我们感谢美津野贵子对IFC和H和E染色的支持，凡妮莎·加西亚对组织切片的支持，以及阿尼卡·钱德拉塞卡兰对手稿准备的帮助。这项工作得到了美国国立卫生研究院（5RO1HL135163-04，PO1HL108793-08）和新基IDEAL联盟的支持。

人体肺组织是从已故组织捐赠者那里获得的，符合国际医学促进会（IIAM）制定的同意程序，并得到锡达斯 - 西奈医学中心内部审查委员会的批准。
1. 用于从气管支气管或小气道/实质（小气道和肺泡）区域分离肺细胞的组织处理
<ol><li>在细胞分离前一天准备并高压灭菌所有解剖仪器，玻璃器皿和适当的溶液。</li>
	<li>接收肺组织后，识别并分离近端和远端区域。气管和支气管被认为是"近端"的。出于该协议的目的，气管和前2-3代支气管被解剖并用于分离"近端"气道上皮。出于本方案的目的，直径为2mm或更小的小气道和周围的实质组织被认为是"远端"肺上皮（图1A）。 注意:所有涉及处理人体肺组织的程序都应在生物安全柜中进行，并使用适当的个人防护设备。</li>
</ol>2. 肺远端组织中小气道和肺泡上皮祖细胞的富集和亚化
<ol><li>远端组织制备<ol>
<li>将远端肺组织置于无菌培养皿（150 x 15 mm）中。将组织切成约1厘米3 块，并放入干净的50 mL管中。</li>
		<li>用冰镇的HBSS清洗组织3x，每次丢弃HBSS洗涤液以去除血液和上皮衬里液。</li>
		<li>将纸巾放入新的培养皿中，用无菌防绒湿巾擦干。使用镊子和剪刀，尽可能多地切除内脏胸膜（覆盖肺部表面的精致透明膜）。</li>
		<li>用剪刀将组织切成直径约2毫米的碎片。将切碎的组织转移到干净的培养皿中，并用无菌单面剃须刀片将其切成大约1毫米的大小，从而进一步切碎。</li>
	</ol></li>
	<li>酶消化 注意:自由酶储备溶液为5毫克/毫升（100倍），DNase储备液为2.5毫克/毫升（100倍）（材料表）。<ol>
<li>在50 mL锥形管中将50μg/ mL自由酶和25μg/ mL脱氢核糖酶加入无菌HBSS中。</li>
		<li>将约2-3g切碎的组织转移到含有25mL HBSS的新50 mL锥形管中，含有自由酶和DNA酶。在37°C下孵育40-60分钟，使用设置为900rpm的混合器连续振荡。孵育30分钟后，使用30mL注射器研磨消化的组织，无需针头以避免形成团块并继续孵育。 注意:酶的孵育时间可能因组织的类型或状况而异。例如，正常组织的酶消化大约需要45分钟。然而，来自特发性肺纤维化样品的纤维化组织可能需要长达60分钟的更长的孵育时间。因此，在此步骤中仔细监测组织，以防止损坏表面标记物，这对于FACS至关重要。</li>
	</ol></li>
	<li>单细胞分离<ol>
<li>通过安装在30 mL注射器上的16 G针头绘制5x来研磨组织。将组织悬浮液吸入宽口径移液器中，并在真空压力下通过一系列细胞过滤器（500μm，300μm，100μm，70μm，40μm）。用20 mL HBSS+缓冲液洗涤过滤器以收集剩余的细胞。HBSS+缓冲液的配方可以在 材料表中找到。
</li>
		<li>在45分钟后向滤液中加入等体积的HBSS +缓冲液以抑制Liberase活性并防止过度消化。</li>
		<li>在4°C下以500 ×g 离心滤液5分钟。 小心地取出并丢弃上清液。向沉淀中加入1mL红细胞（RBC）裂解缓冲液，轻轻摇动管以移出沉淀并在冰上孵育1分钟。 注意:红细胞裂解液中的数量和时间取决于沉淀的大小。重要的是将细胞保持在冰上并仔细监测红细胞裂解溶液中的时间，以防止靶细胞的裂解。如果红细胞裂解不足，请重复该步骤。</li>
		<li>加入10-20 mL HBSS+缓冲液以中和红细胞裂解缓冲液。在4°C下以500 ×g 离心滤液5分钟。</li>
		<li>如果裂解的红细胞（鬼细胞）在细胞沉淀上方形成浑浊层，则将沉淀重悬于10mL HBSS +缓冲液中，并通过70μm细胞过滤器过滤悬浮液以消除幽灵细胞。在4°C下以500 ×g 离心滤液5分钟，然后继续进一步的步骤。</li>
	</ol></li>
	<li>免疫细胞和内皮细胞的消耗（可选步骤）<ol>
<li>根据制造商的方案，使用与单克隆抗人CD31和 CD45抗体（同种型小鼠IgG1）和LS柱偶联的CD31 &GT45微珠，从总细胞池中耗尽CD31 +内皮细胞和CD45 + 免疫细胞（材料表）。</li>
		<li>将主要由上皮细胞和基质细胞组成的流收集在新鲜的无菌管中，并在4°C下以500 ×g 离心5分钟。 执行细胞计数以确定流经的细胞总数。</li>
	</ol></li>
	<li>用于荧光相关细胞分选 （FACS） 的细胞表面染色<ol>
<li>重悬 1 x 107 个细胞/1 mL HBSS+ 缓冲液。以所需浓度加入一抗，并在黑暗中在4°C下孵育细胞30分钟。在这项研究中，除非另有说明，否则使用荧光团偶联的一抗。抗体来源和滴度的详细信息在 材料表中描述。 注意:HTII-280是目前最好的表面反应性抗体，允许将远端肺细胞亚化为主要气道（HTII-280-）和肺泡2型（HTII-280+）细胞级分。这种策略的一个警告是，AT1细胞没有使用这种方法染色，并且由于它们的脆弱性而表现不佳。然而，AT1细胞在远端肺制备中代表性较差，可能是由于它们在FACS选择活细胞期间的脆弱性和损失，因此仅代表气道细胞部分的罕见污染物。</li>
		<li>通过加入3mL HBSS +缓冲液洗涤细胞，并在4°C下以500 ×g 离心5分钟。</li>
		<li>如果使用未偶联的一抗，则加入所需浓度的适当荧光团偶联的二抗，并在冰上孵育30分钟。通过加入3mL HBSS +缓冲液洗去多余的二抗，并在4°C下以500 ×g 离心5分钟。</li>
		<li>弃去上清液，每1×107 个细胞/ mL将细胞重悬于HBSS +缓冲液中。通过过滤器盖将细胞过滤到5 mL聚苯乙烯管中，以确保形成单个细胞悬浮液。加入DAPI（1微克/毫升）以染色可渗透（死）细胞。 注意:必须使用适当的单色和荧光减一 （FMO） 对照（即，抗体染色鸡尾酒减去各一种抗体），以尽量减少 FACS 期间的假阳性。在这项研究中，正极和负极选择珠用于对荧光团之间发射光谱重叠的经验补偿（材料表）。流式细胞仪可富集感兴趣的细胞类型。活的上皮细胞根据其CD45阴性，CD31阴性，CD236阳性细胞表面表型和DAPI阴性染色进行富集。这种上皮细胞级分可以根据细胞类型特异性表面标志物的染色进一步分化，例如AT2细胞富集的HTII-280阳性细胞的特异性染色。相反，HTII-280的阴性选择允许富集小气道上皮细胞，如俱乐部和纤毛细胞（图2）。</li>
	</ol></li>
</ol>3. 气管支气管气道上皮祖细胞的富集和亚化
<ol><li>组织准备<ol>
<li>从肺部切除近端气道（气管/支气管）。使用剪刀沿其长度打开气道以暴露腔，并加入50μg/ mL Liberase以完全覆盖组织。</li>
		<li>在37°C下孵育20分钟，使用设置为900rpm的热混合器连续振荡。</li>
		<li>从离心管中取出近端气道并将其置于无菌培养皿（150×15mm）中。使用手术刀轻轻刮擦气道表面，以完全剥离组织的腔上皮细胞。</li>
		<li>用5 mL无菌HBSS +缓冲液洗涤培养皿，以收集所有脱落的腔上皮细胞，并将脱落的细胞转移到50 mL锥形离心管中。通过将5x穿过16 G针头和18 G针头研磨悬浮液，并安装在10 mL注射器上以获得单细胞悬浮液。</li>
		<li>在4°C下以500 ×g 离心悬浮液5分钟。 将沉淀重悬于新鲜的HBSS +缓冲液中，并将这些腔气道细胞储存在冰上，准备在后续步骤中与切碎的近端气道产生的单细胞悬浮液结合。</li>
		<li>使用剪刀，沿着其环切割剩余的气管支气管组织以产生小条组织，并将条状转移到新鲜的培养皿中。使用单面剃须刀片切碎组织条以制成较小的碎片。 注意:由于近端气道是软骨的，因此不能像远端肺组织那样细碎。</li>
		<li>将切碎的组织转移到C管中，向管中加入2mL自由酶，确保组织被浸没。将C管加载到自动解离器上，并运行人肺协议-2以机械方式进一步解离组织。 注意:该协议中使用的解离器针对此特定应用提供了称为人肺协议-2的优化程序（参见 材料表）。</li>
	</ol>
</li>
	<li>酶消化和单细胞分离<ol>
<li>将约2g切碎的近端组织从C管转移到每个50 mL锥形离心管中，并向每个管中加入50μg/ mL自由酶和25μg/ mL DNase溶液。 注意:为确保有效解离，试管的填充量不应超过 30 mL 标记。</li>
		<li>将切碎的组织在37°C下孵育45分钟，使用设置为900rpm的混合器连续振荡。</li>
		<li>如上所述，将解离的组织悬浮液通过一系列细胞滤网（500μm，300μm，100μm，70μm，40μm）并收集流过。用20 mL HBSS+缓冲液洗涤过滤器以收集剩余的细胞。 注意:由于与远端组织相比，近端组织是软骨和笨重的，因此过滤器堵塞的可能性更高。使用漏斗可以帮助防止液体在通过过滤器时溢出。</li>
		<li>向滤液中加入等体积的HBSS +缓冲液，以抑制自由酶活性并防止过度消化。在此步骤中将分离的腔近端气道细胞从3.1.5加入到细胞悬浮液中。</li>
		<li>将组合的细胞悬浮液以500×g离心10分钟。取出上清液并在HBSS +缓冲液中重复细胞洗涤。如上所述，在2.4（可选步骤）中执行CD45 +免疫细胞和CD31 +内皮细胞的消耗。</li>
		<li>染色方法与远端肺组织相似，按照2.5中的步骤操作。根据 CD45 阴性、CD31 阴性、CD236 阳性细胞表面表型和 DAPI 阴性染色，富集活的上皮细胞。</li>
		<li>进一步子集上皮细胞级数基于细胞类型特异性表面标志物的染色，例如NGFR，允许富集基础（NGFR阳性）和非基础（NGFR阴性，分泌，纤毛，神经内分泌）细胞类型（图3）。</li>
	</ol>
</li>
</ol>4. 类器官培养
<ol><li>将5，000个（可以调整该数字以产生所需密度的上皮类器官）分选的近端或远端上皮细胞与7.5×104 MRC-5细胞（人肺成纤维细胞系）一起分选至无菌的1.5 mL管中。上皮-间充质相互作用对于祖细胞的扩增至关重要。</li>
	<li>在4°C下以500 ×g 离心5分钟。 注意:手动确认从分选机获得的细胞计数非常重要，以确保准确的类器官集落形成效率。</li>
	<li>小心地取出并丢弃上清液，并将细胞沉淀重悬于补充有抗生素的50μL冰冷培养基中。将细胞悬浮液保持在冰上。</li>
	<li>向小瓶中加入50μL冰冷的1x生长因子耗尽的基底膜基质培养基，并轻轻移取冰上的悬浮液以混合。 注意:重要的是使用冰冷的介质并将细胞保持在冰上，以避免基底膜基质介质过早聚合。</li>
	<li>将细胞悬浮液转移到24孔板（1.4 x10 4 细胞/ cm2）中的0.4μm孔径细胞培养插入物上，注意避免引入气泡。</li>
	<li>在37°C下孵育30-45分钟以使基质固化。</li>
	<li>向孔中加入600μL预热生长培养基。 注意:培养基在接种后的前24小时补充抗真菌剂（0.4%）和笔链球菌（1%），在前72小时内补充10μM Rho激酶抑制剂。</li>
	<li>在37°C下在5%CO2 培养箱中培养30天，在此期间应每48小时更换培养基。 注意:可以根据实验的目的更改培养持续时间。较长的终点用于研究分化，而较短的终点（7天，14天等）可用于实验目的，如果实验的目的不是实现完全分化。</li>
	<li>向培养基中加入10μM TGFβ抑制剂15天，以维持细胞处于增殖期并抑制成纤维细胞的过度生长。 注意:结果因用于测定的培养基而异。例如，本文所示的结果使用Pneumacult-ALI培养基产生，其导致从远端肺产生大的类器官，从近端肺产生分化良好和较大的类器官。</li>
</ol>5. 类器官染色
<ol><li>类器官的固定和嵌入<ol>
<li>从上部和下部跨膜插入室中抽吸培养基，并用温热的PBS冲洗一次。</li>
		<li>通过在插入片段中放置300μLPFA（2%w / v）并在37°C下在孔中放置500μL1小时来固定培养物。 取出固定剂并用温热的PBS冲洗，注意不要移开底膜基质塞。 注意:固定的类器官可以在4°C的PBS中浸没储存一到两周，然后再开始进一步的步骤。</li>
		<li>吸出PBS，倒置插入物，并小心地在其外围切割插入膜。使用镊子，取下跨孔膜，注意不要干扰基质塞。</li>
		<li>在培养皿中，点击插入物以恢复基质塞。</li>
		<li>将一滴标本处理凝胶如组胶（保持在37°C）加入基质塞中并保持在4°C，直到凝胶凝固。</li>
		<li>将塞子转移到包埋盒中，通过增加浓度的乙醇（70，90和100%）脱水，在二甲苯中透明并嵌入石蜡中。</li>
		<li>在切片机上切割7μm切片，并收集在带正电荷的载玻片上。</li>
	</ol>
</li>
	<li>类器官的免疫荧光染色<ol>
<li>将载玻片在65°C下放置30分钟至脱蜡。</li>
		<li>通过浸入二甲苯中使切片脱蜡，并通过降低乙醇浓度来再水合。</li>
		<li>使用市售的取栓剂在抗原揭开溶液，柠檬酸碱中进行高温抗原检索，方法是将载玻片浸入溶液中15分钟（材料表）。</li>
		<li>使用pap笔用疏水屏障包围组织。</li>
		<li>通过在封闭缓冲液中孵育，阻断一抗和组织之间的非特异性染色。</li>
		<li>在4°C的孵育室中以适当浓度的一抗稀释的一抗切片在4°C下孵育过夜。</li>
		<li>用洗涤缓冲液在室温下冲洗切片3x。</li>
		<li>在室温下以适当浓度的荧光染料偶联二抗孵育1小时。</li>
		<li>用0.1%吐温20-TBS在室温下冲洗切片3x，将切片在DAPI（1μg/ mL）中孵育5分钟。用0.1%吐温20在TBS（Tris缓冲盐水）中冲洗切片一次，干燥并安装在安装溶液中（图4 和 图5）。 注意:用于免疫荧光染色的一抗和二抗的来源和最佳工作稀释度包含在 材料表中。</li>
	</ol>
</li>
</ol>

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作者没有什么可披露的。

我们描述了一种可靠的方法，用于从人体肺组织中分离出确定的肺细胞亚群，以进行分子或功能分析和疾病建模。方法的关键要素包括实现组织解离和表面表位保存的能力，这允许抗体介导的新鲜分离细胞的富集，以及用于有效生成区域特异性上皮类器官的培养方法的优化。我们专注于恢复和富集上皮祖细胞，当在三维培养中与基质支持细胞重组时能够形成类器官。尽管我们没有定义这些培养物中类器官的克隆性，但使用分离的小鼠肺上皮祖细胞进行的类似研究被证明是克隆基于使用具有不同荧光报告基因的细胞的混合培养物22，23。
本文中描述的方法包括旨在改善细胞从消化的肺组织中恢复的适应性。消化后的样品通过16号针，以进一步破坏任何剩余的未消化团块并实现均匀的细胞悬浮液。通过添加DNase I减轻了由挤出的基因组DNA引起的细胞聚集，DNase I产生了一种均质的细胞制剂，在FACS分离过程中提供不间断的流体。这些简单的修饰共同增强了靶细胞群的恢复，并避免了由于FACS富集过程中的结块而导致的延迟。
以前的方案要求使用弹性蛋白酶，分散酶和曲钦/ 2 mM EDTA进行组织消化，以在细胞分离之前产生单个细胞悬浮液4，5。然而，这种蛋白酶的组合导致表面蛋白的损失，并且需要在紫酚包被的培养皿上培养细胞过夜，以便在抗体染色和FACS之前重新表达表面蛋白。相比之下，Liberase和机械搅拌的组合以轻轻地破坏肺组织提供了一种更有效但更温和的解离方案，可以更快地执行，同时保留表面表位以进行抗体染色和FACS富集。因此，总组织处理时间被压缩，并且可以在组织解离后立即进行FACS分离。
这些方法允许分离和 体外 培养上皮祖细胞，这些祖细胞产生代表其起源区域的特化后代。然而，这些方法可以类似地应用于其他细胞群的鉴定和富集，例如免疫，血管和基质细胞类型。这可能特别适用于区域肺上皮芯片系统的开发，该系统允许对血管和上皮区室进行建模，并引入其他细胞类型，如免疫细胞24，25，26。在最近的一项研究中，我们使用使用这种方法生成的肺泡上皮的3D类器官培养物作为研究肺部COVID 19建模和筛选针对SARS-CoV-2感染的治疗靶点的工具。 28

人体呼吸系统的空气空间大致可分为传导区和呼吸区，分别介导气体的运输及其随后穿过上皮- 微血管屏障的交换。传导气道包括气管、细支气管、细支气管和终末细支气管，而呼吸空气空间包括呼吸细支气管、肺泡管和肺泡。这些空域的上皮衬里沿近端-远端轴改变组成，以适应每个功能独特区域的独特要求。气管支气管气道的假分层上皮由基底、分泌和纤毛三种主要细胞类型组成，此外还有较少的细胞类型，包括刷子、神经内分泌和离子细胞1，2，3。支气管气道具有形态上相似的上皮细胞类型，尽管它们的丰度和功能特性有区别。例如，基底细胞在支气管气道内的丰度较低，分泌细胞包括更大比例的俱乐部细胞，而不是气管支气管气道中占主导地位的浆液细胞和杯状细胞。 呼吸区域的上皮细胞包括呼吸细支气管中定义不明确的立方体细胞类型，以及肺泡导管和肺泡的I型肺泡（ATI）和II型（ATII）细胞1，4。
上皮茎和祖细胞类型的特性有助于每个区域上皮的维持和更新，其特征描述不完整，并且主要从动物模型5，6，7，8中的研究中推断出来。对小鼠的研究表明，假分层气道的基础细胞，或细支气管气道的俱乐部细胞或肺泡上皮的ATII细胞，都可以作为基于无限自我更新和多能分化能力的上皮干细胞7，9，10，11，12.尽管无法进行遗传谱系追踪研究来评估人肺上皮细胞类型的干性，但基于类器官的培养模型的可用性，以评估上皮干细胞和祖细胞的功能潜力，为小鼠和人类13，14，15，16，17之间的比较研究提供了工具。
我们描述了从人肺不同区域分离上皮细胞类型的方法，并使用3D类器官系统来概括区域细胞类型。已经开发了类似的方法，用于来自其他器官系统的上皮细胞的功能分析和疾病建模18，19，20，21。这些方法为鉴定区域上皮祖细胞，进行机械研究以研究其调节和微环境以及实现疾病建模和药物发现提供了平台。尽管在动物模型中进行的肺上皮祖细胞研究可以从 体内 或 体外的分析中受益，但对人类肺上皮祖细胞身份的见解在很大程度上取决于模型生物的外推。因此，这些方法提供了一个桥梁，将人肺上皮细胞类型的特性和行为与他们研究的干细胞/祖细胞的调节联系起来。

<table><tbody><tr><td>Cell Isolation</td><td></td><td></td><td></td></tr><tr><td>10 mL Sterile syringes, Luer-Lok Tip</td><td>Fisher scientific</td><td>BD 309646</td><td></td></tr><tr><td>30 mL Sterile syringes, Luer-Lok Tip</td><td>VWR</td><td>BD302832</td><td></td></tr><tr><td>Biohazard bags</td><td>VWR</td><td>89495-440</td><td></td></tr><tr><td>Biohazard bags</td><td>VWR</td><td>89495-440</td><td></td></tr><tr><td>connecting ring</td><td>Pluriselect</td><td>41-50000-03</td><td></td></tr><tr><td>Deoxyribonuclease (lot#SLBF7798V)</td><td>sigma Aldrich</td><td>DN25-1G</td><td></td></tr><tr><td>Disposable Petri dishes</td><td>Corning/Falcon</td><td>25373-187</td><td></td></tr><tr><td>Funnel</td><td>Pluriselect</td><td>42-50000</td><td></td></tr><tr><td>HBSS</td><td>Corning</td><td>21-023</td><td></td></tr><tr><td>Liberase TM Research Grade</td><td>sigma Aldrich</td><td>5401127001</td><td></td></tr><tr><td>needle 16G</td><td>VWR</td><td>305198</td><td></td></tr><tr><td>needle 18G</td><td>VWR</td><td>305199</td><td></td></tr><tr><td>PluriStrainer 100 &amp;micro;m (Cell Strainer)</td><td>Pluriselect</td><td>43-50100-51</td><td></td></tr><tr><td>PluriStrainer 300 &amp;micro;m (Cell Strainer)</td><td>Pluriselect</td><td>43-50300-03</td><td></td></tr><tr><td>PluriStrainer 40 &amp;micro;m (Cell Strainer)</td><td>Pluriselect</td><td>43-50040-51</td><td></td></tr><tr><td>PluriStrainer 500 &amp;micro;m (Cell Strainer)</td><td>Pluriselect</td><td>43-50500-03</td><td></td></tr><tr><td>PluriStrainer 70 &amp;micro;m (Cell Strainer)</td><td>Pluriselect</td><td>43-50070-51</td><td></td></tr><tr><td>Razor blades</td><td>VWR</td><td>55411-050</td><td></td></tr><tr><td>Red Blood Cell lysis buffer</td><td>eBioscience</td><td>00-4333-57</td><td></td></tr><tr><td>Equipment&amp;rsquo;s</td><td></td><td></td><td></td></tr><tr><td>GentleMACS C Tubes</td><td>MACS Miltenyi Biotec</td><td>130-096-334</td><td></td></tr><tr><td>GentleMACS Octo Dissociator</td><td>MACS Miltenyi Biotec</td><td>130-095-937</td><td></td></tr><tr><td>Leica ASP 300s Tissue processor</td><td></td><td></td><td></td></tr><tr><td>LS Columns</td><td>MACS Miltenyi Biotec</td><td>130-042-401</td><td></td></tr><tr><td>MACS MultiStand**</td><td>Miltenyi Biotech</td><td>130-042-303</td><td></td></tr><tr><td>Thermomixer</td><td>Eppendorf</td><td>05-412-503</td><td></td></tr><tr><td>Thermomixer</td><td>Eppendorf</td><td>05-412-503</td><td></td></tr><tr><td>HBSS+ Buffer</td><td></td><td></td><td></td></tr><tr><td>Amphotericin B</td><td>Thermo fisher scientific</td><td>15290018</td><td>2ml</td></tr><tr><td>EDTA (0.5 M), pH 8.0, RNase-free</td><td>Thermo fisher scientific</td><td>AM9260G</td><td>500&amp;micro;l</td></tr><tr><td>Fetal Bovine Serum</td><td>Gemini Bio-Products</td><td>100-106</td><td>10ml</td></tr><tr><td>HBSS Hank&#039;s Balanced Salt Solution 1X 500 ml</td><td>VWR</td><td>45000-456</td><td>500ml bottle</td></tr><tr><td>HEPES (1 M)</td><td>Thermo fisher scientific</td><td>15630080</td><td>5ml</td></tr><tr><td>Penicillin-Streptomycin-Neomycin (PSN) Antibiotic Mixture</td><td>Thermo fisher scientific</td><td>15640055</td><td>5ml</td></tr><tr><td>List of antibodies for FACS</td><td></td><td></td><td></td></tr><tr><td>Alexa Fluor 647 anti-human CD326 (EpCAM) Antibody</td><td>BioLegend</td><td>369820</td><td>1:50</td></tr><tr><td>BD CompBead Anti-Mouse Ig, K/ Negative control particles set</td><td>Fisher Scientific</td><td>BDB552843</td><td></td></tr><tr><td>CD31 MicroBead Kit, human</td><td>Miltenyi Biotec</td><td>130-091-935</td><td>20&amp;micro;l/ 10&lt;sup&gt;7&lt;/sup&gt; total cells</td></tr><tr><td>CD45 MicroBeads, human</td><td>Miltenyi Biotec</td><td>130-045-801</td><td>20&amp;micro;l/ 10&lt;sup&gt;7&lt;/sup&gt; total cells</td></tr><tr><td>DAPI</td><td>Sigma Aldrich</td><td>D9542-10MG</td><td>1:10000</td></tr><tr><td>FITC anti-human CD235a</td><td>BioLegend</td><td>349104</td><td>1:100</td></tr><tr><td>FITC anti-human CD31</td><td>BioLegend</td><td>303104</td><td>1:100</td></tr><tr><td>FITC anti-human CD45</td><td>BioLegend</td><td>304054</td><td>1:100</td></tr><tr><td>FITC anti-mouse IgM Antibody</td><td>BioLegend</td><td>406506</td><td>1:500</td></tr><tr><td>Mouse IgM anti human HT2-280</td><td>Terrace Biotech</td><td>TB-27AHT2-280</td><td>1:300</td></tr><tr><td>PE anti-human CD271(NGFR)</td><td>BioLegend</td><td>345106</td><td>1:50</td></tr><tr><td>Composition of Organoid Culture mediums</td><td></td><td></td><td></td></tr><tr><td>MRC-5</td><td>ATCC</td><td>CCL-171</td><td></td></tr><tr><td>PneumaCult -ALI Medium</td><td>Stemcell Technologies</td><td>5001</td><td></td></tr><tr><td>Small Airway Epithelial Cell Growth Medium</td><td>PromoCell</td><td>C-21170</td><td></td></tr><tr><td>ThinCert Tissue Culture Inserts, Sterile</td><td>Greiner Bio-One</td><td>662641</td><td></td></tr><tr><td>Y-27632 (ROCK inhibitor) 100mM stock (1000x)</td><td>Stemcell Technologies</td><td>72302</td><td></td></tr><tr><td>Mouse Basal medium:</td><td></td><td></td><td></td></tr><tr><td>Amphotericin B</td><td>Thermo fisher scientific</td><td>15290018</td><td>50 &amp;micro;l</td></tr><tr><td>DMEM/F-12, HEPES</td><td>ThermoFisher scientific</td><td>11330032</td><td>50 ml</td></tr><tr><td>Fetal Bovine Serum</td><td>Gemini Bio-Products</td><td>100-106</td><td>5 ml</td></tr><tr><td>Insulin-Transferrin-Selenium (ITS -G) (100X)</td><td>ThermoFisher scientific</td><td>41400045</td><td>500 &amp;micro;l</td></tr><tr><td>Penicillin-Streptomycin-Neomycin (PSN) Antibiotic Mixture</td><td>Thermo fisher scientific</td><td>15640055</td><td>500 &amp;micro;l</td></tr><tr><td>SB431542 TGF-&amp;beta; pathway inhibitor (stock 100 mM)</td><td>Stem cell</td><td>72234</td><td>5 &amp;micro;l</td></tr><tr><td>List of antibodies for Immunohistochemistry</td><td></td><td></td><td></td></tr><tr><td>Antigen unmasking solution, citric acid based</td><td>Vector</td><td>H-3300</td><td>937 &amp;micro;l in 100ml water</td></tr><tr><td>Histogel</td><td>Thermo Scientific</td><td>HG-4000-012</td><td></td></tr><tr><td>Primary Antibodies</td><td></td><td></td><td></td></tr><tr><td>Anti HT2-280</td><td>Terracebiotech</td><td>TB-27AHT2-280</td><td>1:500</td></tr><tr><td>FOXJ1 Monoclonal Antibody (2A5)</td><td>Thermo Fisher Scientific</td><td>14-9965-82</td><td>1:300</td></tr><tr><td>Human Uteroglobin/SCGB1A1 Antibody</td><td>R and D systems</td><td>MAB4218</td><td>1:300</td></tr><tr><td>Keratin 5 Polyclonal Chicken Antibody, Purified [Poly9059]</td><td>Biolegend</td><td>905901</td><td>1:500</td></tr><tr><td>MUC5AC Monoclonal Antibody (45M1)</td><td>Thermo Fisher Scientific</td><td>MA5-12178</td><td>1:300</td></tr><tr><td>PDPN / Podoplanin Antibody (clone 8.1.1)</td><td>LifeSpan Biosciences</td><td>LS-C143022-100</td><td>1:300</td></tr><tr><td>Purified Mouse Anti-E-Cadherin</td><td>BD biosciences</td><td>610182</td><td>1:1000</td></tr><tr><td>Sox-2 Antibody</td><td>Santa Cruz biotechnologies</td><td>sc-365964</td><td>1:300</td></tr><tr><td>Secondary Antibodies</td><td></td><td></td><td></td></tr><tr><td>Donkey anti-rabbit lgG, 488</td><td>Thermo Fisher Scientific</td><td>A-21206</td><td>1:500</td></tr><tr><td>FITC anti-mouse IgM Antibody</td><td>BioLegend</td><td>406506</td><td>1:500</td></tr><tr><td>Goat anti-Hamster IgG (H+L), Alexa Fluor 594</td><td>Thermo Fisher Scientific</td><td>A-21113</td><td>1:500</td></tr><tr><td>Goat anti-Mouse IgG1 Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td><td>Thermo Fisher Scientific</td><td>A-21121</td><td>1:500</td></tr><tr><td>Goat anti-Mouse IgG2a Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td><td>Thermo Fisher Scientific</td><td>A-21131</td><td>1:500</td></tr><tr><td>Goat anti-Mouse IgG2a Cross-Adsorbed Secondary Antibody, Alexa Fluor 568</td><td>Thermo Fisher Scientific</td><td>A-21134</td><td>1:500</td></tr><tr><td>Goat anti-Mouse IgG2b Cross-Adsorbed Secondary Antibody, Alexa Fluor 568</td><td>Thermo Fisher Scientific</td><td>A-21144</td><td>1:500</td></tr><tr><td>Buffers</td><td></td><td></td><td></td></tr><tr><td>Immunohistochemistry Blocking Solution</td><td></td><td></td><td>3% BSA, o.4% Triton-x100 in TBS (Tris based saline)</td></tr><tr><td>Immunohistochemistry Incubation Solution</td><td></td><td></td><td>3% BSA, ).1% Triton-X100 in TBS</td></tr><tr><td>Immunohistochemistry Washing Solution</td><td></td><td></td><td>TBS with 0.1% Tween 20</td></tr></tbody></table>

isolation and enrichment of human lung epithelial progenitor cells for organoid culture

上皮类器官模型是研究器官系统基础生物学和疾病建模的宝贵工具。当作为类器官生长时，上皮祖细胞可以自我更新并产生分化的后代，其表现出与 体内 对应物相似的细胞功能。在这里，我们描述了一种从人肺中分离区域特异性祖细胞并生成3D类器官培养物作为实验和验证工具的分步方案。我们定义肺的近端和远端区域，目的是分离区域特异性祖细胞。我们利用酶解离和机械解离的组合从肺和气管中分离出总细胞。然后使用基于细胞类型特异性表面标志物的荧光相关细胞分选（FACS）从近端或远端起源细胞中分离出特异性祖细胞，例如用于分选基底细胞的NGFR和用于分选II型肺泡细胞的HTII-280。分离的基础或肺泡II型祖细胞用于生成3D类器官培养物。远端和近端祖细胞均形成类器官，当在第30天接种5000个细胞/孔时，远端区域的集落形成效率为9-13%，近端区域的集落形成效率为7-10%。远端类器官在培养物中维持HTII-280 + 肺泡II型细胞，而近端类器官在第30天分化为纤毛和分泌细胞。这些3D类器官培养物可用作研究肺上皮细胞生物学和上皮间充质相互作用的实验工具，以及开发和验证针对疾病中上皮功能障碍的治疗策略。

源肺组织 气管和肺外支气管（图1A）被用作分离近端气道上皮细胞和随后产生近端类器官的源组织。包括实质和直径小于2mm的小气道（图1A）的远端肺组织用于分离小气道和肺泡上皮细胞（远端肺上皮）和生成小气道或肺泡类器官。由假分层上皮衬里的近端气道包括丰富的基底祖细胞，这些细胞对膜蛋白NGFR具有免疫反应性（图1B，C）。相反，肺泡内壁的上皮细胞包括一个显示与HTII-280单克隆抗体的顶膜免疫反应性的子集，提示其肺泡2型细胞（AT2）身份（图1B，D）。这些表面标志物用于从近端或远端区域分离的上皮细胞的单细胞悬浮液的子集。
组织解离和细胞分级分离 从人肺组织的近端或远端区域分离总细胞的单细胞悬浮液，并使用磁珠和FACS进行分馏，以产生富集的上皮细胞群（图2 和 图3）。分别使用针对CD235a，CD45和CD31的抗体对包括红细胞，免疫细胞和内皮细胞在内的大量污染细胞类型进行染色，然后进行磁性相关细胞分选，以耗尽这些细胞类型从肺细胞的总池中耗尽。在远端（图2E）和近端（图3E）组织样品中，所得的"耗尽"细胞悬浮液对于上皮细胞群显着富集，并相应提高了FACS效率。 使用CD45和CD31微珠耗尽CD235a / CD45 / CD31阳性细胞后，CD31 - / CD45 - / CD235a - 的百分比在远端人群中从14%（图2A，B）增加到51.7%（图2E，F）。FCS进一步消耗CD235a，CD45或CD31阳性染色的细胞，消除DAPI阳性染色的细胞和上皮细胞表面标志物CD326的阳性选择，导致高度富集的远端细胞群占33.5%（图2E，G），而7%（图2A，C）在负人口枯竭之前。远端上皮细胞群的进一步亚化分别通过基于HTII-280单克隆抗体的表面染色（图2D，H）进行分馏来实现。因此，远端肺上皮细胞包括4.3%的HTII-280+ 和2.6%的HTII-280- 亚群（图2D ，不消耗CD31/ CD45 / CD235a）和30%的HTII-280+ 和3.6%的HTII-280- 亚群（图2H 在CD31 / CD45 / CD235a耗尽后）。
使用CD45和CD31微珠耗尽从近端区域分离的CD235a / CD45 / CD31阳性细胞的总细胞，并且CD31 - / CD45 - / CD235a- 的百分比从17%（图3A，B）增加到56.6%（图3E，F）。在从近端区域分离的细胞中对上皮细胞表面标志物CD326的阳性选择，导致高度富集的近端细胞群，占总肺细胞分数的38%（图3E，G），而9.3%（图3A，C）没有消耗阴性群体。近端上皮细胞群的进一步亚化分别通过基于NGFR抗体的表面染色进行分馏来实现（图3D，H）。因此，近端肺上皮细胞包括2.7%的NGFR+ 和6.5%的NGFR- 亚群（图3D 没有CD31 / CD45 / CD235a的消耗），13%是NGFR + 和25%的NGFR- （图3H 在CD31 / CD45 / CD235a耗尽后）。
肺类器官培养 在生长因子耗尽的基底膜基质基质中培养远端肺上皮类器官，这些培养基经过经验测试以优化类器官生长和分化。评估了三种不同的培养基，包括肺气肿-ALI培养基、小气道上皮细胞生长培养基（SAECG培养基）和小鼠基础培养基。使用肺钙-ALI培养基获得最佳的类器官生长，该培养基被选择用于进一步研究。HTII-280 + 远端肺上皮细胞的培养物产生快速扩张的类器官，平均集落形成效率为10%（图4A，B）。使用HTII-280和SPC单克隆抗体对第30天培养物进行免疫荧光染色，发现含有腔内的类器官主要由HTII-280+ 和SPC + 远端肺上皮细胞组成（图4C，C' 和 图4D，D'）。远端肺上皮HTII-280细胞 的培养产生类器官，这些类器官由类似于小气道的假分层上皮组成（未显示）。
将近端肺上皮类器官从接种到基质胶中的NGFR+ 细胞培养，并在肺病-ALI培养基中培养30天。观察到含有大腔类器官（图5D，E，F），平均菌落形成效率为7.8%（图5A，B，C）。类器官由假分层上皮组成，该上皮由自我更新的Krt5 + 和NGFR + 基底细胞组成（图5D，5E）和分化的腔细胞类型，包括FoxJ1 + 纤毛细胞和MUC5AC + 分泌细胞（图5D，E）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61541/61541fig1.jpg"> 图1:人体肺组织取样。 （A）人肺的示意图，显示对近端和远端区域进行采样以进行细胞分离的策略。（B）肺近端和远端区域的H&E染色。（三、四）相应区域的免疫荧光染色在支气管气道基底膜上显示NGFR + 基底祖细胞（红色）和肺泡中的HTII-280 + 肺泡II型II型祖细胞（绿色）。比例尺 = 50 μm。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61541/61541fig1large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61541/61541fig2.jpg"> 图2:远端肺细胞的代表性分选策略。（A，E）在一个生物样本的肺远端区域使用CD31和CD45磁珠耗尽CD45 +和CD31 +群体之前和之后的各种细胞群的百分比。（B，F）FACS图的代表性图像显示了CD31-/CD45-/CD235a-人群在CD31/CD45/CD235a阳性人群（C，G）耗竭前后CD31-/CD45/CD235a阳性人群耗尽前后的门控策略。（D，H）HTII-280 + / - 群体在CD31 / CD45 / CD235a阳性细胞耗尽之前和之后。面板A-D来自同一生物样品，面板E-H来自同一生物样品。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61541/61541fig2large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61541/61541fig3.jpg"> 图3:近端肺细胞的代表性分选策略 （A，E）在肺近端区域使用CD31和CD45磁珠耗尽CD45 +和CD31 +群体之前和之后各种细胞群的百分比。（B，F）FACS图的代表性图像显示了CD31-/CD45-/CD235a-人群在CD31/CD45/CD235a阳性人群耗竭前后的近端CD31/CD45/CD235a阳性人群（C，G）Epcam+人群耗竭前后的门控策略。（D，H）NGFR +/-群体在CD31 / CD45 / CD235a阳性细胞耗尽之前和之后。A-D和E-H面板由两种不同的生物样品制备。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61541/61541fig3large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61541/61541fig4.jpg"> 图4:远端肺类器官的特征。 （A）在肺病培养基（2倍放大）中培养的人远端类器官的代表性图像。（B）在来自两个不同生物样品的类器官的一式三份孔上计算菌落形成效率（%CFE）。（C、 C'）在ALI培养基中培养的相应远端类器官的免疫荧光染色显示HTII-280 + AT 2细胞（绿色）。（德， 德）本研究中用于分离AT2细胞的标记物，HTII-280（绿色）用于另一个表征良好的AT2细胞标志物SPC（红色）。比例尺 = 50um。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61541/61541fig4large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61541/61541fig5.jpg"> 图5: 来自人近端肺的近端类器官的特征。 （一、二）在PneumaCult-ALI中等比例棒50μm中培养的人近端类器官的代表性图像（C）在来自两个不同生物样品的类器官的三份孔上计算集落形成效率百分比（%CFE）。在第30天对分化的近端类器官进行免疫荧光染色，包括（D）Krt5 + 基底细胞（绿色），FoxJ1 + 纤毛细胞（红色）（E）Krt5 + 基底细胞（绿色）和MUC5AC + 高脚杯细胞（红色）。（F）本研究中用于分离基底细胞的标记物，用于表征良好的基底细胞标志物Krt5（红色）的NGFR（绿色）共染色。比例尺 = 50 μm <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61541/61541fig5large.jpg" target="_blank">.请点击此处查看此图的大图。</a>

Watch this Scientific Journal Video about Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture at JoVE.com

Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture

本文提供了组织解离和细胞分馏方法的详细方法，允许从人肺的近端和远端区域富集活的上皮细胞。本文将这些方法应用于通过使用3D类器官培养模型对肺上皮祖细胞进行功能分析。

分离和富集人肺上皮祖细胞用于类器官培养

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, ...

isolation-enrichment-human-lung-epithelial-progenitor-cells-for

Nanyang Technological University

Research

JoVE Journal

Biology

8.0K 次观看.  Cedars-Sinai Medical Center. 本文提供了组织解离和细胞分馏方法的详细方法，允许从人肺的近端和远端区域富集活的上皮细胞。本文将这些方法应用于通过使用3D类器官培养模型对肺上皮祖细胞进行功能分析。

视频: Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and cell-matrix interactions. Due to this complexity, recapitulation of lung development in vitro to promote differentiation of stem cells to lung epithelial cells has been challenging. In this protocol, decellularized lung scaffolds are used to mimic the 3-dimensional environment of the lung and generate stem cell-derived airway epithelial cells. Mouse embryonic stem cell are first differentiated to the endoderm lineage using an embryoid body (EB) culture method with activin A. Endoderm cells are then seeded onto decellularized scaffolds and cultured at air-liquid interface for up to 21 days. This technique promotes differentiation of seeded cells to functional airway epithelial cells (ciliated cells, club cells, and basal cells) without additional growth factor supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. Although there is limited contamination from non-lung endoderm lineages in culture, this protocol only generates airway epithelial populations and does not give rise to alveolar epithelial cells. Airway epithelia generated with this protocol can be used to study cell-matrix interactions during lung organogenesis and for disease modeling or drug-discovery platforms of airway-related pathologies such as cystic fibrosis.

This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting.

用脱细胞支架龙ESC衍生鼠气道上皮细胞的生成

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

科研

生物工程

Primary Human Bronchial Epithelial Cells Grown from Explants

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the function and structure of human epithelial cells. Here we describe in detail the method of growing bronchial epithelial cells from bronchial airway tissue that is harvested by the surgeon at the times of lung surgery (e.g. lung cancer or lung volume reduction surgery). With ethics approval and informed consent, the surgeon takes what is needed for pathology and provides us with a bronchial portion that is remote from the diseased areas. The tissue is then used as a source of explants that can be used for growing primary bronchial epithelial cells in culture. Bronchial segments about 0.5-1cm long and &le;1cm in diameter are rinsed with cold EBSS and excess parenchymal tissue is removed. Segments are cut open and minced into 2-3mm3 pieces of tissue. The pieces are used as a source of primary cells. After coating 100mm culture plates for 1-2 hr with a combination of collagen (30 &mu;g/ml), fibronectin (10 &mu;g/ml), and BSA (10 &mu;g/ml), the plates are scratched in 4-5 areas and tissue pieces are placed in the scratched areas, then culture medium (DMEM/Ham F-12 with additives) suitable for epithelial cell growth is added and plates are placed in an incubator at 37&deg;C in 5% CO2 humidified air. The culture medium is changed every 3-4 days. The epithelial cells grow from the pieces forming about 1.5 cm diameter rings in 3-4 weeks. Explants can be re-used up to 6 times by moving them into new pre-coated plates. Cells are lifted using trypsin/EDTA, pooled, counted, and re-plated in T75 Cell Bind flasks to increase their numbers. T75 flasks seeded with 2-3 million cells grow to 80% confluence in 4 weeks. Expanded primary human epithelial cells can be cultured and allowed to differentiate on air-liquid interface. Methods described here provide an abundant source of human bronchial epithelial cells from freshly isolated tissues and allow for studying these cells as models of disease and for pharmacology and toxicology screening.

Here we describe a detailed method for growing primary human bronchial epithelial cells from explants of human bronchial airway tissue including differentiated growth on an air-liquid interface. This method provides an abundant source of primary cells for investigating the role of the airway epithelium in human lung health and disease.

从外植体生长的首要人权支气管上皮细胞

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the ...

生物学

Generation of 3D Whole Lung Organoids from Induced Pluripotent Stem Cells for Modeling Lung Developmental Biology and Disease

Human lung development and disease has been difficult to study due to the lack of biologically relevant in vitro model systems. Human induced pluripotent stem cells (hiPSCs) can be differentiated stepwise into 3D multicellular lung organoids, made of both epithelial and mesenchymal cell populations. We recapitulate embryonic developmental cues by temporally introducing a variety of growth factors and small molecules to efficiently generate definitive endoderm, anterior foregut endoderm, and subsequently lung progenitor cells. These cells are then embedded in growth factor reduced (GFR)-basement membrane matrix medium, allowing them to spontaneously develop into 3D lung organoids in response to external growth factors. These whole lung organoids (WLO) undergo early lung developmental stages including branching morphogenesis and maturation after exposure to dexamethasone, cyclic AMP and isobutylxanthine. WLOs possess airway epithelial cells expressing the markers KRT5 (basal), SCGB3A2 (club) and MUC5AC (goblet) as well as alveolar epithelial cells expressing HOPX (alveolar type I) and SP-C (alveolar type II). Mesenchymal cells are also present, including smooth muscle actin (SMA), and platelet-derived growth factor receptor A (PDGFR&#945;). iPSC derived WLOs can be maintained in 3D culture conditions for many months and can be sorted for surface markers to purify a specific cell population. iPSC derived WLOs can also be utilized to study human lung development, including signaling between the lung epithelium and mesenchyme, to model genetic mutations on human lung cell function and development, and to determine the cytotoxicity of infective agents.

The article describes step wise directed differentiation of induced pluripotent stem cells to three-dimensional whole lung organoids containing both proximal and distal epithelial lung cells along with mesenchyme.

从诱导多能干细胞生成3D全肺类器官，用于模拟肺发育生物学和疾病

Human lung development and disease has been difficult to study due to the lack of biologically relevant in vitro model systems. Human induced pluripotent ...

发育生物学

Generation of Airway Epithelial Cell Air-Liquid Interface Cultures from Human Pluripotent Stem Cells

Diseases of the conducting airway such as asthma, cystic fibrosis (CF), primary ciliary dyskinesia (PCD), and viral respiratory infections are major causes of morbidity and mortality worldwide. In vitro platforms using human bronchial epithelial cells (HBECs) have been instrumental to our understanding of the airway epithelium in health and disease. Access to HBECs from individuals with rare genetic diseases or rare mutations is a bottleneck in lung research.
Induced pluripotent stem cells (iPSCs) are readily generated by "reprogramming" somatic cells and retain the unique genetic background of the individual donor. Recent advances allow for the directed differentiation of iPSCs to lung epithelial progenitor cells, alveolar type 2 cells, as well as the cells of the conducting airway epithelium via basal cells, the major airway stem cells.
Here we outline a protocol for the maintenance and expansion of iPSC-derived airway basal cells (hereafter iBCs) as well as their trilineage differentiation in air-liquid interface (ALI) cultures. iBCs are maintained and expanded as epithelial spheres suspended in droplets of extracellular matrix cultured in a primary basal cell medium supplemented with inhibitors of TGF-&#223; and BMP signaling pathways. iBCs within these epithelial spheres express key basal markers TP63 and NGFR, can be purified by fluorescence activated cell sorting (FACS), and when plated on porous membranes in standard ALI culture conditions, differentiate into a functional airway epithelium. ALI cultures derived from healthy donors are composed of basal, secretory and multiciliated cells and demonstrate epithelial barrier integrity, motile cilia, and mucus secretion. Cultures derived from individuals with CF or PCD recapitulate the dysfunctional CFTR-mediated chloride transport or immotile cilia, the respective disease-causing epithelial defects.
Here, we present a protocol for the generation of human cells that can be applied for modeling and understanding airway diseases.

Recent advances in human induced pluripotent stem cell differentiation protocols allow for the stepwise derivation of organ-specific cell types. Here, we provide detailed steps for the maintenance and expansion of iPSC-derived airway basal cells and their differentiation into a mucociliary epithelium in air-liquid interface cultures.

从人多能干细胞中产生气道上皮细胞气液界面培养物

Diseases of the conducting airway such as asthma, cystic fibrosis (CF), primary ciliary dyskinesia (PCD), and viral respiratory infections are major ...

<meta charset="utf8"></head><body>用于研究肺屏障完整性、疾病和药物发现的气道模型

iPSC-Derived Epithelial, Mesenchymal, Endothelial, and Immune Cell Co-Culture to Model Airway Barrier Integrity in Lung Health and Disease

Human lung tissue is composed of an interconnected network of epithelium, mesenchyme, endothelium, and immune cells from the upper airway of the nasopharynx to the smallest alveolar sac. Interactions between these cells are crucial in lung development and disease, acting as a barrier against harmful chemicals and pathogens. Current in vitro co-culture models utilize immortalized cell lines with different biological backgrounds, which may not accurately represent the cellular milieu or interactions of the lung. We differentiated human iPSCs into 3D lung organoids (containing both epithelium and mesenchyme), endothelial cells, and macrophages. These were co-cultured in an air-liquid interface (ALI) format to form an epithelial/mesenchymal apical barrier invested with macrophages and a basolateral endothelial barrier (iAirway). iPSC-derived iAirways showed a reduction in barrier integrity in response to infection with respiratory viruses and cigarette toxins. This multi-lineage lung co-culture system provides a platform for studying cellular interactions, signaling pathways, and molecular mechanisms underlying lung development, homeostasis, and disease progression. iAirways closely mimic human physiology and cellular interactions, can be generated from patient-derived iPSC&#39;s, and can be customized to include different cell types of the airway. Overall, iPSC-derived iAirway models offer a versatile and powerful tool for studying barrier integrity to better understand genetic drivers for disease, pathogen response, immune regulation, and drug discovery or repurposing in vitro, with the potential to advance our understanding and treatment of airway diseases.

<meta charset="utf8"></head><body>作者聚焦:使用同基因上皮细胞、内皮细胞和免疫细胞对人类气道重塑和病毒反应进行建模

This article describes the generation of a complex, multi-cellular airway barrier model composed of induced pluripotent stem cell (iPSC)-derived lung epithelium, mesenchyme, endothelial cells, and macrophages in an air-liquid interface culture.

iPSC 衍生的上皮细胞、间充质细胞、内皮细胞和免疫细胞共培养，以模拟肺部健康和疾病中的气道屏障完整性

Human lung tissue is composed of an interconnected network of epithelium, mesenchyme, endothelium, and immune cells from the upper airway of the ...

Isolating Bronchial Epithelial Cells from Resected Lung Tissue for Biobanking and Establishing Well-Differentiated Air-Liquid Interface Cultures

The airway epithelial cell layer forms the first barrier between lung tissue and the outside environment and is thereby constantly exposed to inhaled substances, including infectious agents and air pollutants. The airway epithelial layer plays a central role in a large variety of acute and chronic lung diseases, and various treatments targeting this epithelium are administered by inhalation. Understanding the role of epithelium in pathogenesis and how it can be targeted for therapy requires robust and representative models. In vitro epithelial culture models are increasingly being used and offer the advantage of performing experiments in a controlled environment, exposing the cells to different kinds of stimuli, toxicants, or infectious agents. The use of primary cells instead of immortalized or tumor cell lines has the advantage that these cells differentiate in culture to a pseudostratified polarized epithelial cell layer with a better representation of the epithelium compared to cell lines.
Presented here is a robust protocol, that has been optimized over the past decades, for the isolation and culture of airway epithelial cells from lung tissue. This procedure allows successful isolation, expansion, culture, and mucociliary differentiation of primary bronchial epithelial cells (PBECs) by culturing at the air-liquid interface (ALI) and includes a protocol for biobanking. Furthermore, the characterization of these cultures using cell-specific marker genes is described. These ALI-PBEC cultures can be used for a range of applications, including exposure to whole cigarette smoke or inflammatory mediators, and co-culture/infection with viruses or bacteria. 
The protocol provided in this manuscript, illustrating the procedure in a step-by-step manner, is expected to provide a basis and/or reference for those interested in implementing or adapting such culture systems in their laboratory.

Presented here is a reproducible, affordable, and robust method for the isolation and expansion of primary bronchial epithelial cells for long-term biobanking and the generation of differentiated epithelial cells by culture at the air-liquid interface.

从切除的肺组织中分离支气管上皮细胞用于生物样本库并建立分化的气液界面培养物

The airway epithelial cell layer forms the first barrier between lung tissue and the outside environment and is thereby constantly exposed to inhaled ...

免疫与感染

Mucociliary Epithelial Organoids from Xenopus Embryonic Cells: Generation, Culture and High-Resolution Live Imaging

Mucociliary epithelium provides the first line of defense by removing foreign particles through the action of mucus production and cilia-mediated clearance. Many clinically relevant defects in the mucociliary epithelium are inferred as they occur deep within the body. Here, we introduce a tractable 3D model for mucociliary epithelium generated from multipotent progenitors that were microsurgically isolated from Xenopus laevis embryos. The mucociliary epithelial organoids are covered with newly generated epithelium from deep ectoderm cells and later decorated with distinct patterned multiciliated cells, secretory cells, and mucus-producing goblet cells that are indistinguishable from the native epidermis within 24 h. The full sequences of dynamic cell transitions from mesenchymal to epithelial that emerge on the apical surface of organoids can be tracked by high-resolution live imaging. These in vitro cultured, self-organizing mucociliary epithelial organoids offer distinct advantages in studying the biology of mucociliary epithelium with high-efficiency in generation, defined culture conditions, control over number and size, and direct access for live imaging during the regeneration of the differentiated epithelium.

We describe a simple protocol to develop mucociliary epithelial organoids from deep ectoderm cells isolated from Xenopus laevis embryos. The multipotent progenitors regenerate epithelial goblet cell precursors and allow live tracking of the initiation and progression of the cell transitions on the surface of organoids.

Xenopus胚胎细胞的粘膜异体器官:生成、培养和高分辨率实时成像

Mucociliary epithelium provides the first line of defense by removing foreign particles through the action of mucus production and cilia-mediated ...

Generation and Characterization of Murine Oral Mucosal Organoid Cultures

The mucous lining covering the inside of our mouth, the oral mucosa, is a highly compartmentalized tissue and can be subdivided into the buccal mucosa, gingiva, lips, palate, and tongue. Its uppermost layer, the oral epithelium, is maintained by adult stem cells throughout life. Proliferation and differentiation of adult epithelial stem cells have been intensively studied using in vivo mouse models as well as two-dimensional (2D) feeder-cell based in vitro models. Complementary to these methods is organoid technology, where adult stem cells are embedded into an extracellular matrix (ECM)-rich hydrogel and provided with a culture medium containing a defined cocktail of growth factors. Under these conditions, adult stem cells proliferate and spontaneously form three-dimensional (3D) cell clusters, the so-called organoids. Organoid cultures were initially established from murine small intestinal epithelial stem cells. However, the method has since been adapted for other epithelial stem cell types. Here, we describe a protocol for the generation and characterization of murine oral mucosal organoid cultures. Primary epithelial cells are isolated from murine tongue tissue, embedded into an ECM hydrogel, and cultured in a medium containing: epidermal growth factor (EGF), R-spondin, and fibroblast growth factor (FGF) 10. Within 7 to 14 days of initial seeding, the resulting organoids can be passaged for further expansion and cryopreservation. We additionally present strategies for the characterization of established organoid cultures via 3D whole-mount imaging and gene-expression analysis. This protocol may serve as a tool to investigate oral epithelial stem cell behavior ex vivo in a reductionist manner.

We present a method for the generation and characterization of oral mucosal organoid cultures derived from the tongue epithelium of adult mice.

The mucous lining covering the inside of our mouth, the oral mucosa, is a highly compartmentalized tissue and can be subdivided into the buccal mucosa, ...

分离和富集人肺上皮祖细胞用于类器官培养

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此视频中的章节

用脱细胞支架龙ESC衍生鼠气道上皮细胞的生成

从外植体生长的首要人权支气管上皮细胞

从诱导多能干细胞生成3D全肺类器官，用于模拟肺发育生物学和疾病

从人多能干细胞中产生气道上皮细胞气液界面培养物

iPSC 衍生的上皮细胞、间充质细胞、内皮细胞和免疫细胞共培养，以模拟肺部健康和疾病中的气道屏障完整性

iPSC 衍生的上皮细胞、间充质细胞、内皮细胞和免疫细胞共培养，以模拟肺部健康和疾病中的气道屏障完整性

iPSC 衍生的上皮细胞、间充质细胞、内皮细胞和免疫细胞共培养，以模拟肺部健康和疾病中的气道屏障完整性

从切除的肺组织中分离支气管上皮细胞用于生物样本库并建立分化的气液界面培养物

Xenopus胚胎细胞的粘膜异体器官:生成、培养和高分辨率实时成像

Generation and Characterization of Murine Oral Mucosal Organoid Cultures