>添加10毫升 PBS 和分离的棺通过严厉移组织碎片上下 (至少三次)。收集上清。</li>
	<li>重复步骤2.13 四次。添加培养基以达到50毫升的最终体积。</li>
	<li>通过离心 (300 x g 为5分钟) 将细胞颗粒。</li>
	<li>重10毫升培养基中的颗粒和棺的离心 (80 x g 3 分钟)。</li>
</ol>3. 单细胞制备
<ol><li>丢弃颗粒状棺的上清液, 在1毫升胰蛋白酶样酶中重它们, 连同50µL dnasei (50 µg/毫升)。</li>
	<li>在32° c 的水浴中孵育细胞2分钟。</li>
	<li>添加10毫升培养基。将棺分离成单个细胞, 移至少5次。</li>
	<li>过滤解决方案通过一个40µm 过滤器 (以消除团簇和其他杂质) 和颗粒的单个细胞在 300 x g 5 分钟。</li>
</ol>4. 流式细胞仪和电镀
<ol><li>准备50毫升的1x 汉克的缓冲盐溶液或 HBSS/2%fetal 牛血清 (FCS) 溶液和重每 106细胞1毫升的颗粒</li>
	<li>添加到细胞悬浮 BV421 林- (CD31, CD45, TER119) 在浓度为 1:100, CD24-APC 和 CD117-PE 都在1:250 抗体浓度为30分钟。</li>
	<li>将Lgr5+ 的干细胞和氏细胞分类成单独的低绑定管。有关Lgr5+ 和氏单元格的排序协议的详细信息, 请参阅罗斯et al.7和 Schewe et al.6<ol>
<li>离心分离的细胞在 300 x g 为 5 min. 重100µL 介质中的单元格, 其中的组件为 1.3 (请参见材料目录)。</li>
	</ol>
</li>
	<li>治疗 (例如, 通过化学或基因修饰) 氏细胞 (n = 2000) 或Lgr5+干细胞 (n = 2000)。<ol>
<li>为了治疗细胞, 使用5µL 脂质体介导的转染试剂在总体积100µL 培养基和小干扰 RNA (siRNA) 的浓度 100 nM。孵育30分钟, 在37° c 为 siRNA 或为所选的修改所需的时间。</li>
	</ol>
</li>
	<li>在500µL 培养基中两次冲洗细胞。</li>
	<li>离心机在 300 x g 为 5 min, 重10µL 培养基中的细胞。</li>
	<li>将两个蜂窝组件 (氏或Lgr5+单元格) 集中在20µl 总体积和离心机中, 在 300 x g 处进行5分钟的 RT。</li>
	<li>在 RT 中共孵化氏和Lgr5+单元格10分钟。</li>
	<li>去除10µL 的上清液, 留下一个半月板的液体, 以确保不吸细胞颗粒。</li>
	<li>在 5ml 96 井板中, 将重组基底膜的30µL 添加到细胞中, 总体积为40µL, 重细胞和板。10分钟后, 添加200µL 的完整介质 (请参见材料表)。每48小时更换培养基。</li>
	<li>计数化多样性在天5。</li>
</ol>

<ol>
	<li>Barker, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+stem+cells+in+small+intestine+and+colon+by+marker+gene+Lgr5.">Identification of stem cells in small intestine and colon by marker gene Lgr5.</a> Nature. 449, 1003-1007 (2007).</li><li>Sato, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paneth+cells+constitute+the+niche+for+Lgr5+stem+cells+in+intestinal+crypts.">Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts.</a> Nature. 469, 415-418 (2011).</li><li>Sato, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+Lgr5+stem+cells+build+crypt-villus+structures+in+vitro+without+a+mesenchymal+niche.">Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.</a> Nature. 459, 262-265 (2009).</li><li>Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+Development+and+Disease+with+Organoids.">Modeling Development and Disease with Organoids.</a> Cell. 165, 1586-1597 (2016).</li><li>Cantrell, M. A., Kuo, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoid+modeling+for+cancer+precision+medicine.">Organoid modeling for cancer precision medicine.</a> Genome Med. 7, 32 (2015).</li><li>Schewe, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secreted+Phospholipases+A2+Are+Intestinal+Stem+Cell+Niche+Factors+with+Distinct+Roles+in+Homeostasis,+Inflammation,+and+Cancer.">Secreted Phospholipases A2 Are Intestinal Stem Cell Niche Factors with Distinct Roles in Homeostasis, Inflammation, and Cancer.</a> Cell Stem Cell. 19, 38-51 (2016).</li><li>Roth, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paneth+cells+in+intestinal+homeostasis+and+tissue+injury.">Paneth cells in intestinal homeostasis and tissue injury.</a> PLoS One. 7, e38965 (2012).</li><li>Rodriguez-Colman, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interplay+between+metabolic+identities+in+the+intestinal+crypt+supports+stem+cell+function.">Interplay between metabolic identities in the intestinal crypt supports stem cell function.</a> Nature. 543, 424-427 (2017).</li><li>Dow, L. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apc+Restoration+Promotes+Cellular+Differentiation+and+Reestablishes+Crypt+Homeostasis+in+Colorectal+Cancer.">Apc Restoration Promotes Cellular Differentiation and Reestablishes Crypt Homeostasis in Colorectal Cancer.</a> Cell. 161, 1539-1552 (2015).</li><li>Yilmaz, O. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mTORC1+in+the+Paneth+cell+niche+couples+intestinal+stem-cell+function+to+calorie+intake.">mTORC1 in the Paneth cell niche couples intestinal stem-cell function to calorie intake.</a> Nature. 486, 490-495 (2012).</li><li>Igarashi, M., Guarente, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mTORC1+and+SIRT1+Cooperate+to+Foster+Expansion+of+Gut+Adult+Stem+Cells+during+Calorie+Restriction.">mTORC1 and SIRT1 Cooperate to Foster Expansion of Gut Adult Stem Cells during Calorie Restriction.</a> Cell. , 436-450 (2016).</li></ol>

作者没有竞争的财务利益或其他利益冲突

该方法允许对肠道干细胞生态位的两个基本成分, 即Lgr5+和氏细胞进行精细的功能分析。此方法以前已被我们和其他一些轻微修改6,10,11。在这里, 我们提出了作为一个可重复和标准化的实验室协议的过程。此外, 我们报告的影响, Apc下调在Lgr5+干细胞, 作为一个例子的可能性实施遗传 (或生物化...

肠上皮是哺乳动物体内最快速的更新组织, 因此, 它一直是大量研究的对象, 目的是在隐窝底部的成体干细胞的鉴定和功能表征。Lieberkühn, 由Lgr5基因的表达式指定, 并依赖于标准 Wnt 信号1。值得注意的是, Lgr5+干细胞是由专门的小生境细胞 (氏细胞) 所支持的, 它也依赖于其成熟度 2的 Wnt 信号。同时, 这两种细胞类型的基础上的肠道上皮内膜的自我更新和保持每日的稳态平衡: Lgr5+ 干细胞迅速分化并引起祖细胞和更专门的肠上皮细胞;氏细胞提供必要的利基因素 (如, Dll1, Wnt3, EGF) Lgr5+ 干细胞2。肠道棺的能力, 当镀前体形成组织和更新结构称为 organoids, 或 "mini-guts", 已被作为一个实验工具, 以提供洞察的过程, 如自我更新和正常和病理条件下的分化, 包括癌症4。从老鼠和人类样本中的几个组织, 包括肠道、胰腺、肝脏和肾脏, 都建立了化的培养4。这些化培养物的提取方法和生长因子是组织特异性的, 其目的是促使多分化, 并尽可能地模仿原干细胞生态位在体内.Organoids 可能有潜在的应用, 包括基因疾病的治疗, 对癌症的疗效评估, 药物毒性分析, 或器官的研究体外5。
总的来说, 从组织标本中建立的化文化的主要局限是缺乏细胞特异性。例如, 肠道 organoids 建立从整个肠道棺不允许分析的个别细胞成分包含在组织来源 (如, 整个棺包含Lgr5+, 氏, 和祖细胞)。
在这里, 我们描述了一种新的方法, 称为化, 结合了肠道的优势, 其最基本的组成部分, 即词干 (Lgr5+) 和利基 (氏) 细胞的功能分析。这是通过氏和Lgr5+单元格的独特功能实现的, 当 co-incubated 并引发 organoids2、610时, 它们之间的物理关联。我们利用这一功能, 并预处理两个细胞类型, 然后再允许他们重建 organoids。当这样做时, 每个细胞成分可以暴露在任何给定的药物, 生长因子, 生化抑制剂, 基因改造, 或化学处理之前, 重组和化形成。因此, 使用 "化验" 将允许确定特定药物治疗或基因修饰对干细胞或其生态位对应物是否有特定的影响。

<table><tbody><tr><td>Advanced DMEM F12 *</td><td>Thermo Fisher Scientific</td><td>12634-010</td><td></td></tr><tr><td>Glutamax *</td><td>Thermo Fisher Scientific</td><td>35050061</td><td>Final concentration: 10 mM</td></tr><tr><td>Penicillin/streptomycin *</td><td>Thermo Fisher Scientific</td><td>15140122</td><td>Final concentration: 1%</td></tr><tr><td>Hepes *</td><td>Thermo Fisher Scientific</td><td>15630080</td><td>Final concentration: 10 mM</td></tr><tr><td>Recombinant murine EGF *</td><td>Thermo Fisher Scientific</td><td>PMG8041</td><td>Final concentration: 50 ng/ml</td></tr><tr><td>Recombinant murine Noggin *</td><td>Peprotech</td><td>250-38</td><td>Final concentration: 100 ng/ml</td></tr><tr><td>y27632 *</td><td>Sigma</td><td>Y0503</td><td>Final concentration: 10 &amp;micro;M</td></tr><tr><td>Jagged-1 *</td><td>Anaspec Bio</td><td>AS-61298</td><td>Final concentration: 1 &amp;micro;M</td></tr><tr><td>Recombinant murine R-spondin *</td><td>R&amp;amp;D systems</td><td>3474-RS-050</td><td>Final concentration: 1 mg/ml</td></tr><tr><td>Flexitube Gene solution siRNA APC</td><td>Qiagen</td><td>GS11789</td><td>Final concentration: 100 nM</td></tr><tr><td>Lipofectamine 2000</td><td>Thermo Fisher Scientific</td><td>11668019</td><td></td></tr><tr><td>CD24 APC</td><td>Biolegend</td><td>101814</td><td></td></tr><tr><td>c-kit PE</td><td>Biolegend</td><td>105808</td><td></td></tr><tr><td>BV 421 CD31</td><td>Biolegend</td><td>102424</td><td></td></tr><tr><td>BV 421 CD45</td><td>Biolegend</td><td>103134</td><td></td></tr><tr><td>BV421 TER119</td><td>Biolegend</td><td>116234</td><td></td></tr><tr><td>HBSS</td><td>Thermo Fisher Scientific</td><td>14180046</td><td></td></tr><tr><td>B6.129P2-Lgr5tm1(cre/ERT2)Cle/J</td><td>Jackson Laboratories</td><td>008875</td><td></td></tr><tr><td>Low binding tubes</td><td>Eppendorf</td><td>Z666548-250EA</td><td></td></tr><tr><td>Matrigel</td><td>Corning</td><td>356230</td><td></td></tr><tr><td>* The combination of these reagents constitutes the complete culture medium</td><td></td><td></td><td></td></tr></tbody></table>

the organoid reconstitution assay (ora) for the functional analysis of intestinal stem and niche cells

小肠上皮的特点是以极快的速度流动。在哺乳动物中, 整个上皮衬里在 4-5 天内更新。成体肠道干细胞位于 Lieberkühn 棺的底部, 由Lgr5基因的表达指定, 并通过其特有的高增殖率1保持稳态。在整个小肠中, Lgr5+ 干细胞与称为氏细胞的特殊分泌细胞混合在一起。氏细胞分泌抗菌化合物 (即、溶菌酶和凹/防御), 并对肠道菌群发挥控制作用。最近, 发现了一个新的功能, 氏细胞, 即他们的能力提供利基支持的Lgr5+干细胞通过几个关键配体作为 Wnt3, EGF, 和 Dll12。
当孤立的ex 体内和培养在特定生长因子和细胞外基质成分的存在下, 整个肠道棺产生长寿命和更新3D 结构称为 organoids, 高度类似于地穴绒毛成人小肠的上皮结构3。化的文化, 当建立从整个棺, 允许研究的自我更新和分化的肠道干细胞利基, 但没有解决的贡献, 其各个组成部分, 即Lgr5+和氏细胞。
在这里, 我们描述了一种新的方法, 化分析利用的能力, 氏和Lgr5+细胞关联和形成 organoids 时共。这种方法, 这里称为 "化重构化验" (), 允许遗传和生物化学的修改氏或Lgr5+ 干细胞, 然后重组成 organoids。因此, 它允许功能分析的两个主要组成部分的肠道干细胞利基。

化重构试验允许对小肠上皮细胞的基本生态位和干细胞成分进行单独的功能分析, 这是由Apc基因的小干扰 RNA (siRNA) 证明的。
为了达到这个目的, 我们首先利用荧光活化细胞分类 () 分离 8000 Lgr5+细胞和6000氏细胞从近亲 C57BL6/J 小鼠。在图 1a中, 描述了该方法的流程图。化重组能力的评估...

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

化重构法 (的) 对肠道干细胞和小生境细胞功能分析的研究

肠道化文化是建立在整个棺, 不允许分析自我更新和分化的细胞特有的方式。本协议描述了对排序后的词干 (Lgr5+) 和小生境 (氏) 单元的重构, 这些细胞在进行生物化学和遗传修饰和功能分析之前, 会产生 organoids。

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

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

Unit 1

Stem Cell Biology And Renewal in Epithelial Tissue

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11.3K 次观看.  Erasmus University Medical Center. 肠道化文化是建立在整个棺, 不允许分析自我更新和分化的细胞特有的方式。本协议描述了对排序后的词干 (Lgr5+) 和小生境 (氏) 单元的重构, 这些细胞在进行生物化学和遗传修饰和功能分析之前, 会产生 organoids。

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

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 ...

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癌症研究

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

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

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

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

该<em&gt;离体</em&gt;文化与模式识别小鼠肠道组织体受体刺激

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

免疫与感染

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

从整体组织和活检建立人类上皮Enteroids和Colonoids的

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

医学

Generation, Maintenance, and Characterization of Human Pluripotent Stem Cell-derived Intestinal and Colonic Organoids

Intestinal regional specification describes a process through which unique morphology and function are imparted to defined areas of the developing gastrointestinal (GI) tract. Regional specification in the intestine is driven by multiple developmental pathways, including the bone morphogenetic protein (BMP) pathway. Based on normal regional specification, a method to generate human colonic organoids (HCOs) from human pluripotent stem cells (hPSCs), which include human embryonic stem cells (hES) and induced pluripotent stem cells (iPSCs), was developed. A three-day induction of BMP signaling sufficiently patterns mid/hindgut tube cultures into special AT-rich sequence-binding protein 2 (SATB2)-expressing HCOs containing all of the main epithelial cell types present in human colon as well as co-developing mesenchymal cells. Omission of BMP (or addition of the BMP inhibitor NOGGIN) during this critical patterning period resulted in the formation of human intestinal organoids (HIOs). HIOs and HCOs morphologically and molecularly resemble human developing small intestine and colon, respectively. Despite the utility of HIOs and HCOs for studying human intestinal development, the generation of HIOs and HCOs is challenging. This paper presents methods for generating, maintaining, and characterizing HIOs and HCOs. In addition, the critical steps in the protocol and troubleshooting recommendations are provided.

<meta charset="utf8"></head><body>人多能干细胞来源的肠道和结肠类器官的生成、维持和表征

Here, detailed methods for generating, maintaining, and characterizing human pluripotent stem cell-derived small intestinal and colonic organoids are described. These methods are designed to improve reproducibility, expand scalability, and decrease the working time required for plating and passaging of organoids.

人多能干细胞来源的肠道和结肠类器官的生成、维持和表征

Intestinal regional specification describes a process through which unique morphology and function are imparted to defined areas of the developing ...

发育生物学

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

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

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

结合人体类器官和芯片器官技术，对肠道区域特异性功能进行建模

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

生物工程

Organoid-Derived Epithelial Monolayer: A Clinically Relevant In Vitro Model for Intestinal Barrier Function

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations as the former do not faithfully represent original tissue physiology, and the availability of the latter is limited. Hence, their application hampers fundamental and drug development research. Adult stem-cell-based organoids (henceforth referred to as organoids) are miniatures of normal or diseased epithelial tissue from which they are derived. They can be established very efficiently from different gastrointestinal (GI) tract regions, have long-term expandability, and simulate tissue- and patient-specific responses to treatments in vitro. Here, the establishment of intestinal organoid-derived epithelial monolayers has been demonstrated along with methods to measure epithelial barrier integrity, permeability and transport,&#160;antimicrobial protein secretion, as well as histology. Moreover, intestinal organoid-derived monolayers can be enriched with proliferating stem and transit-amplifying cells as well as with key differentiated epithelial cells. Therefore, they represent a model system that can be tailored to study the effects of compounds on target cells and their mode of action. Although organoid cultures are technically more demanding than cell lines, once established, they can reduce failures in the later stages of drug development as they truly represent in vivo epithelium complexity and interpatient heterogeneity.

Here, we describe the preparation of human organoid-derived intestinal epithelial monolayers for studying intestinal barrier function, permeability, and transport. As organoids represent original epithelial tissue response to external stimuli, these models combine the advantages of expandability of cell lines and the relevance and complexity of primary tissue.

类器官来源的上皮单层:临床相关的肠道屏障功能体外模型

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations ...

生物学

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 ...

化重构法 (的) 对肠道干细胞和小生境细胞功能分析的研究

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小鼠结肠隐窝三维培养离体研究肠道干细胞功能

该

从整体组织和活检建立人类上皮Enteroids和Colonoids的

人多能干细胞来源的肠道和结肠类器官的生成、维持和表征

人多能干细胞来源的肠道和结肠类器官的生成、维持和表征

结合人体类器官和芯片器官技术，对肠道区域特异性功能进行建模

类器官来源的上皮单层:临床相关的肠道屏障功能体外模型

使用肠道器官模型研究猿特异性相互作用的文化方法

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

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