我们感谢 Sumigray 和 Ameen 实验室的成员进行了深思熟虑的讨论。这项工作得到了 Charles H. Hood 基金会儿童健康补助金和囊性纤维化基金会补助金 （004741P222） 的支持，并得到了美国国立卫生研究院国家糖尿病、消化和肾脏疾病研究所的支持，奖励编号为 2R01DK077065-12。

大鼠肠隐窝分离和肠道类器官生成

大鼠肠道类器官模型的开发保留了 体内 器官中发现的重要功能特征，是临床前测试、药物筛选和功能测定的有前途的工具。这种 体外 模型可以与 体内 临床前胃肠病学研究并行使用，对于这些研究，大鼠通常是首选模型，因为它们的肠道尺寸更大，与人类共享生理方面，并且在某些情况下是更好的疾病模型38。在这里，概述了用于分离大鼠肠隐窝、大鼠肠道类器官的生成和长期培养以及下游应用（包括功能性毛喉素肿胀测定、全封地免疫荧光、2D 单层培养和慢病毒基因操作）的稳健分步方案。与小鼠肠道类器官相比，大鼠肠道类器官可能与小鼠肠道类器官的病理生理学不合适的许多疾病环境中相关，并且可能为人类肠道生理学提供更好的模型。
为了建立可以传代和扩增的长寿命类器官培养物，必须确定维持肠上皮增殖所需的关键生长因子。小鼠类器官最常在EGF、R-spondin和Noggin的简单混合物中生长，尽管有报道称Noggin不是肠道类器官培养所必需的39。条件培养基可以替代重组生长因子，最常用的细胞系是 L-WRN，它分泌 Wnt3a、Rspondin-3 和 Noggin39、L-Wnt3a 和 HA-Rspondin1-Fc 293T 细胞40。L-WRN 条件培养基不仅足以支持小鼠肠道39 类器官的生长，而且足以支持来自几种农场动物和伴侣动物（包括狗、猫、鸡、马、牛、羊和猪）的肠道类器官的生长12。然而，人类肠道类器官的生长因子需求有很大不同，因为它们的扩增生长期（即从小球体到大球体的进展）与分化阶段（即分化细胞类型的产生和成熟）需要不同的培养基配方10。大鼠肠道类器官的培养基要求与人肠道类器官的扩增生长培养基非常相似，但值得注意的是，大鼠类器官能够在这种培养基环境中生长和分化，大大简化了其培养要求。虽然我们最初的尝试集中在L-WRN条件培养基中建立和培养大鼠肠道类器官，但长期培养很脆弱，并且大鼠肠道类器官系缺乏稳健性（数据未显示）。这可能是因为 L-WRN 细胞系被设计为分泌 R-spondin 3，而这里推荐的 293T-Rspo1 细胞系被设计为分泌 R-spondin 1。大鼠和人类类器官可能更喜欢 R-spondin 1，这可能是 L-WRN 条件培养基中大鼠类器官系失败的原因。
为了最接近地概括 体内 环境，重要的是开发允许干细胞存活、维持和增殖的类器官培养条件，并能够维持细胞更新和同时分化事件为离散细胞类型。因此，重组蛋白和/或条件培养基中蛋白质的浓度需要严格滴定和控制，以达到这种完美的平衡。特别是，最佳的Wnt水平对于避免肠道类器官培养物的损失至关重要。条件培养基中Wnt太少将无法支持生长，导致干细胞丢失和随后的类器官死亡;Wnt 的过度激活会导致类器官呈囊性和未分化10。虽然这里没有详细说明，但强烈建议使用 Wnt 报告荧光素酶测定法（例如 Topflash 细胞系41）测试每批 L-Wnt3a 和 293T-Rspo1 条件培养基。先前的研究已经描述，与 1% 的 L-Wnt3a 相比，最佳批次的 L-Wnt3a 培养基应导致 15 倍的信号增加 12.5% 和 300 倍的信号增加50% 10。由于大鼠类器官比小鼠类器官对培养要求（特别是 Wnt 活化水平）更敏感，因此这些额外的质量控制步骤极大地有助于提高大鼠类器官培养物的稳健性和可靠性。由于没有类似的报告基因线可用于测试Noggin条件培养基中的Bmp活性和相对Noggin浓度，因此建议尽可能使用重组Noggin来精确控制Noggin水平。虽然小鼠肠道类器官可以在没有Noggin39的情况下生长和维持，但尚未尝试将其用于大鼠肠道类器官培养。
除了细胞培养要求外，大鼠类器官系的成功初始建立主要取决于隐窝分离过程中分化绒毛的有效耗竭。高水平的维拉尔污染导致隐窝死亡，可能是由于来自垂死细胞的信号或基本因子的隔离。为了精确和一致地从上皮制剂中去除这些分化的绒毛，建议在立体镜的帮助下进行上皮分离。对释放的上皮细胞的目视检查提供了何时丢弃PBS并更换PBS的明确提示（图1）。在绒毛充分耗竭之前，不应收集隐窝。Villar细胞是终末分化的，不能在培养物中产生类器官。此外，大鼠肠道类器官的后续传代及其用于任何下游应用都需要细致的护理。在解离试剂中孵育较长时间（10分钟）会导致显着的细胞死亡和类器官系的丢失。
在这里，描述了一种从大鼠类器官生成肠道单层的简单快速的方案。EME 和胶原 I 底物对上皮细胞有不同的影响，可以根据研究目的加以利用。EME允许快速有效地粘附单个细胞并形成细胞突起。相比之下，用胶原蛋白I涂覆表面会延迟这些过程。一旦单层达到大约 80% 的汇合度，在 EME 上生长的细胞就会再次开始生成 3D 类器官结构。然而，它们缺乏足够的物理和化学支持来持续增长。通过将EME中的单层保持在50%-80%的汇合度，可以防止这种恢复到类器官状态。在单层的顶端表面添加稀释的EME可促进从头类器官的快速恢复和形成，从而更快、更容易地产生收敛区域。在I型胶原表面上，细胞可以形成均匀的单层并产生小簇。然而，在单层之上添加胶原I不足以诱导类器官的形成。当添加到单层表面时，必须稀释EME，因为新生的类器官将有更强的机械阻力来克服。然而，这种稀释的EME不允许大类器官的稳健形成。任何从头生成的自然脱离表面的大鼠类器官都必须立即去除并转移到未稀释的EME中，以便恢复结构支持和生长。由于此步骤中类器官的尺寸较小，因此在建立稳健生长之前不建议类器官传代。为什么EME可以支持类器官的重组，但胶原蛋白I是否可以做到这一点，其潜在的生物学意义尚不清楚。然而，有报道称，在 3D 胶原蛋白中生长的细胞不能形成带芽的类器官42,43 或支持长期维持。市售的 EME 产品是细胞外蛋白的异质混合物，主要是层粘连蛋白和胶原 IV44。因此，蛋白质的独特组成以及上皮细胞使用不同细胞复合物与细胞外基质结合的能力可能允许在EME中重塑，但不能在胶原I中重塑。I型胶原衍生的单层是否可以放入EME以支持类器官的形成和生长尚未经过测试。
本文描述了大鼠肠道类器官模型的遗传操作，并概述了3D类器官的慢病毒转导和2D单层瞬时转染的方案。为了克服慢病毒类器官转导效率低的问题，开发了一种用于瞬时转染 2D 单层的方案。单层的扁平形态和暴露的顶端结构域使病毒和含DNA的复合物更容易进入。使用pLJM1-EGFP载体表达EGFP报告基因来验证该技术。24 h后观察GFP报告基因表达，单层维持5-6 d。未来专注于单层慢病毒转导的研究可能比 3D 类器官转导具有更高的效率。使用上述方案，可以从感染的 2D 单层中重整 3D 类器官，以促进稳定细胞系的创建。通过谨慎，大鼠肠道类器官系可以成功维持一年以上，在许多传代中保持稳定，冷冻保存，成功解冻，并使用慢病毒转导进行基因修饰，从而满足对易于访问且易于处理的 体外 肠道类器官模型的需求，该模型保留了与人类的生理相关性。

人体小肠上皮结构和细胞组成复杂，反映了它们的生理功能。小肠的主要作用是从通过其管腔的食物中吸收营养1.为了最大限度地发挥这一功能，肠道表面被组织成称为绒毛的手指状突起，其增加了吸收表面积，以及称为隐窝的杯状内陷，用于容纳和绝缘干细胞。在上皮细胞内，产生各种专门的吸收和分泌细胞类型以执行不同的功能1.由于这种复杂性，很难在高传代转化永生化细胞系中对肠道等组织进行建模。然而，对干细胞，特别是成体干细胞及其分化机制的研究，已经允许开发 3D 肠道类器官培养物。类器官模型的使用改变了该领域，部分原因是它们概括了完整肠道中发现的一些结构成分和细胞类型异质性。肠道类器官可以长期在 体外 培养，因为活性干细胞群的维持2。
肠道类器官已迅速成为研究干细胞生物学、细胞生理学、遗传疾病和营养学的适应性模型3,4，以及开发新型药物递送方法的工具5。此外，患者来源的类器官被用于疾病建模、药物发现和个性化治疗筛选等 6,7,8,9。然而，人类肠道类器官仍然存在挑战。组织可用性、机构审查委员会批准的要求和伦理问题限制了人类样本的广泛使用。此外，由肠隐窝产生的人肠道类器官需要两种不同的培养条件来维持未分化的干细胞或诱导成熟细胞类型的分化10。这与体内形成鲜明对比，在体内，干细胞和成熟分化的细胞类型同时存在并持续产生/维持1。另一方面，小鼠肠道类器官在不太复杂的生长因子混合物中生长，不需要培养基成分的这种转换，并且可以在相同的培养基环境中维持干细胞和分化细胞2,11。然而，与人类相比，小鼠肠道的关键差异可能使小鼠类器官在许多情况下成为次优模型。总体而言，许多来自大型哺乳动物（包括马、猪、绵羊、牛、狗和猫）的肠道类器官已在与小鼠肠道类器官更接近的培养条件下成功生成，而不是人类肠道类器官的培养条件12。小鼠和人类类器官之间生长因子条件的差异可能反映了干细胞生态位组成的差异以及对干细胞存活、增殖和维持的不同要求。因此，需要一种易于访问的模型类器官系统，该系统 1） 与人类肠道细胞组成非常相似，2） 包含具有与人类肠道类器官相似的生长因子要求的干细胞，以及 3） 能够持续维持未分化和分化的区室。理想情况下，该系统将来自常用的临床前动物模型，以便体内和体外实验可以相互关联并协同使用。
大鼠是肠道生理学和药理学研究的常用临床前模型，因为它们的肠道生理学和生物化学与人类非常相似13，特别是在肠道通透性方面14。与小鼠相比，它们的体型相对较大，使它们更适合外科手术。虽然有时会使用包括猪在内的大型动物模型，但大鼠是一种更实惠的模型，需要更少的饲养空间，并且具有现成的市售标准品系15。使用大鼠模型的一个缺点是，与小鼠相比，用于体内研究的遗传工具包没有得到很好的开发，并且产生新的大鼠系，包括敲除、敲入和转基因，通常成本过高。强大的大鼠肠道类器官模型的开发和优化将允许在一个可访问的模型中进行基因操作、药物治疗和更高的通量研究，该模型保留了与人类的关键生理相关性。然而，一种啮齿类器官模型相对于另一种啮齿类器官模型的优势高度依赖于所研究的特定过程或基因;在人类中发现的某些基因可能是小鼠的假基因，但不是大鼠的假基因16,17。此外，单细胞RNAseq18,19,20越来越多地揭示了物种特异性细胞亚型。最后，大鼠和小鼠肠道疾病模型通常在表型21,22上显示出相当大的差异，因此必须选择更接近人类症状和疾病过程的模型进行下游工作。大鼠肠道类器官模型的生成为研究人员提供了额外的灵活性和选择，以选择最适合其情况的模型系统。在这里，现有的方案23,24被扩展为产生大鼠肠道类器官，并概述了从十二指肠或空肠产生和维持大鼠肠道类器官的方案。此外，还描述了几种下游应用，包括慢病毒感染、全贴片染色和毛喉素肿胀测定。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3-D Culture Matrix Rat Collagen I</td>
			<td>Cultrex/R&#38;D Systems</td>
			<td>3447-020-01</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;m cell strainer</td>
			<td>Corning/Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Gibco/Thermo Fisher</td>
			<td>12634010</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Sigma Aldrich</td>
			<td>A2942-20ML</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement (50X), serum free</td>
			<td>Thermo Fisher</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Cayman Chemical</td>
			<td>13122</td>
			<td></td>
		</tr>
		<tr>
			<td>CryoStor</td>
			<td>Stem Cell Technologies</td>
			<td>100-1061</td>
			<td></td>
		</tr>
		<tr>
			<td>Cultrex HA-Rspondin1-Fc 293T cells</td>
			<td>R &#38; D Systems</td>
			<td>3710-001-01</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td>Molecular Probes/Thermo Fisher</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco/Thermo Fisher</td>
			<td>16-000-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Gastrin I (human)</td>
			<td>Sigma Aldrich</td>
			<td>G9145</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentle Cell Dissociation Reagent</td>
			<td>Stem Cell Technologies</td>
			<td>100-0485</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Thermo Fisher</td>
			<td>35-050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Growth factor-reduced Matrigel, phenol red-free</td>
			<td>Corning</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>AmericanBio</td>
			<td>AB06021</td>
			<td></td>
		</tr>
		<tr>
			<td>Lanolin</td>
			<td>Beantown Chemical</td>
			<td>144255-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco/Thermo Fisher</td>
			<td>A2916801</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Wnt3a cells</td>
			<td>ATCC</td>
			<td>CRL-2647</td>
			<td></td>
		</tr>
		<tr>
			<td>N-2 Supplement (100X)</td>
			<td>Thermo Fisher</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetylcysteine</td>
			<td>Sigma Aldrich</td>
			<td>A9165-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide&#160;</td>
			<td>Sigma Aldrich</td>
			<td>N0636</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM I Reduced Serum Medium</td>
			<td>Gibco/Thermo Fisher</td>
			<td>31985070</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>Fisher Scientific</td>
			<td>P31-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Sigma Aldrich</td>
			<td>P7793</td>
			<td>transparent film</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Thermo Fisher</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco/Thermo Fisher</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>pLJM1-EGFP</td>
			<td>Addgene</td>
			<td>19319</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>Millipore</td>
			<td>TR-1003-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine hydrochloride (PEI)</td>
			<td>Sigma Aldrich</td>
			<td>764965</td>
			<td></td>
		</tr>
		<tr>
			<td>p-phenylenediamine&#160;</td>
			<td>Acros Organics/Thermo Fisher</td>
			<td>417481000</td>
			<td></td>
		</tr>
		<tr>
			<td>Puromycin</td>
			<td>VWR</td>
			<td>J593-25mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human FGF2 protein</td>
			<td>Peprotech</td>
			<td>100-18B-250ug</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human IGF-1 protein</td>
			<td>Biolegend</td>
			<td>B356441</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human Noggin protein</td>
			<td>R &#38; D Systems</td>
			<td>6057-NG-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant mouse EGF protein</td>
			<td>Thermo Fisher</td>
			<td>PMG8041</td>
			<td></td>
		</tr>
		<tr>
			<td>Sprague Dawley rat</td>
			<td>Charles River Laboratories</td>
			<td>Strain 001</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>American Bioanalytical</td>
			<td>AB02025-00500</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express Enzyme</td>
			<td>Gibco/Thermo Fisher</td>
			<td>12604013</td>
			<td></td>
		</tr>
		<tr>
			<td>Y27632 dihydrochloride</td>
			<td>Sigma Aldrich</td>
			<td>Y0503</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation and manipulation of rat intestinal organoids

当使用类器官评估生理学和细胞命运决策时，使用一个紧密概括 体内 环境的模型是很重要的。因此，患者来源的类器官被用于疾病建模、药物发现和个性化治疗筛选。小鼠肠道类器官通常用于了解肠道功能/生理学和干细胞动力学/命运决定的各个方面。然而，在许多疾病背景下，大鼠通常比小鼠更受欢迎，因为它们在疾病病理生理学方面与人类的生理相似性更高。由于 缺乏体内可用的遗传工具，大鼠肠道类器官已被证明是脆弱的，难以长期培养。在这里，我们建立在先前发表的方案的基础上，从十二指肠和空肠稳健地产生大鼠肠道类器官。我们概述了利用大鼠肠道类器官的几种下游应用，包括功能性肿胀测定、全贴片染色、2D 肠样单层的生成和慢病毒转导。大鼠类器官模型为 该领域对体外 模型的需求提供了一种实用的解决方案，该模型保留了与人类的生理相关性，可以快速进行基因操纵，并且很容易获得，而没有获取人类肠道类器官所涉及的障碍。

The human small intestinal epithelial architecture and cellular composition are complex, reflecting their physiological functions. The primary role of the small intestine is to absorb nutrients from food passing through its lumen1. To maximize this function, the intestinal surface is organized into finger-like protrusions called villi, which increase the absorptive surface area, and cup-like invaginations called crypts, which house and insulate the stem cells. Within the epithelium, various specialized absorptive and secretory cell types are generated to perform distinct functions1. Because of this complexity, it has been difficult to model tissues like the intestine in high passage-transformed immortalized cell lines. However, the study of stem cells, especially adult stem cells and their differentiation mechanisms, has allowed for the development of 3D intestinal organoid cultures. The use of organoid models has transformed the field, in part due to their recapitulation of some architectural components and cell type heterogeneity found in the intact intestine. Intestinal organoids can be cultured long-term in vitro due to the maintenance of the active stem cell population2.
Intestinal organoids have rapidly become an adaptable model to study stem cell biology, cell physiology, genetic disease, and nutrition3,4, as well as a tool to develop novel drug delivery methods5. In addition, patient-derived organoids are being utilized for disease modeling, drug discovery, and personalized treatment screening, among others6,7,8,9. However, human intestinal organoids still present challenges. Tissue availability, requirements for Institutional Review Board approval, and ethical issues limit the widespread use of human samples. Additionally, human intestinal organoids generated from intestinal crypts require two distinct culture conditions for the maintenance of undifferentiated stem cells or to induce the differentiation of mature cell types10. This contrasts with in vivo, where stem cells and mature differentiated cell types are simultaneously present and continuously generated/maintained1. On the other hand, mouse intestinal organoids, which are grown in a less complex cocktail of growth factors, do not require this switch in media composition and can maintain stem cells and differentiated cells in the same media context2,11. However, key differences in mouse intestine when compared to humans can make mouse organoids a suboptimal model in many instances. Overall, many intestinal organoids from larger mammals, including horses, pigs, sheep, cows, dogs, and cats, have been successfully generated in culture conditions more closely aligned with mouse intestinal organoids than the culture conditions of human intestinal organoids12. The differences in growth factor conditions between mouse and human organoids likely reflect differences in stem cell niche composition and differing requirements for stem cell survival, proliferation, and maintenance. Therefore, there is a need for an easily accessible model organoid system that 1) closely resembles human intestinal cell composition, 2) contains stem cells with growth factor requirements like those of human intestinal organoids, and 3) is capable of continuously maintaining undifferentiated and differentiated compartments. Ideally, the system would be from a commonly used preclinical animal model, such that in vivo and in vitro experiments can be correlated and used in tandem.
Rats are a commonly used preclinical model for intestinal physiology and pharmacology studies due to their very similar intestinal physiology and biochemistry to humans13, particularly with regards to intestinal permeability14. Their relatively larger size compared to mice makes them more amenable to surgical procedures. While large animal models, including pigs, are sometimes used, rats are a more affordable model, require less space for husbandry, and have readily commercially available standard strains15. A drawback of using rat models is that the genetic toolkit for in vivo studies is not well developed compared to mice, and the generation of novel rat lines, including knockouts, knock-ins, and transgenics, is often cost-prohibitive. The development and optimization of a robust rat intestinal organoid model would allow for genetic manipulation, pharmacological treatments, and higher throughput studies in an accessible model that retains key physiological relevance to humans. However, the advantages of one rodent organoid model versus another is highly dependent on the particular process or gene being studied; certain genes found in humans may be pseudogenes in mice but not rats16,17. Additionally, species-specific cell subtypes are increasingly being unveiled by single-cell RNAseq18,19,20. Finally, rat and mouse intestinal disease models often display considerable variations in phenotypes21,22 such that the model that more closely recapitulates the symptoms and disease process seen in humans must be selected for downstream work. The generation of a rat intestinal organoid model provides additional flexibility and choice for researchers in selecting a model system most appropriate for their circumstances. Here, existing protocols23,24 are expanded upon for the generation of rat intestinal organoids and a protocol is outlined for the generation and maintenance of rat intestinal organoids from the duodenum or jejunum. In addition, several downstream applications, including lentiviral infection, whole mount staining, and forskolin swelling assays, are described.

作者聚焦：大鼠肠道类器官的产生和操作  

大鼠肠道类器官的产生和操作

In this study we established a rat intestinal organoid model that is robust in long-term cell culture. We also demonstrate the first successful genetic manipulations using both lentiviral infection and transient transfection. This model is an excellent tool for manipulating intestinal biology in the rat where the genetic tools available in vivo are limited.Rat intestinal organoids have previously been difficult to culture long-term. By generating a robust, genetically-manipulatable rat organoid model, we are providing researchers additional flexibility and choice to select an organoid model best suited for their particular circumstances. Mouse intestinal organoids are used to study stem cell behavior, cell fate and physiology, but they can be a suboptimal model.Since there are key differences between mouse and human biology. On the other hand, human intestinal organoids can be difficult to obtain, and have complex cell culture requirements. The rat organoid model we describe here retains key physiological relevance to human biology, while being considerably easier to access and care for than human intestinal organoids.Developing and optimizing the rat intestinal organoid model allows for genetic manipulation, pharmacological treatments, and higher throughput studies of intestinal biology and accessible model with key physiological relevance to humans. Additionally, these rat organoids will allow for the study of species-specific physiology, cell fates, and phenotypic variations and disease models in the intestine.

使用第 2 节中概述的方案生成大鼠十二指肠和空肠类器官。在隐窝分离步骤中，从PBS中有效去除绒毛非常重要。如果在EME中接种过多的绒毛和隐窝，则可能导致整个培养物死亡，并且无法建立类器官系。因此，在解剖镜下分离隐窝是有用的，可以目视确认维拉尔的耗竭。图 1 描绘了具有代表性的维拉尔碎片和隐窝（图1A）。请注意，与绒毛相比，隐窝的尺寸明显更小（图1B）。电镀后，隐窝将在接下来的几天内扩展为球状体，并在第 4-7 天开始发芽和分化（图 2）。一旦类器官达到广泛萌芽阶段，它们就应该被传代。在传代过程中，重要的是破坏类器官以将隐窝芽分开，以便类器官数量可以扩大（图3B）。
类器官在冷冻后的成功回收在很大程度上取决于它们被冷冻的状态。处于高度增殖未分化状态的类器官以最高效率恢复。因此，我们建议诱导它们呈球形和囊形，而不是出芽和分化。为了实现这一点，可以通过增加培养基中 Wnt 配体 R-spondin 的量和在培养基中加入烟酰胺来过度激活 Wnt，烟酰胺已被证明在多种培养系统中支持类器官形成和细胞存活30,31。图 3A 显示了解冻后仅 2 天的健康类器官培养物。在解冻过程中在培养基中加入 BSA 也有助于大鼠肠道类器官培养物的存活，事实证明，这种培养物比小鼠肠道类器官更脆弱。
虽然 3D 类器官培养通常是首选，因为它概括了一些正常的肠道结构，但它使其他方法（包括活成像、转染和慢病毒转导）在技术上更具挑战性。使用由3D类器官32 生成的2D单层（图4）可以更有效地引入质粒。虽然 3D 肠道类器官传统上对瞬时转染具有抗性，但使用基于脂质的转染方法可以成功引入编码 EGFP 的质粒。步骤6.1概述了使用PEI的最具成本效益的方法（图5），但电穿孔和市售转染试剂也产生了类似的结果（数据未显示）。未来的研究将集中在这些方法是否可用于将CRISPR构建体引入单层中。
重要的是，能够在转染后从 2D 单层重组 3D 类器官，以便它们可以保持为具有隐窝 3D 结构组件的可传代系。有趣的是，当EME被加回细胞顶部时，铺在EME上的2D单层很容易重整成小球状体，而胶原I基质不足以重整3D结构（图6）。
虽然瞬时转染对许多研究有用，但稳定细胞系的形成通常更有用，需要将慢病毒引入细胞中。通过修改先前发表的方案，大鼠肠道类器官感染慢病毒（图7）。该协议的一个关键步骤是将类器官分解成小聚集体或细胞簇。如果培养物没有被有效破坏并且类器官保持完整，慢病毒颗粒就不会进入细胞。感染后，类器官必须恢复和再生。这里概述的方案允许在选择前将病毒颗粒摄取10%-48%（平均值：19.4%±6.5%）的细胞。
由于EME残基去除不完全或抗体外显率不完全，类器官的全贴片染色可能很困难。这里概述的方案允许对类器官进行稳健的染色。如果类器官离盖玻片太远，在共聚焦显微镜上对类器官的可视化也可能很困难。通过使用VALAP，可以创建一个具有一定高度的孔，使类器官不会被盖玻片压碎，但仍允许沉降在盖玻片附近，以便于成像。针对顶端阴离子通道囊性纤维化跨膜传导调节因子 （CFTR） 和鬼笔环肽的代表性染色以标记 F-肌动蛋白如图 8 所示。
最后，类器官在功能测定中具有实用性。来自囊性纤维化患者的患者来源类器官已被用于筛选 CFTR 功能，因为用 cAMP 激动剂毛喉素治疗可诱导 CFTR 介导的强力液体分泌，导致类器官肿胀 29,33-37。这项工作的一个目标是确定和开发一种可以与体内临床前研究并行使用的类器官模型。因此，我们旨在确定大鼠肠道类器官是否发生毛喉素诱导的肿胀。事实上，在毛喉素处理后30分钟内，大鼠类器官肿胀，在120分钟内观察到最大效果（图9）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65343/65343fig01.jpg"> 图 1：上皮分离过程中的 Villar 片段和隐 窝。 （A）隐窝分离方案中EDTA溶液中维拉尔片段的代表性图像。黄色箭头标记维拉尔碎片。红色箭头描绘了附着在维拉尔碎片上的地穴。注意相对大小的差异。（B）单个隐窝的更高放大倍率图像（红色箭头），以便可视化形态。比例尺：100 μm。 <a href="https://www.jove.com/files/ftp_upload/65343/65343fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65343/65343fig02.jpg"> 图2：大鼠肠道类器官进展。 分离后立即将大鼠空肠隐窝接种在EME中（A，B）。在2天内，隐窝变成了球状体（C，D）。到第 5 天，它们开始启动隐窝芽 （E，F），并在第 7 天 （G） 详细发展和生长。比例尺：100 μm。 <a href="https://www.jove.com/files/ftp_upload/65343/65343fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65343/65343fig03.jpg"> 图 3：解冻和传代后的类器官。 （A）冷冻保存后，按照概述的方案解冻大鼠空肠类器官。请注意，解冻后仅 2 天就同时存在球状体和出芽类器官。（B） 按照概述的方案传代后立即在 A 中描绘的相同类器官系。请注意 A 和 B 中结构之间的相对大小差异以及 B 中存在单个隐窝状结构域。比例尺：100 μm。 <a href="https://www.jove.com/files/ftp_upload/65343/65343fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65343/65343fig04.jpg"> 图 4：3D 类器官形成的 2D 单层结构。 （A-C） EME 上的 2D 单层进展。（D-F） I.型胶原蛋白的2D单层进展。到第 5 天，每种条件产生 ~80% 的汇合度。比例尺：100 μm。 <a href="https://www.jove.com/files/ftp_upload/65343/65343fig04large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65343/65343fig05.jpg"> 图 5：2D 单层的瞬时转染。 使用 PEI 瞬时转染 pLJM1-EGFP 质粒的 EME 上生长的 2D 单层的代表性图像。（A）明场，（B）荧光（GFP），（C）叠加。红色虚线标记单层边界。比例尺：100 μm。 <a href="https://www.jove.com/files/ftp_upload/65343/65343fig05large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65343/65343fig06.jpg"> 图 6：EME 上 2D 单层的 3D 类器官重整。 （A） 在 EME 上生长的 2D 单层形成 3D 类器官。将EME添加到单层顶端表面后5天有效形成的类器官。注意小 3D 球体周围大量死细胞。（B） 将胶原I添加到胶原I上生长的2D单层的顶端表面5天后2D单层的持久性。 比例尺：100μm。 <a href="https://www.jove.com/files/ftp_upload/65343/65343fig06large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/65343/65343fig07.jpg"> 图 7：3D 类器官的慢病毒感染。 使用概述的方案用核GFP慢病毒颗粒感染大鼠空肠类器官。恢复生长5天后，固定类器官并用DAPI复染。（A） DAPI：灰色;nuclearGFP：绿色。（B）核GFP：绿色。红色虚线标记类器官边界。比例尺：50 μm。 <a href="https://www.jove.com/files/ftp_upload/65343/65343fig07large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/65343/65343fig08.jpg"> 图8：大鼠肠道类器官的全封套免疫荧光 。 （A） CFTR、（B） 鬼笔环肽和 （C） 合并大鼠空肠类器官的全封套免疫荧光。注意类器官中CFTR染色的顶端富集（ A为灰色， C为品红色）。鬼笔环肽标记F-肌动蛋白，并突出标记顶端刷状边界（ B为灰色， C为青色）。比例尺：25 μm。 <a href="https://www.jove.com/files/ftp_upload/65343/65343fig08large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/65343/65343fig09.jpg"> 图 9：大鼠肠道类器官在毛喉素刺激下肿胀。 添加 cAMP 激动剂毛喉素后大鼠肠道类器官肿胀的代表性时间过程。0分钟时间表示加入10μM毛喉素之前的时间点。图像以 30 分钟的时间间隔显示相同的类器官。在添加毛喉素后120分钟观察到最大肿胀。红色虚线勾勒出类器官边界。类器官腔中间的深色物质由死细胞组成。比例尺：100 μm。 <a href="https://www.jove.com/files/ftp_upload/65343/65343fig09large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
表 1：AdDMEM+ 配方。 制作标准 AdDMEM+ 培养基的成分，这是此处所示方法中的基础培养基。 <a href="https://www.jove.com/files/ftp_upload/65343/Table1_Re.xlsx" target="_blank">请按此下载此表格。</a>
表2：大鼠肠道类器官培养基（rIOM）配方。 标准大鼠肠道类器官培养基的详细配方，包括重组蛋白的溶剂和储存条件。 <a href="https://www.jove.com/files/ftp_upload/65343/Table2_RE.xlsx" target="_blank">请按此下载此表格。</a>
表 3：解决方案。 制作整个协议中使用的其他溶液的配方和说明。 <a href="https://www.jove.com/files/ftp_upload/65343/Table3_resub2_Re.xlsx" target="_blank">请按此下载此表格。</a>
表 4.用于 2D 单层培养 （rIOM2D） 的大鼠肠道类器官培养基。 针对单层 2D 生长优化的类器官培养基的改良配方。 <a href="https://www.jove.com/files/ftp_upload/65343/Table4_resub2_RE.xlsx" target="_blank">请按此下载此表格。</a>

Watch this Scientific Journal Video about Generation and Manipulation of Rat Intestinal Organoids at JoVE.com

在这里，我们提出了一种产生大鼠肠道类器官并将其用于几种下游应用的方案。大鼠通常是首选的临床前模型，强大的肠道类器官系统满足了对 体外 系统伴随 体内 研究的需求。

When using organoids to assess physiology and cell fate decisions, it is important to use a model that closely recapitulates in vivo contexts. ...

在这项研究中，我们建立了一个大鼠肠道类器官模型，该模型在长期细胞培养中具有鲁棒性。我们还展示了首次使用慢病毒感染和瞬时转染的成功基因操作。该模型是操纵大鼠肠道生物学的绝佳工具，其中体内可用的遗传工具有限。大鼠肠道类器官以前很难长期培养。通过生成一个强大的、可遗传操作的大鼠类器官模型，我们为研究人员提供了额外的灵活性和选择，以选择最适合其特定情况的类器官模型。小鼠肠道类器官用于研究干细胞行为、细胞命运和生理学，但它们可能是一个次优模型。由于小鼠和人类生物学之间存在关键差异。另一方面，人类肠道类器官可能难以获得，并且具有复杂的细胞培养要求。我们在这里描述的大鼠类器官模型保留了与人类生物学的关键生理相关性，同时比人类肠道类器官更容易获得和护理。开发和优化大鼠肠道类器官模型可以进行基因操作、药物治疗和肠道生物学的更高通量研究，以及与人类具有关键生理相关性的可访问模型。此外，这些大鼠类器官将允许研究肠道中物种特异性的生理学、细胞命运、表型变异和疾病模型。

generation-and-manipulation-of-rat-intestinal-organoids

Research

JoVE Journal

Biology

Generation and Manipulation of Rat Intestinal Organoids

2.6K 次观看.  Yale School of Medicine. 在这项研究中，我们建立了一个大鼠肠道类器官模型，该模型在长期细胞培养中具有鲁棒性。我们还展示了首次使用慢病毒感染和瞬时转染的成功基因操作。该模型是操纵大鼠肠道生物学的绝佳工具，其中体内可用的遗传工具有限。大鼠肠道类器官以前很难长期培养。通过生成一个强大的、可遗传操作的大鼠类器官模型，我们为研究人员提供了额外的灵活性和选择，以?...

视频: 作者聚焦：大鼠肠道类器官的产生和操作  

When using organoids to assess physiology and cell fate decisions, it is important to use a model that closely recapitulates in vivo contexts. Accordingly, patient-derived organoids are used for disease modeling, drug discovery, and personalized treatment screening. Mouse intestinal organoids are commonly utilized to understand aspects of both intestinal function/physiology and stem cell dynamics/fate decisions. However, in many disease contexts, rats are often preferred over mice as a model due to their greater physiological similarity to humans in terms of disease pathophysiology. The rat model has been limited by a lack of genetic tools available in vivo, and rat intestinal organoids have proven fragile and difficult to culture long-term. Here, we build upon previously published protocols to robustly generate rat intestinal organoids from the duodenum and jejunum. We provide an overview of several downstream applications utilizing rat intestinal organoids, including functional swelling assays, whole mount staining, the generation of 2D enteroid monolayers, and lentiviral transduction. The rat organoid model provides a practical solution to the need of the field for an in vitro model which retains physiological relevance to humans, can be quickly genetically manipulated, and is easily obtained without the barriers involved in procuring human intestinal organoids.

Here, we present a protocol to generate rat intestinal organoids and use them in several downstream applications. Rats are often a preferred preclinical model, and the robust intestinal organoid system fills the need for an in vitro system to accompany in vivo studies.

科研

生物学

Establishment and Passaging of Small Intestine Organoids

To begin, place the euthanized rat onto the dissection surface with the ventral side up. Pinch the skin layer with sterile forceps and make cuts at the surface level, avoiding damage to internal organs. Using large sharp dissection scissors, make a longitudinal surface level cut in the center of the abdomen.Then stemming from that cut, make two shorter horizontal cuts one on each side. Using forceps, peel the skin away to expose the abdominal cavity. Cut through the peritoneal membrane to fully expose the internal organs in the abdominal cavity with ready access to the intestine.Using scissors and forceps, locate the stomach and identify the duodenum, approximately two to three centimeters distal to it which appears as a yellowish segment. The proximal jejunum is located approximately four to five centimeters distal to the ligament of Treitz. A landmark between the duodenum and jejunum.Place the isolated intestinal fragment into a 10 centimeter Petri dish. Flush the desired intestinal segment of mesentery with 10 milliliters of ice cold PBS until cleared of luminal contents. On a paper towel, cut the intestinal segment into two centimeter long pieces.Then open each intestinal piece longitudinally to expose the epithelium. Scrape the exposed luminal surface using a glass microscope slide to remove villi. Place the intestinal pieces into the EDTA solution on ice and rotate it four degrees Celsius for 30 minutes on a tube revolver set to 10 RPM.At the dissecting microscope, pour the contents of the tube into a 10 centimeter Petri dish. Add in an additional five milliliters of ice cold PBS. Using fine forceps, hold an intestinal segment and shake vigorously to observe the epithelium release into the PBS.Initially, the PBS will contain mostly villi. Shake the intestinal fragments continuously, periodically discard the PBS containing villi, and add 10 milliliters of fresh PBS to the intestinal fragments. Continue to shake the fragments and repeat the PBS washing step until villi are no longer released into the PBS.But instead, the PBS primarily contains crypts. Discard the remaining intestinal fragments and enrich the remaining PBS in the Petri dish for intestinal crypts. In a tissue culture hood, collect the PBS containing crypts and filter through a 70 micron cells drainer.Centrifuge the filtrate at 250 x g for five minutes. Then remove their supernatant and resuspend the pellet in five millimeters of advanced DMEM plus. Centrifuge again at 250 x g for five minutes.And remove the supernatant, leaving 50 microliters of media with the pellet. Resuspend the pellet in the remaining media and add it to the aliquot of the extracellular matrix extract or EME on ice. Gently pipet up and down to suspend the crypts evenly throughout the EME avoiding making bubbles.Place 50 microliters of the EME domes into a 35 millimeter tissue culture dish. An incubated 37 degrees Celsius with 5%carbon dioxide for 20 minutes. After incubation, add a two milliliters of rat intestinal organoid media, or RIOM, containing 10 micromolar each of Y27632 and CHIR99021.Next, to passage rat intestinal organoids, aspirate the medium from a plate containing organoids and add one milliliter of dissociation reagent to release the organoids from the EME domes. Transfer the dissociation reagent with fragmented organoids to a 15 milliliter conical tube. Wash the culture plate with two milliliters of advanced DMEM plus and add it to the 15 milliliter conical tube containing the fragmented organoids.Using a glass pasture pipette, gently pipette up and down 15 to 20 times to fragment the organoids. Centrifuge the organoids at 350 x g for two minutes and add 50 microliters of solution from the bottom of the tube into the EME. After mixing the solution, plate 50 microliters of the EME domes into a 35 millimeter tissue culture dish and incubate at 37 degrees Celsius with 5%carbon dioxide for 20 minutes.After incubation, add two milliliters of RIOM containing 10 micromolar each of Y27632 and CHIR99021. Representative images of villi fragments and crypts are shown. Crypts are smaller in size compared to villi.Plated crypts expand over the next few days and begin to bud and differentiate by days four to seven. After two days of thawing, a healthy organoid culture showed the presence of both spheroids and budded organoids. The same organoid line after passaging showed the presence of single crypt-like domains.

小肠类器官的建立和传代

Generation of Rat Intestinal 2D Monolayers from 3D Organoids

To coat the plate with EME, dilute EME 1 to 20 in cold Advanced DMEM+For coating with collagen, dilute five milligrams per milliliter of collagen I in Advanced DMEM+to 100 micrograms per milliliter. Coat with the plate with 200 microliters of diluted EME or collagen to cover the well surface entirely and incubate for one to two hours at 37 degrees Celsius. To generate monolayers, aspirate the media from a 35 millimeter plate containing organoids.Add one milliliter of PBS and disrupt the EME in the wells with a P1000 tip, pipetting up and down approximately 20 times to loosen all the EME. Transfer the contents to a 15 milliliter conical tube. Add one milliliter of PBS to the plate to collect additional organoids and transfer them to the same conical tube.Centrifuge the sample at 350 G for two minutes and remove the supernatant, including any EME residue. Then, add one milliliter of trypsin solution to the organoid pellet and incubate at 37 degrees Celsius for two minutes. Pipette up and down 10 times using a P1000 tip and add two milliliters of Advanced DMEM+to neutralize trypsin.Centrifuge at 350 G for five minutes and aspirate the supernant before resuspending the pellet in 4.8 milliliters of rIOM2D. Remove excess EME or collagen in Advanced DMEM+from the wells. Then, add 200 microliters of organoids in rIOM2D and 10 micromolar Y27632 to each pre-coated well.After 4 to 16 hours, collect the media and centrifuge at 1, 000 G for one minute. Transfer the supernatant into a new 15 milliliter conical tube and discard the pellet. Wash each well with 300 microliters of PBS before adding 200 microliters of the centrifuged rIOM2D to each well.Change the rIOM2D every two to three days, suspending the use of Y27632.2D monolayers plated on the EME readily reformed into small spheroids when EME was added back to the top of the cells. In contrast, a collagen I substrate was insufficient to reform 3D structures.