作者感谢 U19 AI157984、U01 DK103168、U19 AI144297、P30 DK56338、P01 AI057788、U19 AI116497 赠款和 NASA 合作协议通知/TRISH NNX16AO69A。

此处使用的类器官系来自德克萨斯医学中心消化疾病中心 GEMS Core。简而言之，为了最初建立类器官系，对供体组织样品进行洗涤和酶消化以释放肠隐窝。隐窝包埋在基底膜中并在培养基中培养。贝勒医学院机构审查委员会批准了获取组织样本的研究方案，从中建立类器官系，并获得所有供体的知情同意，以从捐赠组织中建立类器官系。
1. 3D HIO 的传代以扩展以进行分化
<ol><li>用 P1000 移液管从 24 孔板（固体基底膜周围）中生长的类器官孔中取出并丢弃培养基。向孔中加入 300 μL 0.05% 胰蛋白酶-EDTA，并使用 P1000 移液管上下移液 5 次，机械分解类器官。</li>
	<li>将板在 37 °C 下孵育 4 分钟。向每个孔中加入 500 μL 不含生长因子的完全培养基（含 10% FBS）的 CMGF-培养基（表 1）。使用 P1000 移液管和移液管 CMGF 培养基和 HIO 混合物，在孔中来回 10 次。将整个体积转移到 15 mL 试管中。将多个孔的通道合并到同一个 15 mL 管中。每管混合的孔数不要超过 10 个。</li>
	<li>使用 100 x g 的摆动转子离心机在 4 °C 下旋转细胞 5 分钟。去除所有培养基，将沉淀保持在试管底部。将试管放在冰上。</li>
	<li>计算铺板 HIO 数量所需的基底膜量，并使用冷 P200 移液器吸头将 HIO 沉淀重悬于 30 μL/孔的基底膜中。使用冷 P200 移液器吸头将 HIO 基底膜混合物作为液滴移液到 24 孔板的中心。</li>
	<li>将板转移至 37 °C 培养箱中。让基底膜凝固 5-10 分钟，然后加入 500 μL 含有 WNT、R-spondin、noggin、EGF 和 ROCK 抑制剂 （WRNE+Y; 表 1）到每个孔中，并在 37 °C 培养箱中培养。每隔一天使用 WRNE+Y 刷新区域性，持续 7 天。 注意：健康的 HIO 将是囊性的，带有闪亮的出芽上皮（图 2）。无法看到囊肿中间的细胞。不健康的 HIO 看起来暗淡无光，上皮很厚，中间很坚实。最重要的特征是闪亮的外观。</li>
</ol>2. HIO 差异化：3D 格式
<ol><li>培养 1 周后，将 1 个 3D HIO 孔用于 3 个 3D 分化 HIO 孔。</li>
	<li>从孔中取出培养基。使用 P1000 移液管分散基底膜和 HIO，每孔加入 500 μL 的冷 CMGF-，并上下移液至少 5 次。使用摆动转子离心机以 100 x g 的速度在 4 °C 下旋转 HIO 5 分钟。去除所有培养基，将沉淀保持在试管底部。</li>
	<li>每孔添加 30 μL 被铺板的 HIO 基底膜。将含有 HIO 的 30 μL 基底膜液滴移液到 24 孔板的孔中。在 37 °C 下孵育 10 分钟。添加 500 μL WRNE+Y/Diff （50/50） 培养基。</li>
	<li>24 小时后将培养基切换到分化培养基 3 天，每隔一天刷新一次培养基。每个样品准备 3 个孔进行混合，以减少技术差异。</li>
</ol>3. HIO 区分：单层形式
<ol><li>用 100 μL/孔的人胎盘胶原蛋白 IV（1mg/ml 在 100 mM 乙酸中）包被在 96 孔板的膜细胞培养小室或孔中，在冷 H2O （1：30） 中稀释。在 37 °C 下孵育至少 1.5 小时或最多过夜。对于膜细胞培养插入物，在板空间中添加无菌 H2O 以加湿板;对于 96 孔板，在未使用的孔中加入无菌 H2O。</li>
	<li>通过去除培养基并通过添加 500 μL/孔的冷 0.5 mM EDTA（0.5 M EDTA 在无菌 PBS 中以 1：1000 稀释）来回收基底膜和细胞，收集在 WRNE 中培养 7 天的 3D HIO。使用 P1000 移液管上下移液类器官和溶液 5 次，然后转移到 15 mL 微量离心管中。每管混合的类器官孔不要超过 10 个。在 4 °C 的摆动桶转子中以 300 x g 离心 5 分钟。 注：要在 96 孔板上制备 3 个孔，请使用 1 个 3D HIO 孔。要制备 1 个膜细胞培养小室，请使用 1 孔 3D HIO（96 孔板的单细胞接种密度约为 1-2 x 105 个细胞/孔，膜细胞培养小室为 2.5-3 x 105 个细胞/孔）。</li>
	<li>去除 500 μL 培养基，将沉淀留在试管底部。通过加入 500 μL 0.05% 胰蛋白酶/0.5 mM EDTA 并在 37 °C 下孵育 4-5 分钟来解离 HI。 使用 P1000 用力上下移液 ~50 次。避免通过靠着试管侧面移液来产生气泡。</li>
	<li>通过添加 1 mL 含 10% FBS 的 CMGF 灭活胰蛋白酶。用 1 mL 含 10% FBS 的 CMGF 润湿 40 μm 细胞过滤器，并将细胞添加到细胞过滤器的顶部，过滤掉大块未消化的细胞。利用重力让细胞通过过滤器，并丢弃含有细胞团块的细胞过滤器。</li>
	<li>通过在室温下在摆动桶转子中以 400 x g 离心 5 分钟来收集细胞。</li>
	<li>从胶原包被的膜细胞培养小室或 96 孔板中取出液体。将 HIO 沉淀重悬于含有 10 μM Y-27632 的 100 μL/孔 WRNE 中，然后移液到膜细胞培养小室的上隔室或 96 孔板中。对于膜细胞培养小室，将 600 μL 含有 10 μM Y-27632 的 WRNE 添加到孔的下室中。</li>
	<li>24 小时后，从膜细胞培养小室的上部和下部或 96 孔板中取出培养基，并更换为分化培养基 5 天。每个样品准备 3 个孔进行混合，以减少技术差异。</li>
</ol>4. 从分化的 3D HIO 制备单细胞悬液，用于单细胞转录组学
<ol><li>将酶细胞消化试剂置于 37 °C 的 CO2 培养箱中，在 3D HIO 消化开始前 30 分钟解冻和加热。使用 10 倍明场显微镜检查 HIO 以确保细胞健康。</li>
	<li>从孔中取出培养基，向孔中加入 500 μL 冷细胞回收溶液。使用 P1000 移液器和标准移液器吸头上下移液几次，以从基底膜中释放细胞。将细胞悬液转移至 1.5 mL 微量离心管中。 注：使用重复孔可确保足够的细胞数量并减少孔间差异。在下面的步骤 4.7 之前不要合并孔。</li>
	<li>将细胞管在冰上孵育 10 分钟，每 5 分钟上下吹打一次。通过在 4 °C 下以 400 x g 离心 3 分钟来收集细胞。</li>
	<li>从试管中取出液体，注意不要干扰细胞沉淀。将细胞重悬于 500 μL 预热的酶细胞消化试剂中。使用 P1000 上下移液至少 10 次。</li>
	<li>将 HIO 在 37 °C 下在 CO2 培养箱中孵育 30 分钟。每 10 分钟，使用 P1000 移液器将整个体积移液 10 次，以帮助解离细胞。避免将气泡引入细胞悬液中。</li>
	<li>在 4 °C 下以 400 x g 的离心沉淀细胞 3 分钟。 去除上清液，将细胞重悬于 1 mL 分化培养基中，并储存在冰上。 注意：从此时起，HIO 必须始终保持在冰上。</li>
	<li>使用 P1000 快速上下移液细胞 50 次，以确保 HIO 解离成单个细胞。注意在此步骤中不要引入气泡，因为它会导致细胞损失。</li>
	<li>使用 P1000 移液器吸头通过 70 μm 吸头过滤器过滤整个体积。使用 1.5 mL 微量离心管收集洗脱液。 注意：如果消解时间足够，过滤器尖端不会堵塞。如果过滤器堵塞，请使用多个过滤器尖端;不要强行使音量通过过滤器。</li>
	<li>用 40 μm 尖端过滤器重复步骤 4.8。将样品储存在冰上。</li>
	<li>在 5 μL 细胞悬液中加入 5 μL 0.4% 台盼蓝，使用台盼蓝排除法在细胞计数室中对活的单细胞进行计数。根据需要将样品稀释至 1000 个细胞/μL。如果细胞密度小于 1000 个细胞/μL，则在 4 °C 下以 400 x g 沉淀细胞 2 分钟，除去原始培养基，并重悬于细胞培养基中。该方案通常产生 5 x 105-1 x 106 个细胞。 注：制备高质量悬浮液（90% 活细胞和 90% 单细胞）对于高质量单细胞数据至关重要。然而，经验表明，在治疗（例如感染或小分子应用）影响活力的情况下，可以接受低至 75% 的活力。</li>
	<li>根据制造商的方案为单细胞转录分析平台准备 DNA 文库。根据 scRNAseq 数据分析的最佳实践方案处理数据13,14。</li>
</ol>5. 从膜细胞培养小室上分化的 HIO 制备单细胞悬液
<ol><li>将酶细胞消化试剂置于 37 °C 的 CO2 培养箱中解冻和加热，然后开始 HIO 膜细胞培养小室消化前 30 分钟。将预热的酶细胞消化试剂分成 200 μL 等分试样，装在 1.5 mL 微量离心管中，每个膜细胞培养小室 1 管。将试管保存在 37 °C 培养箱中，直到准备好使用。</li>
	<li>向 5 mL PBS 中加入 5 μL 0.5 M EDTA 来制备 PBS-EDTA。将 500 μL PBS-EDTA 分装到 24 孔板中，每个膜细胞培养小室 1 孔。</li>
	<li>用 PBS-EDTA 洗涤细胞：将膜细胞培养小室从含有生长培养基的孔转移到含有 PBS-EDTA 的孔中。从顶端取出细胞培养基，并用 100 μL 的 PBS-EDTA 替换。注意不要打扰细胞层。</li>
	<li>立即丢弃添加到膜细胞培养小室顶端的 PBS-EDTA。从孔中取出膜细胞培养小室，用纸巾轻轻接触边缘吸干，然后将其倒置在工作台上。使用锋利的手术刀刀片，通过切割膜的内圆周，从插入物中取出膜。快速工作以防止干燥。</li>
	<li>使用镊子将膜转移到预热的 1.5 mL 微量离心管中，该离心管中装有酶细胞消化试剂，并确保膜完全浸没。对所有其他膜细胞培养小室重复此操作。 注：将酶细胞消化试剂的体积增加到 500 μL 可能有助于解离致密的单层。</li>
	<li>通过在 CO2 培养箱中于 37 °C 下孵育带膜的试管来解离细胞。用 200 μL 移液器每 10 分钟轻轻移液整个体积 5 次，总共 30 分钟，进行混合。避免气泡和膜的吸力以保持活力。</li>
	<li>每 10 分钟将一小份细胞 （1-2 μL） 转移到载玻片中，通过定性评估单个细胞的百分比来评估解离。可能需要长达 50 分钟的额外移液才能达到 80%-90% 的单细胞，具体取决于 HIO 类型。在混合步骤中，用移液器吸头轻轻刮擦膜以促进解离，特别是对于难以解离的细胞，但刮擦可能会影响活力。</li>
	<li>将每种条件的 3 个重复孔合并到单个 1.5 mL 微量离心管（总共 600 μL）中。添加 400 μL 细胞培养基 （diff） + 10 μM ROCKi（1 mL 总体积）。通过移液轻轻混合。 注：使用重复孔可确保足够的细胞数量并减少孔间差异。在此步骤中，应汇集 Wells。</li>
	<li>使用 1 mL 移液器吸头通过 70 μm 吸头过滤器过滤整个体积。将流出物收集在新的 1.5 mL 微量离心管中。 注意：如果消解时间足够，过滤器尖端不会堵塞。如果过滤器堵塞，请使用多个过滤器尖端;不要强行通过音量。</li>
	<li>用 40 μm 尖端过滤器重复步骤 5.9。将样品储存在冰上。</li>
	<li>在 5 μL 细胞悬液中加入 5 μL 0.4% 台盼蓝，使用台盼蓝排除法在细胞计数室中对活的单细胞进行计数。根据需要将样品稀释至 1000 个细胞/μL。如果细胞密度小于 1000 个细胞/μL，则在 4 °C 下以 400 x g 旋转 2 分钟以沉淀细胞，去除原始培养基，并以所需的细胞密度重悬于 WRNE+Y 中。该方案通常产生 4 x 105 - 6 x 105 个细胞。 注：制备高质量悬浮液（90% 活细胞和 90% 单细胞）对于高质量单细胞数据至关重要。然而，经验表明，在治疗（例如感染或小分子应用）影响活力的情况下，可以接受低至 75% 的活力。</li>
	<li>根据制造商的方案为单细胞转录分析平台准备 DNA 文库。根据 scRNAseq 数据分析的最佳实践方案处理数据13,14。</li>
</ol>6. 从分化为 96 孔单层的 HIO 制备单细胞悬液
<ol><li>将酶细胞消化试剂置于 37 °C 的 CO2 培养箱中，在开始单层 HIO 消化前 30 分钟解冻和加热。</li>
	<li>向 5 mL PBS 中加入 5 μL 0.5 M EDTA 来制备 PBS-EDTA。丢弃 96 孔板中的细胞培养基，并用 100 μL 的 PBS-EDTA 替换。注意不要打扰细胞层。</li>
	<li>解离细胞：弃去 PBS-EDTA，向每个孔中加入 100 μL 温热的酶细胞消化试剂。将板置于 37 °C 的 5% CO2 培养箱中，每 10 分钟用 200 μL 移液器吸头轻轻移液整个体积 5 次，持续 20-30 分钟，混合。避免气泡以保持活力。 注：将酶消化试剂的体积增加到 500 μL 可能有助于解离致密的单层。</li>
	<li>每 10 分钟将一小份细胞 （1-2 μL） 转移到载玻片中，以评估解离情况。可能需要长达 50 分钟才能达到 80%-90% 的单细胞，具体取决于 HIO 类型。在混合步骤中，用移液器吸头轻轻刮擦板以促进解离，特别是对于难以解离的细胞;但是，刮擦可能会影响活力。</li>
	<li>将每种条件的 3 个重复孔合并到单个 1.5 mL 微量离心管（总共 300 μL）中。添加 700 μL 细胞培养基 （diff） + 10 uM ROCKi（1 mL 总体积）。通过移液轻轻混合。 注：使用重复生长的孔可确保足够的细胞数量并减少孔间差异。</li>
	<li>使用 1 mL 移液器吸头通过 70 μm 吸头过滤器过滤整个体积。将流出物收集在新的 1.5 mL 微量离心管中。 注意：如果酶细胞消化试剂消化时间足够，过滤头不会堵塞。如果过滤器堵塞，请使用多个过滤器尖端;不要强行通过音量。</li>
	<li>用 40 μm 尖端过滤器重复步骤 6.6。将样品储存在冰上。</li>
	<li>在 5 μL 细胞悬液中加入 5 μL 0.4% 台盼蓝，使用台盼蓝排除法在细胞计数室中对活的单细胞进行计数。根据需要稀释至 1000 个细胞/μL。如果细胞密度小于 1000 个细胞/μL，则在 400 x g 4 °C 下旋转 2 分钟以沉淀细胞，去除原始培养基，并以所需的细胞密度重悬于细胞培养基 + ROCKi 中。该方案通常产生 2 x 105 - 6 x 105 个细胞。 注：制备高质量悬浮液（90% 活细胞和 90% 单细胞）对于高质量单细胞数据至关重要。然而，经验表明，在治疗（例如感染或小分子应用）影响活力的情况下，可以接受低至 75% 的活力。</li>
	<li>根据单细胞转录分析平台制造商的方案准备 DNA 文库。根据 scRNAseq 数据分析的最佳实践方案处理数据13,14。</li>
</ol>

将单细胞转录组学与人肠道类器官整合

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W., 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=Organization+of+the+human+intestine+at+single-cell+resolution.">Organization of the human intestine at single-cell resolution.</a> Nature. 619 (7970), 572-584 (2023).</li><li>Burclaff, 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=A+proximal-to-distal+survey+of+healthy+adult+human+small+intestine+and+colon+epithelium+by+single-cell+transcriptomics.">A proximal-to-distal survey of healthy adult human small intestine and colon epithelium by single-cell transcriptomics.</a> Cell Mol Gastroenterol Hepatol. 13 (5), 1554-1589 (2022).</li><li>Triana, 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=Single-cell+transcriptomics+reveals+immune+response+of+intestinal+cell+types+to+viral+infection.">Single-cell transcriptomics reveals immune response of intestinal cell types to viral infection.</a> Mol Syst Biol. 17 (7), e9833 (2021).</li><li>Triana, 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=Single-cell+analyses+reveal+SARS-CoV-2+interference+with+intrinsic+immune+response+in+the+human+gut.">Single-cell analyses reveal SARS-CoV-2 interference with intrinsic immune response in the human gut.</a> Mol Syst Biol. 17 (4), e10232 (2021).</li><li>Fujii, 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=Human+intestinal+organoids+maintain+self-renewal+capacity+and+cellular+diversity+in+niche-inspired+culture+condition.">Human intestinal organoids maintain self-renewal capacity and cellular diversity in niche-inspired culture condition.</a> Cell Stem Cell. 23 (6), 787-793 (2021).</li><li>Beumer, 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=BMP+gradient+along+the+intestinal+villus+axis+controls+zonated+enterocyte+and+goblet+cell+states.">BMP gradient along the intestinal villus axis controls zonated enterocyte and goblet cell states.</a> Cell Rep. 38 (9), 110438 (2022).</li><li>Beumer, 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=High-resolution+mRNA+and+secretome+atlas+of+human+enteroendocrine+cells.">High-resolution mRNA and secretome atlas of human enteroendocrine cells.</a> Cell. 182 (4), 1062-1064 (2020).</li><li>Busslinger, G. A., 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=Human+gastrointestinal+epithelia+of+the+esophagus,+stomach,+and+duodenum+resolved+at+single-cell+resolution.">Human gastrointestinal epithelia of the esophagus, stomach, and duodenum resolved at single-cell resolution.</a> Cell Rep. 34 (10), 108819 (2021).</li><li>Luecken, M. D., Theis, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+best+practices+in+single-cell+RNA-seq+analysis:+a+tutorial.">Current best practices in single-cell RNA-seq analysis: a tutorial.</a> Mol Syst Biol. 15 (6), e8746 (2019).</li><li>Heumos, L., 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=Best+practices+for+single-cell+analysis+across+modalities.">Best practices for single-cell analysis across modalities.</a> Nat Rev Genet. 24 (8), 550-572 (2023).</li></ol>

作者没有利益冲突。

使用单细胞基因组学平台，可以分解复杂的生物系统，例如模拟肠上皮的组织来源的 HIO 培养物，从而产生对整体生物反应的单个细胞贡献 4,5,6。细胞异质性和稀有细胞群也可以被识别和询问。需要优化细胞输入，以使用基于单细胞转录组学的平台最大限度地提高输出。在这里，我们描述了将产生高度活力和高质量单细胞悬液?...

小肠上皮有两个不同的区域：容纳肠道干细胞 （ISC） 的隐窝和由分泌和吸收谱系的分化细胞组成的绒毛。上皮的区域特异性增加了这种复杂性，它在小肠区域之间提供了独特的功能特性。开创性工作建立了培养条件，其中人类小肠隐窝和绒毛区都可以从手术组织或组织活检中 离体 产生1。这些培养物弥合了动物研究和临床试验之间的差距，并揭示了以前未被认识的胃肠道细胞生物学、生物化学和生理学。HIO 使用含有促进干细胞活力的生长因子和细胞外支持基质的培养基以 3D 球形结构繁殖。这些条件导致主要由祖细胞和干细胞组成的隐窝样 HIO 模型。去除生长因子促进 ISC 分化（绒毛样模型）和成熟肠上皮细胞（杯状、肠内分泌、簇状、肠上皮细胞）的产生，以及细胞增殖和分化、极化、屏障完整性、区域特异性特征和适当的生理反应2.HIO 培养物在遗传上是稳定的，可以在其隐窝样状态下无限增殖。允许以单层形式生长的培养条件提供了易于进入这些培养物顶端表面的机会 3。HIO 还允许在生物分析中考虑宿主个体的变异性，例如遗传、年龄、性别、种族和疾病状态。分析和功能评估工具与以转化细胞系为中心的方法中使用的工具相同，包括多种分子技术，如流式细胞术、显微镜、转录组学、蛋白质组学和代谢组学。
单细胞转录组学通过深入了解每种细胞类型对生物过程的个体贡献，正在彻底改变我们对小肠生物学和生理学的理解。使用这项技术的开创性工作提供了天然人类肠道中存在的细胞类型的景观 4,5,6。单细胞转录分析平台允许探索单个细胞的转录景观，使细胞异质性成为科学探索的最前沿。在一些单细胞转录分析平台中，微流控分配和条形码用于从每个样品中多达 10,000 个细胞获得的细胞多聚腺苷酸化 mRNA 生成 cDNA 文库。在该平台上，含有单细胞、条形码寡核苷酸、逆转录试剂和油的液滴形成反应囊泡，导致来自单个细胞的所有 cDNA 具有相同的条形码。然后，可以使用下一代测序对条形码 cDNA 进行高效测序。生成的数据可以通过软件和可视化工具进行处理，这些工具将条形码序列转换为可视化图谱和单细胞转录图谱。可以使用公开可用的人类小肠上皮数据库来识别细胞群 4,5,6。尽管许多研究利用该平台在单细胞水平上询问小鼠肠道类器官，但对 HIO 的单细胞转录反应的分析落后于 7,8,9,10,11,12。
在这里，我们提供了从小肠 HIO 中分离活细胞以在单细胞转录分析平台上进行处理的分步指南（图 1）。我们提供培养和分化指南以及针对上皮分化优化的培养基成分。我们概述了三种不同培养形式的细胞回收方法：在塑料或膜细胞培养小室上进行三维 （3D） 和单层培养。我们提供使用开源软件获得的样本聚类数据，以得出每个聚类中的差异表达基因。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>[Leu15]-Gastrin I</td>
			<td>Sigma-Aldrich</td>
			<td>G9145</td>
			<td>10 nM</td>
		</tr>
		<tr>
			<td>0.05% Trypsin-EDTA</td>
			<td>Invitrogen</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>0.4% Trypan blue</td>
			<td>Millipore-Sigma</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 M EDTA</td>
			<td>Corning</td>
			<td>46-034-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>1x PBS Ca- Mg-</td>
			<td>Corning</td>
			<td>21-040-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>24 mm Transwell</td>
			<td>Costar</td>
			<td>3412</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well Nunclon delta surface tissue culture dish</td>
			<td>Thermo Scientific</td>
			<td>142475</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#181;m cell strainer</td>
			<td>Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#181;m Flowmi tip strainer</td>
			<td>SP Bel-Art Labware</td>
			<td>H13680-0040</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;m Flowmi tip strainer</td>
			<td>SP Bel-Art Labware</td>
			<td>H13680-0070</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well plate</td>
			<td>Corning</td>
			<td>3595</td>
			<td></td>
		</tr>
		<tr>
			<td>A-83-01</td>
			<td>Tocris</td>
			<td>2939</td>
			<td>500 nM</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>StemCell Technologies</td>
			<td>7920</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Invitrogen</td>
			<td>12634-028</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td>1X</td>
		</tr>
		<tr>
			<td>Chromium Next GEM Single Cell 3&#8217; GEM, Library &#38; Gel Bead Kit v3.1</td>
			<td>10x Genomics</td>
			<td>PN-1000128</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen IV</td>
			<td>Sigma-Aldrich</td>
			<td>C5533-5MG</td>
			<td>33 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Corning Cell Recovery Solution&#160;</td>
			<td>VWR</td>
			<td>354253</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS (Mg2-, Ca2-)</td>
			<td>Invitrogen</td>
			<td>14190-136</td>
			<td>1X</td>
		</tr>
		<tr>
			<td>GlutaMAX-I</td>
			<td>Invitrogen</td>
			<td>35050-061</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>HEPES 1M</td>
			<td>Invitrogen</td>
			<td>15630-080</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>L-WRN conditioned media</td>
			<td>ATCC</td>
			<td>CRL-3276</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel, GFR, phenol free</td>
			<td>Corning</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse recombinant EGF</td>
			<td>Invitrogen</td>
			<td>PMG8043</td>
			<td>50 ng/mL</td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Invitrogen</td>
			<td>17502-048</td>
			<td>1X</td>
		</tr>
		<tr>
			<td>N-Acetylcysteine</td>
			<td>Sigma-Aldrich</td>
			<td>A9165-5G</td>
			<td>500 &#181;M</td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma-Aldrich</td>
			<td>N0636</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>SB202190</td>
			<td>Sigma-Aldrich</td>
			<td>S7067</td>
			<td>10 &#181;M</td>
		</tr>
		<tr>
			<td>Transwell</td>
			<td>Corning</td>
			<td>3413</td>
			<td></td>
		</tr>
		<tr>
			<td>Y27632</td>
			<td>Stem Cell Technologies</td>
			<td>72308</td>
			<td>10 &#181;M</td>
		</tr>
	</tbody>
</table>

using human intestinal organoids to understand the small intestine epithelium at the single cell transcriptional level

单细胞转录组学彻底改变了我们对人体细胞生物学的理解。最先进的人小肠类器官培养物提供了 离体 模型系统，弥合了动物模型和临床研究之间的差距。单细胞转录组学在人肠道类器官 （HIO） 模型中的应用揭示了以前未被认识的胃肠道细胞生物学、生物化学和生理学。先进的单细胞转录组学平台使用微流控分配和条形码生成 cDNA 文库。这些条形码 cDNA 可以通过下一代测序平台轻松测序，并被各种可视化工具用于生成图谱。在这里，我们描述了以不同形式培养和区分人小肠 HIO 的方法和程序，用于从这些形式中分离活细胞，这些形式适用于单细胞转录分析平台。这些协议和程序有助于使用小肠 HIO，以加深对在各种不同环境背景下人类肠上皮细胞在转录水平上的细胞反应的理解。

The small intestinal epithelium has two distinct zones: the crypt that houses the intestinal stem cell (ISC) and the villus, which is comprised of differentiated cells of the secretory and absorptive lineages. Adding to this complexity is the regional specificity of the epithelium that provides unique functional properties between the regions of the small intestine. Pioneering work established culture conditions in which both the human small intestinal crypt and villus zones can be generated ex vivo from surgical tissues or tissue biopsies1. These cultures are bridging the gap between animal studies and clinical trials and are revealing previously unrecognized cell biology, biochemistry, and physiology of the gastrointestinal tract. The HIOs are propagated as 3D spherical structures using a media with growth factors that promote stem cell viability and extracellular support matrices. These conditions result in a crypt-like HIO model that consists mostly of progenitors and stem cells. Removal of the growth factors promotes ISC differentiation (villus-like model) and production of mature intestinal epithelial cells (goblet, enteroendocrine, tuft, enterocyte) in appropriate ratios along with cell proliferation and differentiation, polarization, barrier integrity, regional specific features, and appropriate physiological responses2. HIO cultures are genetically stable and can be propagated indefinitely in their crypt-like state. Easy access to the apical surface of these cultures is provided by culture conditions that allow growth in a monolayer format3. HIOs also allow considerations of host individual variability such as genetics, age, sex, ethnicity, and disease status to be included in biological analyses. Analytic and functional assessment tools are identical to those used in approaches centered on transformed cell lines and include a variety of molecular techniques such as flow cytometry, microscopy, transcriptomics, proteomics, and metabolomics.
Single cell transcriptomics is revolutionizing our understanding of the biology and physiology of the small intestine by providing insight into the individual contributions of each cell type to a biological process. Pioneering work using this technology has provided a landscape of the cell types present in the native human intestine4,5,6. Single cell transcriptional profiling platforms allow exploration of the transcriptional landscape of individual cells, allowing cell heterogeneity to come to the forefront of scientific exploration. In some single cell transcriptional profiling platforms, microfluidic partitioning and barcoding are used to generate cDNA libraries from cellular polyadenylated mRNAs obtained from up to 10,000 cells per sample. On this platform, droplets containing single cells, barcoded oligonucleotides, reverse transcription reagents, and oil form a reaction vesicle that results in all cDNAs from a single cell having the same barcode. The barcoded cDNAs can then be efficiently sequenced using next generation sequencing. Data generated can be handled through software and visualization tools, which convert the barcoded sequences into visualization maps and single cell transcriptional profiles. Cell populations can be identified using publicly available databases of human small intestinal epithelium4,5,6. Although many studies have utilized this platform to interrogate murine intestinal organoids at the single cell level, the analysis of single cell transcriptional responses of HIOs has lagged behind7,8,9,10,11,12.
Here, we provide a step-by-step guide to isolating viable cells from small intestinal HIOs for processing on single cell transcriptional profiling platforms (Figure 1). We provide culturing and differentiation guidelines along with media components that have been optimized for epithelial differentiation. We outline cell recovery methods for three different culture formats: three dimensional (3D) and monolayer cultures either on plastic or on membrane cell culture inserts. We provide sample clustering data obtained using open-source software to derive differentially expressed genes in each cluster.

作者聚焦：将单细胞转录组学与类器官培养相结合，以获得高级研究和治疗见解

使用人类肠道类器官在单细胞转录水平上了解小肠上皮

We are integrating single-cell transcriptomics and organoid technology to better understand the biology of the small intestine. These technologies are allowing us deeper insight into the cellular heterogeneity that exists within that tissue. The tools that improve the throughput, sensitivity, and the resolutions are the keys that advanced research in the field.Some examples are improved computational methods and the technological innovations. Cost reductions also a major focus. The process of preparing and sequencing the libraries of samples can cause transcription variations, which is one challenge.For organoids, specific challenges include limited spatial context or information about temporal dynamics. Single-cell analysis of the human intestinal organoids provides previously unattainable information into the cell types that make up the intestinal epithelium. This, thereby, opens the window to the transcriptional landscape of those cells.We are using these technologies to identify biomarkers and mechanisms of human intestinal diseases. These biomarkers and mechanisms will advance our ability to utilize personalized medicine-based approaches to treat human disease and improve human health overall.

从 2-3 个膜细胞培养插入室、单层和 3D HIO 孔中混合单细胞悬液，以确保足够的细胞产量并减少孔间差异。使用单细胞转录分析平台专用试剂制备单细胞文库。并在下一代测序平台上使用配对末端读数进行测序，30,000 个读数/细胞。使用单细胞基因组学分析工具对读数进行定位、计数和分析。线粒体读取超过 20% 或计数基因少于 200 个的低质量细胞被排除在分析之外。为了评估细胞的质量，我们绘制...

该方案将人类肠道类器官技术与单细胞转录组学分析相结合，为以前未曾探索的肠道生物学提供了重要的见解。

Single cell transcriptomics has revolutionized our understanding of the cell biology of the human body. State-of-the-art human small intestinal organoid ...

using-human-intestinal-organoids-to-understand-small-intestine

Research

JoVE Journal

Biology

Using Human Intestinal Organoids to Understand the Small Intestine Epithelium at the Single Cell Transcriptional Level

396 次观看.  Baylor College of Medicine. 我们正在整合单细胞转录组学和类器官技术，以更好地了解小肠的生物学特性。这些技术使我们能够更深入地了解该组织内存在的细胞异质性。提高通量、灵敏度和分辨率的工具是推进该领域研究的关键。一些例子是改进的计算方法和技术创新。降低成本也是一个主要重点。样本文库的制备和测序过程可能会导致转录变异，这是一个挑战。

视频: 作者聚焦：将单细胞转录组学与类器官培养相结合，以获得高级研究和治疗见解

Single cell transcriptomics has revolutionized our understanding of the cell biology of the human body. State-of-the-art human small intestinal organoid cultures provide ex vivo model systems that bridge the gap between animal models and clinical studies. The application of single cell transcriptomics to human intestinal organoid (HIO) models is revealing previously unrecognized cell biology, biochemistry, and physiology of the GI tract. The advanced single cell transcriptomics platforms use microfluidic partitioning and barcoding to generate cDNA libraries. These barcoded cDNAs can be easily sequenced by next generation sequencing platforms and used by various visualization tools to generate maps. Here, we describe methods to culture and differentiate human small intestinal HIOs in different formats and procedures for isolating viable cells from these formats that are suitable for use in single-cell transcriptional profiling platforms. These protocols and procedures facilitate the use of small intestinal HIOs to obtain an increased understanding of the cellular response of human intestinal epithelium at the transcriptional level in the context of a variety of different environments.

The protocol combines human intestinal organoid technology with single cell transcriptomic analysis to provide significant insight into previously unexplored intestinal biology.

科研

生物学

Preparation of Single Cell Suspensions from Differentiated 3D Human Intestinal Organoids for Single Cell Transcriptomics

To begin, thaw the enzymatic cell digestion reagent at 37 degrees Celsius for 30 minutes. Under a brightfield microscope at 10x magnification, observe the differentiated three-dimensional human intestinal organoids to ensure cell health. Replace the media from the wells of a 24-well plate containing differentiated organoids with 500 microliters of cold cell recovery solution.Pipette up and down to release the cells from the basement membrane. Transfer the cell suspension to a 1.5-milliliter microcentrifuge tube. Incubate the tube on ice for 10 minutes, pipetting up and and down every five minutes.Centrifuge the cell suspension at 400 g for three minutes at four degrees Celsius. Remove the supernatant from the tube without disturbing the pellet. Resuspend the pellet in 500 microliters of prewarmed enzymatic cell digestion reagent, pipetting up and down 10 times.Place the organoids for 30 minutes at 37 degrees Celsius in a carbon dioxide incubator. Every 10 minutes, pipette the entire volume 10 times using a P1000 pipette to help dissociate the cells. Centrifuge the cell suspension at 400 g for three minutes at four degrees Celsius, and resuspend the pellet in one milliliter of differentiation media.Rapidly pipette the cells up and down 50 times on ice to dissociate organoids into single cells. Filter the entire volume through a 70 micron tip strainer and collect the eluate in a fresh 1.5-milliliter tube. Then filter the eluate obtained through a 70 micron strainer on a 40 micron tip strainer.Add five microliters of 0.4%Trypan Blue to five microliters of cell suspension, and count viable single cells using Trypan Blue Exclusion. Dilute the sample with cell culture media to obtain 1000 cells per microliter. A total of 4, 402 single cells were isolated from differentiated ileal three-dimensional human intestinal organoids, representing expected cell types, including absorptive enterocytes and secretory cells.

从分化的 3D 人肠道类器官制备单细胞悬液，用于单细胞转录组学

Preparation of Single Cell Suspensions from Human Intestinal Organoids Differentiated on Membrane Cell Culture Insert

To begin, place the 15 milliliter tube containing prewarmed enzymatic cell digestion reagent in a 37 degree Celsius incubator until use. Aliquot 500 microliters of freshly prepared PBS/EDTA into the wells of a 24 well plate. Under a brightfield microscope, observe the growth of differentiated three dimensional human intestinal organoids and transwells.Transfer the cell culture inserts from the growth media to the PBS/EDTA solution. Remove the culture media from the apical side and replace it with 100 microliters of PBS/EDTA without disturbing the cell layer. After discarding PBS/EDTA, remove the insert from the well, blot it gently onto the edge of a paper towel and inverted on the bench top.Using a sharp scalpel blade, cut around the inner circumference of the membrane to remove the membrane from the insert. Transfer the membrane with forceps to the 1.5 milliliter tube containing 200 microliters of prewarmed enzymatic cell digestion reagent. Place the membrane for 30 minutes at 37 degrees Celsius in a carbon dioxide incubator.Every 10 minutes, pipette the entire volume five times using a P 200 pipette. Transfer, one to two microliters of cell suspension to a glass slide every 10 minutes to assess dissociation by qualitatively assessing the percentage of single cells. After dissociation, combine three replicate wells per condition in a single 1.5 milliliter micro centrifuge tube.Add 400 microliters of cell culture differentiation medium containing 10 micromolar ROCK inhibitor and mix gently by pipetting. Filter the entire volume through a 70 micron tip strainer, collecting the flowthrough in a fresh 1.5 milliliter tube. Then filter the flowthrough obtained through a 70 micron strainer on a 40 micron tip strainer.Add five microliters of 0.4%trypan blue to five microliters of cell suspension and count viable single cells. Dilute the sample with WRNE growth medium to obtain 1, 000 cells per microliter. A total of 5, 623 single cells were isolated from differentiated jejunal human intestinal organoids grown on membrane cell culture inserts.

从膜细胞培养小室上分化的人肠道类器官制备单细胞悬液

Preparation of Single Cell Suspensions from Human Intestinal Organoids Differentiated as 96 Well Monolayers

To begin, prepare enzymatic cell digestion reagent and PBS/EDTA for human intestinal organoids digestion. Under a brightfield microscope, confirm the growth of differentiated three-dimensional human intestinal organoids as monolayers. Discard the cell culture media from monolayer culture and replace it with 100 microliters of PBS/EDTA.After removing PBS/EDTA, add 100 microliters of warm enzymatic cell digestion reagent to each well. Place the plate at 37 degrees Celsius in 5%carbon dioxide incubator and pipette the entire volume five times every 10 minutes for 20 to 30 minutes. Transfer one to two microliters of cell suspension to a glass slide every 10 minutes to assess dissociation by observing the percentage of single cells.After dissociation, combine three replicate wells per condition in a single 1.5 milliliter microcentrifuge tube. Add 700 microliters of cell culture differentiation medium containing 10 micromolar ROCK inhibitor, and mixed gently by pipetting. Filter the entire volume through a 70 micron tip strainer, collecting the flow-through in a fresh 1.5 milliliter tube.Filter the flow-through obtained through a 70 micron strainer on a 40 micron tip strainer. Add five microliters of 0.4%Trypan Blue to five microliters of cell suspension, and count viable single cells. Dilute the sample with cell culture media containing ROCK inhibitor to obtain 1, 000 cells per microliter.A total of 9, 952 single cells were isolated from differentiated intestinal human intestinal organoids grown in 96 well plates.