Name: generation of human nasal epithelial cell spheroids for individualized cystic fibrosis transmembrane conductance regulator study
Uploaded: 2018-04-11T14:45:00.000+00:00
Duration: 8 min
Description: Watch this Scientific Journal Video about Generation of Human Nasal Epithelial Cell Spheroids for Individualized Cystic Fibrosis Transmembrane Conductance Regulator Study at JoVE.com

这项工作得到了囊性纤维化基础疗法, 格兰特号 CLANCY14XX0, 并通过囊性纤维化基金会的支持, CLANCY15R0 号。作者希望感谢克里斯蒂娜. 雷在病人招募和监管监督方面的协助。作者还要感谢他的工作组, 该组织在囊性纤维化基金会的支持下, 帮助培养他文化能力: 普雷斯顿 Bratcher, 卡尔文棉花, 玛蒂娜 Gentzsch, 伊丽莎白 Joseloff, 迈克尔 Myerburg, 戴夫尼科尔斯,斯科特 Randell, 史蒂夫罗, g. 所罗门, 凯瑟琳. Tuggle。

他样本是从辛辛那提儿童医院医学中心的研究中心获得的。这里描述的所有方法都已被辛辛那提儿童医院医疗中心的机构审查委员会批准。在测试之前从所有科目获得书面同意。
1. 准备扩张介质和抗生素介质
<ol><li>收集表 1中列出的媒体组件。在室温下解冻冷冻材料, 并根据 "扩展介质" 在表 1中所示, 做出必要的库存解决方案。 注意: 处理霍乱毒素时要小心, 如果摄取有毒物质, 是皮肤、眼睛和呼吸道刺激性的。在安全柜的清洁条件下制作并组合所有的解决方案和介质。</li>
	<li>使用50毫升血清吸管, 从一个容器中除去并丢弃50毫升的基介质 (Dulbecco 修饰鹰 Medium/F12), 以补偿其他材料的添加。将所有基介质手工倒入蒸压1升玻璃瓶中。</li>
	<li>根据需要使用50毫升血清吸管或1毫升吸管, 将所有剩余的元件从表 1的 "扩展介质" 部分转移到包含介质的蒸压玻璃瓶瓶中。用手轻轻地旋转瓶子混合。</li>
	<li>将介质过滤成新鲜的、无菌的1升、0.22 µm 聚醚砜 (PES) 过滤瓶, 使用制造商的说明和注释与 "扩展介质" 和日期。</li>
	<li>
准备抗生素介质。如表1所示, 在 "抗生素介质" 下做必要的抗生素库存解决方案。
	<ol>
<li>使用50毫升血清吸管, 转移150毫升的膨胀介质到一个新鲜的250毫升蒸压玻璃瓶。</li>
		<li>使用1毫升吸管, 将表 1中 "抗生素介质" 部分中的所有组件添加到包含介质的蒸压玻璃瓶瓶中。用手旋匀, 直到介质在外观上均匀。</li>
		<li>过滤介质进入新鲜, 无菌250毫升, 0.22 µm PES 过滤瓶使用制造商的指示和注释与 "抗生素介质" 和日期。</li>
	</ol>
</li>
	<li>将介质存储在摄氏4摄氏度的一个月内, 如果多云, 则会丢弃, 暗示污染。</li>
</ol>2. 准备差异化媒体
<ol><li>收集表 2中列出的媒体组件。在室温下解冻冷冻材料, 并按照表 2中的说明做出必要的库存解决方案。 注意: 在处理维甲酸时要小心, 这是一种生殖毒素, 如果吞下会中毒, 导致皮肤发炎。制作, 整除, 并结合所有解决方案和媒体在清洁的条件下, 在安全柜。</li>
	<li>从一个容器中除去并丢弃52毫升的基介质, 以补偿其他材料的添加。将所有基介质倒入蒸压1升玻璃瓶中。</li>
	<li>根据需要使用25毫升血清吸管或1毫升吸管, 将所有剩余的组件从表 2转移到包含介质的蒸压玻璃瓶中, 并用手轻轻地旋转以混合。</li>
	<li>将介质过滤成新鲜的, 不育的1升, 0.22 µm 聚醚砜 (PES) 过滤瓶使用制造商的指示和注释与 "差异媒体" 和日期。</li>
	<li>将介质存储在摄氏4摄氏度的一个月内, 如果多云, 则会丢弃, 暗示污染。</li>
</ol>3. 涂层培养皿和板料成纤维细胞
注: 在安全柜内的清洁条件下执行所有步骤。
<ol><li>将0.5 毫升胶原蛋白和37.5 毫升不育水混合在50毫升锥形管中。</li>
	<li>将4-5 毫升胶原溶液注入每个清洁的 P100 培养皿, 确保整个表面覆盖。</li>
	<li>在37摄氏度和 5% CO2上, 用盖子和孵化过夜。</li>
	<li>在生物安全柜中, 取出所有碟盖。涡流溶液覆盖盘, 然后吸。</li>
	<li>用紫外线 (紫外线) 光照射1小时消毒, 将盖子放回盘子上, 用铝箔叠住盘子。</li>
	<li>将涂装的菜肴存放在4摄氏度3-6 月, 在紫外线照射下, 在细胞培养使用前重新杀菌15-30 分钟。</li>
	<li>24 h 在获得他样品之前, 对预涂的盘子和标签作为小鼠胚胎成纤维细胞 (MEF) 和日期进行消毒。</li>
	<li>添加扩展媒体覆盖板 (约5毫升) 与血清吸管。</li>
	<li>解冻一小瓶 2 x 106辐照 MEFs 在37°c 水浴器。一旦 MEFs 解冻, 将一半的瓶子 (大约 106细胞) 添加到盘子里, 用1毫升的吸管, 用手旋转以驱散。</li>
	<li>在37摄氏度和 5% CO2上孵化一夜, 准备他文化。 注: 如有必要, 步骤 3.7-3.10 可以在电镀细胞前60分钟进行, 尽管过夜是理想的。</li>
</ol>4. 获取和处理他样品
<ol><li>确保为所有科目获得 IRB 批准的同意书。</li>
	<li>有病人擤鼻涕, 以清除松散的粘液。</li>
	<li>使用鼻内窥镜可视化下鼻甲。确保不存在任何可能阻止标本收集的病灶或息肉。</li>
	<li>
通过刮除或刷牙从第一鼻孔收集鼻腔细胞。
	<ol>
<li>刮除: 弯曲刮匙 (在其中点, 以创建一个轻微的, ~135 °的角度与顶点远离刮匙 (杯。将刮匙 (插入鼻孔, 在下鼻甲下推进刮匙 (杯。轻轻按下陀螺刮匙 (杯, 并通过拉刮匙 (向前刮陀螺, 重复3-4 倍的总和。</li>
		<li>刷子: 通过下面的陀螺下的细胞学刷, 然后旋转和移动的刷子在和出略4-5 倍, 不退出鼻孔。</li>
	</ol>
</li>
	<li>放置刮匙 (/刷子在一个15毫升圆锥填充抗生素介质。对其他鼻孔重复步骤 4.3-4.4, 将刮匙 (/刷子放入同一锥形管中。</li>
	<li>在冰上储存锥形, 直到准备好加工。确保在样品采集4小时内进行处理, 但如有必要, 可在购置后的24小时内发生。</li>
</ol>5. 在菜肴中他细胞的加工和扩展
注: 在安全柜内的清洁条件下执行所有步骤。
<ol><li>使用1毫升的吸管和介质从样品圆锥, 洗涤所有细胞刮匙/细胞学刷子到同一锥管。一旦清洁, 丢弃刮匙/刷子。</li>
	<li>离心机圆锥在 360 x g 和4°c 为5分钟。</li>
	<li>吸入上清液, 注意不要扰乱细胞颗粒。</li>
	<li>在3毫升细胞脱离溶液中重新悬浮细胞颗粒, 吹打1毫升融汇的混合物。</li>
	<li>离心机圆锥在 360 x g 和4°c 为5分钟。</li>
	<li>重复步骤 5.3-5.5 次。</li>
	<li>吸入剩余的上清, 注意不要扰乱细胞颗粒。</li>
	<li>在1毫升的抗生素培养基中重新悬浮颗粒, 用 hemocytometer 计数细胞。</li>
	<li>使用1毫升的吸管, 板细胞滴状到涂层, 成纤维细胞种子的菜肴在步骤3.10 准备。添加抗生素介质, 以充分覆盖的盘子和分散的细胞在表面。标签菜与内容和日期和孵化在37°c 和 5% CO2。</li>
	<li>48小时后, 确保细胞附着在培养皿上。吸入介质, 用足够的新鲜抗生素介质代替盘子。</li>
	<li>每周更换媒体 3x, 用真空线和巴斯德吸管吸旧介质, 用足够的新鲜介质替换盘子。经过5天的抗生素介质, 将介质改成扩展介质, 每周持续更换三次。 注意: 在文化的第一周, 污染风险很高。在单独的孵化器中保存新鲜的样品, 以减少交叉污染的风险。</li>
	<li>每周检查细胞生长数次。一旦细胞大约70-80% 汇合, 进入通道细胞。</li>
</ol>6. 通道他细胞
<ol><li>
在乙二胺四乙酸 (EDTA) 中制备0.1% 胰蛋白酶溶液。 注意: 使用小心处理胰蛋白酶, 这是皮肤, 呼吸和眼部刺激性。<ol>
<li>用锥形管从猪胰腺中重15毫克的胰蛋白酶。</li>
		<li>在一个单独的锥形管中重56毫克 EDTA。</li>
		<li>在生物安全柜中, 将胰蛋白酶和 EDTA 转化为蒸压250毫升玻璃瓶。使用小整除无菌磷酸盐缓冲盐水 (PBS) 冲洗胰蛋白酶/EDTA 管, 以确保完全转移。</li>
		<li>添加无菌 PBS, 使溶液总数达150毫升。紧紧地关上盖子。</li>
		<li>孵育胰蛋白酶溶液在37°c 水浴器直到完全溶解。检查每15分钟, 并及时删除一旦溶解。 注意: 不要在长时间内不加检查 (例如,过夜), 因为溶液中的胰蛋白酶如果加热太长会变性。</li>
		<li>清洁瓶2-3 喷雾70% 乙醇, 并返回安全柜。</li>
		<li>过滤0.1% 胰蛋白酶-EDTA 溶液成新鲜, 不育250毫升, 0.22 µm PES 过滤瓶使用制造商的说明和注释的内容和日期。</li>
		<li>贮存胰蛋白酶-EDTA 溶液在4摄氏度, 长达一个月, 如果多云, 丢弃。</li>
	</ol>
</li>
	<li>引入分化培养基, 0.1% 胰蛋白酶-EDTA 溶液, 不育 PBS, 室温30分钟以上, 清洁70% 乙醇, 转到清洁生物安全柜。</li>
	<li>在生物安全柜中, 使用真空线和巴斯德吸管, 从组织培养皿中吸取和丢弃培养基。用干净的血清吸管加入、旋转和吸5毫升的 PBS 来洗涤盘子。</li>
	<li>使用5毫升血清吸管, 加入5毫升0.1% 胰蛋白酶-EDTA 溶液的组织培养皿, 并返回到孵化器在37°c 和 5% CO2 5 分钟。</li>
	<li>在4-10X 的倒置显微镜下可视化细胞。检查细胞是否从盘子中分离, 变成圆形, 漂浮。轻轻地点击培养皿的一侧, 以去除细胞, 并/或重新孵化5分钟, 直到大多数细胞分离。 注意: 请小心快速移除从培养皿中分离出来的细胞;长时间接触0.1% 胰蛋白酶-EDTA 会损害细胞活力。</li>
	<li>使用10毫升血清吸管, 添加5毫升的膨胀介质到盘中, 并收集所有液体含量 (10 毫升总) 成一个标签, 无菌50毫升圆锥管。</li>
	<li>
如果细胞仍然附着在培养皿上, 重复步骤6.4 至6.6 两次, 收集在同一圆锥管的内容。
	<ol>
<li>如果细胞在三轮胰蛋白酶溶液暴露后仍然附着在培养皿上, 使用无菌细胞刮刀轻轻地除去其余部分。刮完盘子后, 用5毫升的膨胀介质清洗刮刀和盘子, 并在同一标记的锥形管上收集。 注意: 如果传代到球体, 请修改此过程以执行差异 trypsinization。加入胰蛋白酶溶液后 (步骤 6.4), 经常检查盘子, 直到成纤维细胞分离 (通常为1-2 分钟), 只留下与盘子相连的多边形上皮细胞。收集并预留这上清, 这可以传代向前扩展。重复步骤 6.4-6.6 使用相同的胰蛋白酶溶液, 只收集其余的上皮细胞为球形培养。</li>
	</ol>
</li>
	<li>离心机圆锥在 360 x g 为5分钟和4°c。从锥形中吸取介质。</li>
	<li>并用重悬细胞颗粒在1毫升的膨胀介质和计数细胞使用 hemacytometer。</li>
	<li>一旦量化, 细胞可以被分割, 如果必要的, 或者传代向前到一个新的培养基 (包括制备成纤维细胞共培养, 步骤 3, 和电镀细胞为扩张, 步骤 5.9-5.12) 或培养为球体 (步骤 7)。 注: 如果传代到新的培养皿进行扩展, 建议在 P100 盘中使用 0.5-1 x 106单元格的播种密度。</li>
</ol>7. 他球形培养的种子细胞
注: 执行本步骤中的所有步骤, 除离心外, 在清洁的生物安全柜内。
<ol><li>在每个制造商的指示 (100 µL 每球体井) 解冻地下室膜基体在冰。 注: 考虑在收据上制作基底膜基质的无菌500µL 整除数;这个数量将种子一个单一的4井板。</li>
	<li>分离适当数量的差异 trypsinized, 传代细胞 (从步骤 6.9), 以最终浓度50万细胞每毫升的矩阵。对于4井板, 使用20万细胞。收集1.5 毫升管。</li>
	<li>离心细胞和媒介混合物在 360 x g 5 分钟在4°c。完全, 但小心地吸入和丢弃介质使用1毫升吸管, 注意不要扰乱细胞颗粒。</li>
	<li>使用1毫升吸管尖端, 增加100µL 每个计划好的基底膜基质到1.5 毫升管包含细胞。把管子放在冰上。</li>
	<li>吸管向上和向下仔细和彻底并用重悬细胞在基底膜基质。快速移动以避免基体凝固。避免完全清空吸管尖端, 以确保气泡没有引入基质/细胞混合。</li>
	<li>
种子100µL 基质整除数到4井试管受精培养板的每个井中。
	<ol>
<li>使用干净的剪刀, 修剪的远端3-4 毫米的200µL 吸管提示。</li>
		<li>使用修剪过的尖端, 小心地将细胞/基质混合物的100µL 注入每个井的中心。吸管慢慢地, 目标是使一个单一的矩阵 "下降" 的中心每个井。下落应该保持球形, 并且不应该触碰井的边。移动盘子时, 要小心, 以免干扰这些水滴。</li>
		<li>孵化基底膜基质下降37°c 和 5% CO2 30 分钟, 直到固体。</li>
		<li>小心地将500µL 的分化介质注入每一个井中, 注意不要干扰基质的下落。确保介质只覆盖矩阵的下落, 并在必要时添加更多。</li>
	</ol>
</li>
</ol>8. 区分和成熟他球体
<ol><li>使用1毫升吸管尖端, 轻轻地吸入所有介质从良好;避免吸走矩阵, 这将摧毁球体在该部分的矩阵, 虽然任何球体遗留下来可能仍然可行。</li>
	<li>使用新鲜的1毫升吸管尖端, 轻轻地添加500µL 的分化介质, 以周围的基质。不要用吸管接触基体, 避免干扰基体的下落。</li>
	<li>将球体返回到孵化器 37 oC 和 5% CO2以进行持续增长。每周重复步骤8.1 至8.3 三次。</li>
	<li>在20X 的倒置显微镜下, 对球形形态学进行每日评估。球体应在电镀3-5 天内形成, 并在大约10天内达到成熟度。</li>
</ol>9. 预处理、刺激和图像他球体分析
<ol><li>预处理球体在像以前描述的30之前所希望的那样。 注: 对于 VX809 预处理, 在成像前将3µM 的 VX809 添加到介质中48-72 小时。混合1µL 10 毫米 VX809 的股票 (见材料表) 在3.3 毫升的介质, 以此浓度。</li>
	<li>将球体的每一个井改成1毫升的新鲜分化介质, 包括预处理 (步骤 8.1-8.3), 然后再成像球体。</li>
	<li>准备新鲜刺激药物解决方案 (佛司可林, 3-异丁基 1-甲基黄嘌呤 [IBMX], VX770 和 CFTR 抑制剂 172 [Inh172]) 从股票 (见材料表)。对于佛司可林/IBMX, 添加1µL 的每一个股票到98µL 的无菌水在一个标签1.5 毫升管。对于 VX770 和 Inh172, 添加1µL 的每一个股票到99µL 的无菌水在单独标记1.5 毫升管。制作一个总体积, 允许50µL 的解决方案, 为每一个测试良好。在生物安全柜无菌条件下准备解决方案。</li>
	<li>将球形板转移到孵化室设置为37°c 和 5% CO2, 安装在倒置显微镜上, 配有电子图像采集软件和 XY 调整的机械级。打开显微镜和照相机, 以及成像计算机。</li>
	<li>
在一个井中捕获球体的基线图像。
	<ol>
<li>打开图像捕获软件 (Slidebook 5.5, 以获取以下方向)。初始化后, 通过单击 "文件", 然后 "新建" 来打开一个新文件。相应地命名该文件, 然后单击 "保存"。</li>
		<li>仔细删除的文化板块盖和中心一个矩阵下降的目标。</li>
		<li>从矩阵的顶部开始, 在网格中移动以在显微镜目镜的20x 放大倍数中找到球体。集中和向下捕捉球体在其最宽的点。在成像后, 在矩阵下落的地图上标注每个球体的位置以重新识别。</li>
		<li>在软件中, 单击 "图像捕获"。为了曝光, 选择 "手动" 并输入50毫秒. 确保其他设置适当 (Bin 因子: 1x1, W: 512, H: 512, X 和 Y 偏移量: 0). 在 "名称" 框 (例如,球体1基线) 中为每个图像输入适当的名称。单击 "开始" 以查看实时图像, 并 "保存" 以捕获基线图像。</li>
		<li>重复步骤 9.5. 1-9. 5.4, 直到达到目标号, 或整个矩阵下降被映像。 注: 组差异可以检测到3-5 球体每一个条件, 但是, 它已经成为我们的做法, 图像10或更多球体每一个条件, 以增加权力。分析的可变性在 "代表性结果" 一节的图 2中显示, 每个主题的样本为8-10 球体作为示例。</li>
	</ol>
</li>
	<li>
刺激球体。
	<ol>
<li>使用200µL 吸管, 将50µL 适当的刺激药物放入井内介质中, 注意不要干扰基质。 注: 经过这10倍稀释 (50 µL 成500µL 的培养基), 最终药物浓度将为: 佛司可林10µM, IBMX 100 µM, VX770 1 µM, Inh172 10 µM。</li>
		<li>仅限于营地, 只添加佛司可林/IBMX。</li>
		<li>营 + VX770, 先加佛司可林/IBMX, 再 VX770 2 分钟。</li>
		<li>对于抑制条件, 先添加 Inh172, 然后在2分钟后佛司可林/IBMX。</li>
	</ol>
</li>
	<li>刺激后立即通过 timelapse 成像监测球形肿胀1小时。在软件中, 单击 "图像捕获"。单击 "Timelapse", 并将间隔设置为三十年代, 工期为60分钟. 输入适当的名称, 然后单击 "保存" 以启动 Timelapse。 注: 标准 timelapse 是每三十年代捕捉一个球体的图像, 以确保高质量的视频。或者, 对整个井 (例如, 4X) 执行低放大率捕获, 如果需要, 则捕获的图像越少。如果使用自动阶段, 则使用预定义的坐标执行所有球体的 timelapse;否则, 使用球体子集的 timelapse 提供对肿胀的定性审查, 并确保在成像过程中不会出现系统问题 (例如,从井中的矩阵脱离)。</li>
	<li>
在完成 1 h timelapse 映像后, 立即使用步骤9.5.3 中创建的映射捕获所有球体 (每步 9.5) 的刺激后图像。
	<ol>
<li>参考预刺激图像, 以确保对后续图像使用相同的焦点平面 (周围细胞、球体或碎片的焦质量极大地促进了这一过程)。</li>
		<li>从步骤 9.5.4 (例如,球体1后刺激) 中保存具有配对注释的文件。 注: 在 timelapse 后立即完成此步骤, 因为球体可能继续膨胀。</li>
	</ol>
</li>
	<li>用新的分化介质替换成像井中的介质。</li>
	<li>对每个井/条件重复步骤9.5 至 9.9, 调整刺激药物, 如实验条件所示。</li>
	<li>所有成像完成后, 返回球体到孵化器37°c 和 5% CO2。 注: 根据孵化显微镜室的不育性, 球体后的刺激污染是相当普遍的。在单独的孵化器中保持成像球体, 以最小化交叉污染风险, 或者在成像后立即丢弃球体。</li>
</ol>10. 分析他球体图像
<ol><li>将所有捕获的图像导出为与图像分析软件兼容的格式。在软件中, 单击 "查看", 悬停在 "导出" 上, 然后选择 "所有图像的默认视图为 TIFF..."。保存到硬盘驱动器或闪存驱动器进行分析。 注意: 商业分析软件 (材料表) 和. tiff 文件的性能良好, 并在这里描述, 但分析也可以使用其他平台执行。在命名文件时, 工作人员分析图像时应对实验条件视而不见, 但要知道哪些图像是配对的 (前和后刺激) 以确保等效分析。</li>
	<li>
利用分析软件, 勾勒出各球体图像的腔区面积, 导出为数据分析软件。
	<ol>
<li>开放分析软件。单击 "文件", 然后 "打开", 然后导航到包含球体图像的文件文件夹。选择所有图像进行分析, 然后单击 "打开"。</li>
		<li>单击 "测量" 并选择 "区域测量"。在 "区域测量" 对话框中, 选择 droptab "配置", 并确保选中 "图像名称" 和 "区域" 框。</li>
		<li>单击 "日志" 并选择 "打开数据日志"。在 "数据日志" 对话框和相应的名称文件中单击 "确定" (例如, 条件 X, 日期)。这将将所有度量值传输到电子表格。</li>
		<li>选择 "跟踪区域" 工具按钮, 仔细地在第一个图像中跟踪球体的流明以进行分析。在 "区域测量对话框" 中, 单击 "记录数据" 将测量记录到 Excel 中。</li>
		<li>重复步骤 10.2. 2-10.2. 4, 直到对所有球体进行分析。</li>
	</ol>
</li>
	<li>计算每个球体图像对的基线, 并编译数据进行分析时, 在腔区区域的百分比变化 (100 * [后传感基线] ÷基线)。</li>
</ol>

<ol>
	<li>Rowe, S. M., Miller, S., Sorscher, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystic+fibrosis.">Cystic fibrosis.</a> N Engl J Med. 352 (19), 1992-2001 (2005).</li><li>Rommens, J. 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=Identification+of+the+cystic+fibrosis+gene:+chromosome+walking+and+jumping.">Identification of the cystic fibrosis gene: chromosome walking and jumping.</a> Science. 245 (4922), 1059-1065 (1989).</li><li>Anderson, M. P., 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=Nucleoside+triphosphates+are+required+to+open+the+CFTR+chloride+channel.">Nucleoside triphosphates are required to open the CFTR chloride channel.</a> Cell. 67 (4), 775-784 (1991).</li><li>Boucher, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+airway+ion+transport.+Part+one.">Human airway ion transport. Part one.</a> Am J Respir Crit Care Med. 150 (1), 271-281 (1994).</li><li>Ehre, C., Ridley, C., Thornton, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystic+fibrosis:+an+inherited+disease+affecting+mucin-producing+organs.">Cystic fibrosis: an inherited disease affecting mucin-producing organs.</a> Int J Biochem Cell Biol. 52, 136-145 (2014).</li><li>Accurso, F. 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=Effect+of+VX-770+in+persons+with+cystic+fibrosis+and+the+G551D-CFTR+mutation.">Effect of VX-770 in persons with cystic fibrosis and the G551D-CFTR mutation.</a> N Engl J Med. 363 (21), 1991-2003 (2010).</li><li>De Boeck, K., 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=Efficacy+and+safety+of+ivacaftor+in+patients+with+cystic+fibrosis+and+a+non-G551D+gating+mutation.">Efficacy and safety of ivacaftor in patients with cystic fibrosis and a non-G551D gating mutation.</a> J Cyst Fibros. 13 (6), 674-680 (2014).</li><li>Moss, R. B., 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=Efficacy+and+safety+of+ivacaftor+in+patients+with+cystic+fibrosis+who+have+an+Arg117His-CFTR+mutation:+a+double-blind,+randomised+controlled+trial.">Efficacy and safety of ivacaftor in patients with cystic fibrosis who have an Arg117His-CFTR mutation: a double-blind, randomised controlled trial.</a> Lancet Respir Med. 3 (7), 524-533 (2015).</li><li>Wainwright, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lumacaftor-Ivacaftor+in+Patients+with+Cystic+Fibrosis+Homozygous+for+Phe508del+CFTR.">Lumacaftor-Ivacaftor in Patients with Cystic Fibrosis Homozygous for Phe508del CFTR.</a> N Engl J Med. , (2015).</li><li>Brewington, J. J., McPhail, G. L., Clancy, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lumacaftor+alone+and+combined+with+ivacaftor:+preclinical+and+clinical+trial+experience+of+F508del+CFTR+correction.">Lumacaftor alone and combined with ivacaftor: preclinical and clinical trial experience of F508del CFTR correction.</a> Expert Rev Respir Med. 10 (1), 5-17 (2016).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> CFTR2 Database. , (2017).</li><li>Mou, H., Brazauskas, K., Rajagopal, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Personalized+medicine+for+cystic+fibrosis:+establishing+human+model+systems.">Personalized medicine for cystic fibrosis: establishing human model systems.</a> Pediatr Pulmonol. 50 Suppl 40, S14-S23 (2015).</li><li>Randell, S. H., Fulcher, M. L., O'Neal, W., Olsen, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+epithelial+cell+models+for+cystic+fibrosis+research.">Primary epithelial cell models for cystic fibrosis research.</a> Methods Mol Biol. 742, 285-310 (2011).</li><li>Whitcutt, M. J., Adler, K. B., Wu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+biphasic+chamber+system+for+maintaining+polarity+of+differentiation+of+cultured+respiratory+tract+epithelial+cells.">A biphasic chamber system for maintaining polarity of differentiation of cultured respiratory tract epithelial cells.</a> In Vitro Cell Dev Biol. 24 (5), 420-428 (1988).</li><li>Worthington, E. N., Tarran, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+ASL+measurements+and+mucus+transport+rates+in+cell+cultures.">Methods for ASL measurements and mucus transport rates in cell cultures.</a> Methods Mol Biol. 742, 77-92 (2011).</li><li>Van Goor, F., 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=Correction+of+the+F508del-CFTR+protein+processing+defect+in+vitro+by+the+investigational+drug+VX-809.">Correction of the F508del-CFTR protein processing defect in vitro by the investigational drug VX-809.</a> Proc Natl Acad Sci U S A. 108 (46), 18843-18848 (2011).</li><li>Hug, M. J., Tummler, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+current+measurements+to+diagnose+cystic+fibrosis.">Intestinal current measurements to diagnose cystic fibrosis.</a> J Cyst Fibros. 3 Suppl 2, 157-158 (2004).</li><li>van Barneveld, A., Stanke, F., Ballmann, M., Naim, H. Y., Tummler, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+biochemical+analysis+of+CFTR+in+human+rectal+biopsies.">Ex vivo biochemical analysis of CFTR in human rectal biopsies.</a> Biochim Biophys Acta. 1762 (4), 393-397 (2006).</li><li>Dekkers, J. F., 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+functional+CFTR+assay+using+primary+cystic+fibrosis+intestinal+organoids.">A functional CFTR assay using primary cystic fibrosis intestinal organoids.</a> Nat Med. 19 (7), 939-945 (2013).</li><li>Dekkers, J. F., 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=Optimal+correction+of+distinct+CFTR+folding+mutants+in+rectal+cystic+fibrosis+organoids.">Optimal correction of distinct CFTR folding mutants in rectal cystic fibrosis organoids.</a> Eur Respir J. 48 (2), 451-458 (2016).</li><li>Dekkers, R., 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+bioassay+using+intestinal+organoids+to+measure+CFTR+modulators+in+human+plasma.">A bioassay using intestinal organoids to measure CFTR modulators in human plasma.</a> J Cyst Fibros. 14 (2), 178-181 (2015).</li><li>Zomer-van Ommen, D. D., 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=Limited+premature+termination+codon+suppression+by+read-through+agents+in+cystic+fibrosis+intestinal+organoids.">Limited premature termination codon suppression by read-through agents in cystic fibrosis intestinal organoids.</a> J Cyst Fibros. , (2015).</li><li>Bridges, M. A., Walker, D. C., Davidson, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystic+fibrosis+and+control+nasal+epithelial+cells+harvested+by+a+brushing+procedure.">Cystic fibrosis and control nasal epithelial cells harvested by a brushing procedure.</a> In Vitro Cell Dev Biol. 27a (9), 684-686 (1991).</li><li>Di Lullo, A. 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=An+&amp;#34;ex+vivo+model&amp;#34;+contributing+to+the+diagnosis+and+evaluation+of+new+drugs+in+cystic+fibrosis.">An &amp;#34;ex vivo model&amp;#34; contributing to the diagnosis and evaluation of new drugs in cystic fibrosis.</a> Acta Otorhinolaryngol Ital. 37 (3), 207-213 (2017).</li><li>Pranke, I. 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=Correction+of+CFTR+function+in+nasal+epithelial+cells+from+cystic+fibrosis+patients+predicts+improvement+of+respiratory+function+by+CFTR+modulators.">Correction of CFTR function in nasal epithelial cells from cystic fibrosis patients predicts improvement of respiratory function by CFTR modulators.</a> Sci Rep. 7 (1), 7375 (2017).</li><li>Devalia, J. L., Davies, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+nasal+and+bronchial+epithelial+cells+in+culture:+an+overview+of+their+characteristics+and+function.">Human nasal and bronchial epithelial cells in culture: an overview of their characteristics and function.</a> Allergy Proc. 12 (2), 71-79 (1991).</li><li>Devalia, J. L., Sapsford, R. J., Wells, C. W., Richman, P., Davies, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+and+comparison+of+human+bronchial+and+nasal+epithelial+cells+in+vitro.">Culture and comparison of human bronchial and nasal epithelial cells in vitro.</a> Respir Med. 84 (4), 303-312 (1990).</li><li>Thavagnanam, 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=Nasal+epithelial+cells+can+act+as+a+physiological+surrogate+for+paediatric+asthma+studies.">Nasal epithelial cells can act as a physiological surrogate for paediatric asthma studies.</a> PLoS One. 9 (1), e85802 (2014).</li><li>Jorissen, M., Van der Schueren, B., Van den Berghe, H., Cassiman, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+preservation+and+regeneration+of+cilia+on+human+nasal+epithelial+cells+cultured+in+vitro.">The preservation and regeneration of cilia on human nasal epithelial cells cultured in vitro.</a> Arch Otorhinolaryngol. 246 (5), 308-314 (1989).</li><li>Brewington, J. 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=Detection+of+CFTR+function+and+modulation+in+primary+human+nasal+cell+spheroids.">Detection of CFTR function and modulation in primary human nasal cell spheroids.</a> J Cyst Fibros. , (2017).</li><li>Sun, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tgf-beta+downregulation+of+distinct+chloride+channels+in+cystic+fibrosis-affected+epithelia.">Tgf-beta downregulation of distinct chloride channels in cystic fibrosis-affected epithelia.</a> PLoS One. 9 (9), e106842 (2014).</li><li>Boucher, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+airway+surface+dehydration+as+the+initiating+event+in+CF+airway+disease.">Evidence for airway surface dehydration as the initiating event in CF airway disease.</a> J Intern Med. 261 (1), 5-16 (2007).</li></ol>

作者没有什么可透露的。

该协议描述了病人衍生的鼻细胞球形培养的产生, 能够产生一个个性化的, 特定的 CFTR 功能模型。在这个过程中有几个关键步骤, 应该密切关注, 以避免困难。首先是一个很好的样本采集从病人的鼻子。一个好的样本应该有 > 5万细胞, 有限的粘液/碎片, 并准备在4小时内处理 (虽然成功也很容易实现与隔夜航运冰)。练习用刮匙 (或刷子来获取样品是必要的, 这有助于通过将早期样品采集集中到一个或两个提供者手中, 以最大限度地提高他们的舒适度。第二, 良好的清洁/无菌技术在所有组织培养步骤是必不可少的。在我们的经验中, 对所有他文化的主要失败模式是细菌或真菌污染, 这会使孵化器中的主要样本和其他生长的文化复杂化。这种风险只会随着慢性感染的受试者的文化而增加 (例如, CF), 因此成功主要取决于保持文化清洁和分离的能力。我们的做法是在最初的5-7 天内将一个孵化器单独保存在感染易发生的文化中, 保护那些年龄较大、成功的文化免受意外污染。第三, 在扩张阶段密切观察细胞并避免 overconfluence 是很重要的。如果细胞被允许在盘子上完全汇合, 那么就有一种风险, 那就是文化要么衰老, 要么细胞开始死亡和分离。最后, 当电镀细胞为球体时, 必须通过基质彻底分散细胞, 并将其均匀分布在样品上。无论是过度或人口不足的基质下降将导致文化的失败, 和大量的细胞将不会产生成功的球体。
在实施这项议定书时, 调查人员可能会遇到一些共同的问题。如上所述, 如果没有粘液和清洁的养殖技术, 就最好避免污染。如果文化通过扩张成功, 但没有形成分化的球体, 可能会出现几个问题。如果矩阵出现的主要是空的, 很可能是细胞被播种的密度过低;增加后续尝试的播种密度大约20%。反之, 如果矩阵看起来是 "肮脏的" 与大量的细胞碎片, 这是可能的细胞被播种在太高的密度, 随后的尝试应该使用的播种密度减少约20%。播种后喂养的常见并发症, 维护是基质从培养容器中分离出来的。这是由于过度积极的媒体交换, 可以避免谨慎和手动改变介质与1毫升的吸管, 而不是壁吸力。如果遇到, 球体仍然可以被刺激和成像, 虽然这可能是困难的, 如果矩阵 "漂浮" 在井中成像, 需要谨慎映射的球体在井内。
根据实验室的需要, 调查人员可能会对该协议进行几项修改。对于更高分辨率的球体成像, 我们以前用光学玻璃选项取代了4井板, 包括35毫米的玻璃底碟或会议厅幻灯片。这允许高分辨率, 实时成像的球体, 但可能降低吞吐量。另外, 为了增加吞吐量, 矩阵中较小的整除数细胞可以播种到其他容器中 (例如, 24 井培养板)。在我们的经验中, 球体以 "水滴" 的形式在基体上生长得最好, 而不是在井的底部形成一张纸;因此, 我们取得了最好的成功与水滴至少25µL. 调查人员可能希望通过稀释介质来改变基体的密度。这就减少了所需的基质总量, 提高了化验的成本。然而, 这也可能改变球体组成。在我们以前的经验中, 基底膜基质的低浓度导致了球状结构的改变, 在 "细胞-先出" 形态中, 球体的部分或全部形成。因此, 在尝试进行功能测试之前, 应谨慎地评估矩阵集中的任何协议更改所产生的球体。最后, 可以使用不同的刺激或抑制药物, 或不同浓度的这些药物。佛司可林、IBMX 和 Inh172 是根据阿里文化中以前的经验31为我们的研究选择的。使用其他药物 (例如,治疗刺激的异丙肾上腺素, GlyH101 抑制) 可能更好地适用于调查员的研究。同样地, 使用不同浓度的佛司可林、IBMX 或 Inh172 可以改变试验的动态范围, 但是, 我们没有系统地测试这些选项。
鉴于现有的前体患者派生的 CFTR 检测的数量, 所描述的模型因几个主要原因而显著。首先, 它利用鼻腔细胞作为一种容易获得的呼吸组织来源。他采购可以安全地进行, 在几乎任何设置 (诊所, 或, 研究访问) 在所有年龄组25最低限度的培训。这有助于稳健的样品采购, 并在必要时重复取样, 因为增长困难或污染。与肠道 organoids 相比, 他球体的特征较不明显, 目前形式的动态范围较小, 但是, 使用呼吸代替胃肠组织可能是一个关键的好处, 因为一些 CF 疾病驱动程序 (例如黏膜纤毛间隙、上皮钠通道表达) 不等同于肠道。相对于他细胞的平面、阿里文化, 球体的生长速度更快, 可能更能代表体内条件, 测量生理过程 (体液动态平衡), 这可能比单独的电生理学32更重要。通过从样品采购到测试的时间的提高, 污染潜力减少, 从而增加了文化成功的可能性。最后, 模型的3维性质可能适合于在阿里文化中不可行的气道发展或形态学研究的新颖和/或互补线 (例如, 腔内粘液跟踪, 快速分化研究,等)。
他球体作为 CFTR 功能的检测有几个关键的局限性。首先, 这种化验方法仍然比较低吞吐量。使用当前的方法, 图像采集在每种文化条件下花费一个小时, 分析每一个条件需要额外的20-30 分钟。由于某些条件之间的重叠程度 (如图 2A中所示), 这就加剧了这种情况, 这就需要大量的测量 (这种重叠本身也可能代表了此检测灵敏度的限制)。因此, 一项4-6 条件的试验需要将近两天的时间才能完成。采用自动图像采集和分析的方法可以提高吞吐量;这种适应目前正在进行中。其次, 这个模型需要大量的矩阵, 这可能会变得昂贵。如上所述, 稀释矩阵可能有助于克服这一障碍, 但必须谨慎采取, 因为增长矩阵的改变可能会导致球体形态学的改变。第三, 这个模型只依赖于 XY 平面上的球体测量, 而忽略了 Z 平面上的膨胀, 这可能会引入偏倚。尽管以前的重现性分析已经得到了保证, 但通过使用 Z 平面扫描的自动成像来计算体积30, 可以克服这一缺点。最后, 最重要的是, 对单个主体的体内药物反应的球体反应的广泛工作尚未完成。在没有这种相关性的情况下, 模型的预测价值--虽然前景看好--还不清楚。
他球体的生成和分析允许对单个 CFTR 活动和调制进行前体分析, 这可能最终成为药物反应的临床前模型。此外, 考虑到 CF 疾病严重程度的广泛变化, 这种个性化模型可以提供对一个学科独特的气道微环境的洞察力。这样, 这种模型可能有助于研究更广泛的 CFTR 生物学和帮助了解疾病的异质性。该模型的未来应用以及其他类似的个性化 CFTR 功能模型, 有望更好地了解实验室中的 CFTR 生物学, 并在临床上驱动个性化和精确的医学。

囊性纤维化 (CF) 是一种致命的, 常染色体隐性疾病, 影响全球7万多人1。这种寿命缩短的遗传疾病是由囊性纤维化跨膜电导调节蛋白 (CFTR) 的突变引起的2。CFTR 是三磷酸腺苷结合盒系列的成员, 作为离子通道, 允许氯和碳酸氢在多个极化上皮的顶端膜上运动, 包括胃肠道、汗腺、和呼吸树, 其中其他3,4。因此, 功能失调的 CFTR 导致多系统上皮功能障碍, 大多数死亡率来自呼吸系统疾病的 1.在 CF 肺, 失去 CFTR 驱动的气道表面液体 (手语) 调节和粘液释放导致增厚的粘液, 气道梗阻, 慢性感染, 和进步气道重塑和丧失肺功能1,5。
尽管 CFTR 功能障碍的鉴别作为疾病的病因, 在 CF 的治疗传统上侧重于缓解症状 (例如, 胰酶替代疗法, 气道清除疗法)1。这种方法最近被革命性的新疗法, 称为 "CFTR 调制器", 直接针对功能失调的 CFTR。这种方法已将临床景观从症状管理转移到疾病修改护理, 但带有若干限制6、7、8、9、10。调制器活动是特定的蛋白质缺陷伴随着每个 CFTR 突变和限制选择基因种群11。这种限制是由蛋白质缺陷的异质性所驱动的, 也是罕见突变组临床试验的不切实际。此外, 在研究良好基因型的学科中 (例如, F508del/F508del CFTR, 最常见的 CFTR 突变), 疾病负担和调制器响应的范围有很大的变异性6,7,8 ,9,11。
为了克服这两个问题, 调查人员建议使用个性化模型进行临床前测试12。这个概念利用患者特定的组织生成一个个性化的前体模型系统进行复合测试, 预测体内对治疗的个性化方式的反应。一旦经过验证, 这种模型可以被临床医生用来驱动精确治疗, 不管病人的潜在 CFTR 基因型。
在肺移植时从外植体组织获得的人支气管上皮 (HBE) 细胞建立了这样一种模型的可能性 CF13,14。HBEs 生长在空气液界面 (阿里) 允许功能 CFTR 量化直接通过电生理学测试或间接通过措施的手语稳定13,15。这个模型对于理解 CFTR 生物学是至关重要的, 是 CFTR 调制器16的关键驱动因素。不幸的是, HBE 模型不成立作为一个个性化的模型, 由于侵入性质的采集 (肺移植或支气管刷牙) 和缺乏样本的那些罕见的突变或轻度疾病。反之, 肠道组织, 从直肠或十二指肠活检标本, 可用于肠道电流测量 (ICM) 或肿胀为基础的 organoid 试验研究个性化 CFTR 功能17,18,19. Organoid 化验, 特别是, 是非常敏感的模型 CFTR 活动20,21,22。两种模型均受组织来源 (肠道组织, 而大多数疾病病理学是呼吸道) 和半侵入性采集方法的限制。另外, 一些调查人员研究了人鼻上皮 (他) 细胞, 以模拟 CFTR 恢复23,24,25。HNEs 可以通过刷子或刮除在任何年龄的主题, 并在阿里养殖时, 重述 HBEs25,26,27,28的许多特征的安全收获。他单层培养传统上受鳞状转变限制, 长时间成熟29。此外, 报告的短路电流测量在 HNEs 比 HBEs, 建议一个较小的窗口, 以检测治疗效果25。
鉴于需要一个个性化的, 非侵入性的, 呼吸组织培养模型的 CFTR 功能, 我们试图合并的敏感性, 肿胀基础, organoid 检测与非侵入性和呼吸性质的 HNEs。在这里, 我们描述了我们的方法, 生成3维 "球形" 文化的 HNEs 的个性化 CFTR 研究在肿胀为基础的化验30。他球体极化可靠与上皮尖向球形中心, 或流明。该模型概括了下呼吸道上皮的许多特征, 并且比阿里文化更快地成熟。作为一种功能性检测, 他球体可靠地量化了 CFTR 功能的范围, 以及在研究的突变组 (例如、F508del CFTR) 中的调制。这种基于肿胀的化验方法利用了 CFTR 的离子/盐传输特性, 间接地测量了液体流入球体的量, 因为水跟随顶端的盐流出。以这种方式, 刺激球体与完全功能 CFTR 膨胀强劲, 而那些功能失调的 CFTR 膨胀较少或萎缩。通过对前、1小时刺激后球体的图像分析, 测量腔区面积, 确定百分比变化, 对此进行量化。然后, 可以将这一措施与实验组进行比较, 以特定于病人的方式筛查药物的生物活性。

<table><tbody><tr><td>1.5 mL Eppendorf Tube</td><td>USA Scientific&amp;nbsp;</td><td>4036-3204</td><td></td></tr><tr><td>150 mL Filter Flask</td><td>Midsci&amp;nbsp;</td><td>TP99150</td><td>To filter Media</td></tr><tr><td>15 mL Conical Tube</td><td>Midsci&amp;nbsp;</td><td>TP91015</td><td></td></tr><tr><td>1 L Filter Flask</td><td>Midsci&amp;nbsp;</td><td>TP99950</td><td>To filter Media</td></tr><tr><td>35 mm Glass-Bottom Dish</td><td>MatTek Corporation</td><td>P35G-0-20-C</td><td>Optional</td></tr><tr><td>3-Isobutyl-1-Methylxanthine (IBMX)</td><td>Fisher Scientific&amp;nbsp;</td><td>AC228420010&amp;nbsp;</td><td>Prepare a 100 mM stock solution of 22.0 mg in 1 mL of DMSO</td></tr><tr><td>50 mL Conical Tube</td><td>Midsci&amp;nbsp;</td><td>&amp;nbsp;TP91050</td><td></td></tr><tr><td>Accutase</td><td>Innovative Cell Technologies, Inc.</td><td>AT-104</td><td>Cell detachment solution</td></tr><tr><td>Adenine</td><td>Sigma-Aldrich</td><td>A2786-25G</td><td>See Table 1</td></tr><tr><td>Amphotericin B</td><td>Sigma-Aldrich</td><td>A9528-100MG</td><td>See Table 1</td></tr><tr><td>Bovine Brain Extract (9mg/mL)</td><td>Lonza&amp;nbsp;</td><td>CC-4098</td><td>See Table 2</td></tr><tr><td>Ceftazidime hydrate</td><td>Sigma-Aldrich</td><td>C3809-1G</td><td>See Table 1</td></tr><tr><td>Cell Scrapers 20 cm&amp;nbsp;</td><td>Midsci&amp;nbsp;</td><td>TP99010</td><td></td></tr><tr><td>CFTR Inh172</td><td>Tocris Bioscience</td><td>3430</td><td>Prepare a 10 mM stock solution of 4.0 mg in 1 mL of DMSO</td></tr><tr><td>Cholera Toxin B (From Vibrio cholerae)</td><td>Sigma-Aldrich</td><td>C8052-.5MG</td><td>See Table 1</td></tr><tr><td>CYB-1</td><td>Medical Packaging Corporation</td><td>CYB-1</td><td>Cytology brush</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D5879-500ML</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Media (DMEM)/F12 Hepes&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>11330-057</td><td>Base Medium; See Tables 1 and 2</td></tr><tr><td>Epidermal Growth Factor (Recombinant Human Protein, Animal-Origin Free)</td><td>Thermo Fisher Scientific</td><td>PHG6045</td><td>See Table 1</td></tr><tr><td>Epinephrine</td><td>Sigma-Aldrich</td><td>E4250-1G</td><td>See Table 2</td></tr><tr><td>Ethanol</td><td>Fisher Scientific&amp;nbsp;</td><td>2701</td><td></td></tr><tr><td>Ethanolamine</td><td>Sigma-Aldrich</td><td>E0135-500ML</td><td>16.6 mM solution; See Table 2</td></tr><tr><td>Ethylenediaminetetraacetic Acid (EDTA)</td><td>TCI America&amp;nbsp;</td><td>E0084</td><td></td></tr><tr><td>Fetal Bovine Serum (high performance FBS)</td><td>Invitrogen&amp;nbsp;</td><td>10082147</td><td>See Table 1</td></tr><tr><td>Forskolin</td><td>Sigma-Aldrich</td><td>F6886-50</td><td>Prepare a 10 mM stock solution of 4.1 mg in 1 mL of DMSO</td></tr><tr><td>Growth Factor-Reduced Matrigel</td><td>Corning, Inc.</td><td>356231</td><td>Corning Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, Phenol Red-Free, LDEV-Free, 10 mL.</td></tr><tr><td>Hemacytometer</td><td>Hausser Scientific</td><td>1483</td><td></td></tr><tr><td>Human Collagen Solution, Type I (VitroCol; 3 mg/mL)</td><td>Advanced BioMatrix</td><td>5007-A</td><td>Collagen solution</td></tr><tr><td>HyClone (aka FetalClone II)&amp;nbsp;</td><td>GE Healthcare&amp;nbsp;</td><td>SH30066.03HI</td><td>See Table 2</td></tr><tr><td>Hydrocortisone</td><td>StemCell Technologies</td><td>07904</td><td>See Tables 1 and 2</td></tr><tr><td>Insulin, human recombinant, zinc solution</td><td>Life Technologies&amp;nbsp;</td><td>12585014</td><td>4 mg/mL solution; see Table 2</td></tr><tr><td>IVF 4-Well Dish, Non-treated</td><td>NUNC (via Fisher Scientific)</td><td>12566350</td><td>4-well plate for spheroids; similar well size to a 24-well plate</td></tr><tr><td>MEF-CF1-IRR</td><td>Globalstem</td><td>GSC-6001G</td><td>Irradiated murine embryonic fibroblasts</td></tr><tr><td>Metamorph 7.7</td><td>Molecular Devices</td><td></td><td>Analysis Software; https://www.moleculardevices.com/systems/metamorph-research-imaging/metamorph-microscopy-automation-and-image-analysis-software for a quote</td></tr><tr><td>Olympus IX51 Inverted Microscope</td><td>Olympus Corporation</td><td>Discontinued</td><td>Imaging Microscope. Replacment: Olympus IX53, https://www.olympus-lifescience.com/pt/microscopes/inverted/ix53/&amp;nbsp; for a quote</td></tr><tr><td>Pen Strep</td><td>Life Technologies&amp;nbsp;</td><td>15140122</td><td>See Table 2</td></tr><tr><td>Phsophoryletheanolamine</td><td>Sigma-Aldrich</td><td>P0503-5G</td><td>See Table 2</td></tr><tr><td>Retinoic Acid</td><td>Sigma-Aldrich</td><td>R2625-50MG</td><td>See Table 2</td></tr><tr><td>Rhinoprobe</td><td>Arlington Scientific, Inc.</td><td>96-0905</td><td>Nasal curette</td></tr><tr><td>Slidebook 5.5</td><td>3i, Intelligent Imaging Innovations</td><td>Discontinued</td><td>Imaging Software. Replacement: Slidebook 6, https://www.intelligent-imaging.com/slidebook for a quote</td></tr><tr><td>Sterile Phosphate Buffered Saline (PBS)</td><td>Thermo Fisher Scientific</td><td>20012050</td><td></td></tr><tr><td>Sterile Water</td><td>Sigma-Aldrich</td><td>W3500-6X500ML</td><td></td></tr><tr><td>Tissue Culture Dish 100</td><td>Techno Plastic Products</td><td>93100</td><td>Tissue culture dish for expansion</td></tr><tr><td>Tobramycin</td><td>Sigma-Aldrich</td><td>T4014-100MG</td><td>See Table 1</td></tr><tr><td>Transferrin (Human Transferrin 0.5 mL)</td><td>Lonza&amp;nbsp;</td><td>CC-4205</td><td>See Table 2</td></tr><tr><td>Triiodothryonine</td><td>Sigma-Aldrich</td><td>T6397-1G</td><td>3,3&amp;prime;,5-Triiodo-L-thyronine sodium salt&amp;nbsp; [T3]; See Table 2</td></tr><tr><td>Trypsin from Porcine Pancreas</td><td>Sigma-Aldrich</td><td>T4799-10G</td><td></td></tr><tr><td>Ultroser-G</td><td>Crescent Chemical (via Fisher Scientific)</td><td>NC0393024</td><td>20 mL lypophilized powder; See Table 2</td></tr><tr><td>Vancomycin hydrochloride from Streptomyces orientalis</td><td>Sigma-Aldrich</td><td>V2002-5G</td><td>See Table 1</td></tr><tr><td>VX770</td><td>Selleck Chemicals</td><td>S1144</td><td>Prepare a 1 mM stock solution of 0.4 mg in 1 mL of DMSO</td></tr><tr><td>VX809</td><td>Selleck Chemicals</td><td>S1565</td><td>Purchase or prepare a 10 mM stock solution of 4.5 mg in 1 mL of DMSO</td></tr><tr><td>Y-27632 Dihydrochloride&amp;nbsp; ROCK inhibitor&amp;nbsp;</td><td>Enzo LifeSciences&amp;nbsp;</td><td>ALX-270-333-M025&amp;nbsp;</td><td>See Table 1</td></tr></tbody></table>

generation of human nasal epithelial cell spheroids for individualized cystic fibrosis transmembrane conductance regulator study

囊性纤维化跨膜电导调节剂 (CFTR) 调制药物的引入改变了囊性纤维化 (CF) 的护理, 目前使用的基因型定向治疗模型有若干局限性。首先, 罕见或理解突变组被排除在明确的临床试验。此外, 随着额外的调制药物进入市场, 它将变得难以优化的调制器选择一个单独的主题。这两个问题都是通过使用病人衍生的, 个性化的临床前模型系统的 CFTR 功能和调制。人鼻上皮细胞 (HNEs) 是一种容易获得的呼吸道组织的来源, 这种模式。在此, 我们描述了一个三维球体模型的 CFTR 函数和调制使用初级 HNEs。HNEs 是以微创的方式与受试者隔绝, 在有条件的重新编程条件下扩展, 并播种到球体文化中。在播种2周内, 球体培养产生他球体, 可刺激与 3 ', 5 '-循环腺苷 (阵营) 产生的激动剂激活 CFTR 功能。球体肿胀被量化为 CFTR 活动的代表。他球体利用微创, 但鼻腔细胞的呼吸道起源, 生成一个可访问的, 个性化的模型, 与反映疾病发病率和死亡率的上皮细胞有关。与空气液界面他培养相比, 球体的成熟度相对快, 降低了整体污染率。在目前的形式, 模型是有限的吞吐量低, 虽然这是抵消了相对容易的组织获取。他球体可以用来可靠地量化和表征 CFTR 活动的个人水平。目前正在进行的一项研究将这种量化与体内药物反应联系起来, 将确定他球体是否是患者对 CFTR 调制的反应的真正的临床前预测因子。

HNEs 应附着在培养皿上, 在72小时内形成小岛细胞;一个星期的好和糟糕的岛屿形成的例子分别显示在图 1A和1B中。这些岛屿应该在15-30 天内扩大到覆盖这道菜。小的或次优的样品可能需要更长的时间, 而且往往不会产生有用的球体。感染性药物的污染是由深黄色/混浊介质、细胞无法附着在培养皿上, 以及/或真菌/细菌的直接可视化所证明的。任何受污染的文化都应立即丢弃, 以避免交叉污染。
在基底膜基质培养的前3-4 天内, 小囊性结构应开始在基质中形成。这些将在大约10天成熟到完整的球体展示一个薄壁和一个腔壁表面。如果镀在描述的密度, 成功的文化将包含50-100 球体每矩阵下降。流明可能比较清晰 (图 1C) 或填充细胞碎片和粘液 (图 1D);前者更常见于球体与野生型 CFTR (wtCFTR), 后者在 CF 球体。在图 1E和1F中分别演示了图 1C和1D中球体的腔内区域的掩蔽。在图 1G和1H中提供了不成功的球形区域性的示例。
野生类型和 F508del CFTR 纯合他球体的代表性功能数据分别显示在图 2A和2B中;这些数据代表 > 10 独特的他样本野生类型和 F508del CFTR 纯合主题30。总之, 球体功能 CFTR 膨胀, 而那些功能失调的 CFTR 肿胀显著减少, 或可能萎缩。具体地说, 球体从 wtCFTR 的受试者应该在刺激时膨胀一个多小时, 如果在 CFTR 抑制剂 Inh172 的存在刺激下, 就会膨胀的更少或收缩。相反, 球体从一个主题纯合为 F508del CFTR 应该收缩或膨胀非常轻微, 增加肿胀 (或减少收缩), 当药理纠正 CFTR 调制器 VX809 和 VX770。以往对同一基因型主体内部和之间球体可靠性的分析表明, CFTR 基因型的功能分离和重复测量中的适度变异性30。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>媒体组件</td>
			<td>库存解决方案</td>
			<td>量</td>
			<td>存储</td>
		</tr>
<tr>
<td colspan="4">扩展媒体</td>
		</tr>
<tr>
<td>DMEM/F-12 营养混合物 "基地媒介"</td>
			<td>使用情况</td>
			<td>2x 500 毫升容器</td>
			<td>存储在4ºC 到制造商到期日期</td>
		</tr>
<tr>
<td>胎牛血清</td>
			<td>使用情况</td>
			<td>50毫升</td>
			<td>存储在-20 ºC 到制造商到期日期</td>
		</tr>
<tr>
<td>霍乱毒素</td>
			<td>10毫克1毫升无菌水</td>
			<td>1µL</td>
			<td>商店库存在-20 ºC 六月以上</td>
		</tr>
<tr>
<td>表皮生长因子</td>
			<td>500µg 1 毫升无菌水</td>
			<td>20µL</td>
			<td>商店库存在-20 ºC 六月以上</td>
		</tr>
<tr>
<td>松</td>
			<td>0.4 毫克在400µL 无菌水中</td>
			<td>整个400µL 整除</td>
			<td>每个批次都要新鲜。在室温下贮存粉末直至制造商到期日</td>
		</tr>
<tr>
<td>腺 嘌呤</td>
			<td>24毫克1毫升无菌水</td>
			<td>整个1毫升整除</td>
			<td>每个批次都要新鲜。在室温下贮存粉末直至制造商到期日</td>
		</tr>
<tr>
<td>Y-27632</td>
			<td>3.2 毫克1毫升无菌水</td>
			<td>整个1毫升整除</td>
			<td>每个批次都要新鲜。贮存粉在-20 ºC 到制造商到期日</td>
		</tr>
<tr>
<td colspan="4">抗菌介质</td>
		</tr>
<tr>
<td>两性霉素 B</td>
			<td>使用情况</td>
			<td>1.2 毫升</td>
			<td>存储在4ºC 到制造商到期日期</td>
		</tr>
<tr>
<td>啶</td>
			<td>15毫克1毫升无菌水</td>
			<td>整个1毫升整除</td>
			<td>每个批次都要新鲜。贮存粉在-20 ºC 到制造商到期日</td>
		</tr>
<tr>
<td>妥布霉</td>
			<td>15毫克1毫升无菌水</td>
			<td>整个1毫升整除</td>
			<td>每个批次都要新鲜。贮存粉在-20 ºC 到制造商到期日</td>
		</tr>
<tr>
<td>万古 霉 素</td>
			<td>15毫克1毫升无菌水</td>
			<td>整个1毫升整除</td>
			<td>每个批次都要新鲜。贮存粉在-20 ºC 到制造商到期日</td>
		</tr>
</tbody></table>表 1: 膨胀和抗菌介质的成分。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>媒体组件</td>
			<td>库存解决方案</td>
			<td>量</td>
			<td>存储</td>
		</tr>
<tr>
<td>DMEM/F-12 营养混合物 "基地媒介"</td>
			<td>使用情况</td>
			<td>2x 500 毫升容器</td>
			<td>存储在4ºC 到制造商到期日期</td>
		</tr>
<tr>
<td>Ultroser-G</td>
			<td>20毫升的无菌水在一个单一的, 20 毫升的冻干 Ultroser-G</td>
			<td>整个20毫升整除</td>
			<td>每个批次都要新鲜。贮存粉在4ºC 到制造商到期日</td>
		</tr>
<tr>
<td>胎儿克隆 II</td>
			<td>使用情况</td>
			<td>20毫升</td>
			<td>存储在-20 ºC 到制造商到期日期</td>
		</tr>
<tr>
<td>笔链球菌</td>
			<td>使用情况</td>
			<td>10毫升</td>
			<td>存储在-20 ºC 到制造商到期日期</td>
		</tr>
<tr>
<td>牛脑提取物</td>
			<td>使用情况</td>
			<td>2.48 毫升</td>
			<td>存储在-20 ºC 到制造商到期日期</td>
		</tr>
<tr>
<td>转 铁 蛋白</td>
			<td>使用情况</td>
			<td>250µL</td>
			<td>存储在-20 ºC 到制造商到期日期</td>
		</tr>
<tr>
<td>胰岛素</td>
			<td>使用情况</td>
			<td>250µL</td>
			<td>存储在-20 ºC 到制造商到期日期</td>
		</tr>
<tr>
<td>乙醇 胺</td>
			<td>使用情况</td>
			<td>15µL</td>
			<td>在室温下贮存至制造商到期日</td>
		</tr>
<tr>
<td>肾上腺素</td>
			<td>2.75 毫克1毫升无菌水</td>
			<td>整个1毫升整除</td>
			<td>每个批次都要新鲜。贮存粉在4ºC 到制造商到期日</td>
		</tr>
<tr>
<td>甲状腺</td>
			<td>8.4 毫克在50µL 亚砜</td>
			<td>整个50µL 整除</td>
			<td>每个批次都要新鲜。贮存粉在-20 ºC 到制造商到期日</td>
		</tr>
<tr>
<td>松</td>
			<td>7.24 毫克在1毫升乙醇</td>
			<td>1µL</td>
			<td>商店库存在-20 ºC 六月以上</td>
		</tr>
<tr>
<td>Phsophoryletheanolamine</td>
			<td>35.25 毫克1毫升无菌水</td>
			<td>1µL</td>
			<td>商店库存在-20 ºC 六月以上</td>
		</tr>
<tr>
<td>甲酸</td>
			<td>3毫克在1毫升亚砜</td>
			<td>1µL</td>
			<td>商店库存在-20 ºC 六月以上</td>
		</tr>
</tbody></table>表 2: 分化介质的成分。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57492/57492fig1.jpg"> 图 1: 他扩展和他球体的结构特征.Brightfield 图像的他扩展文化采取七天后, 电镀证明成功 (白色箭头, 面板A) 和不成功 (面板B) 他殖民地形成在馈线成纤维细胞的背景。成功的 wtCFTR 和 F508del 纯合球体分别显示在面板C和D中。在面板E和F中分别提供了用于从 c/c++ 中描绘球体的腔区的掩蔽。不成功的球体文化的特点是小的, 无序的细胞碎片 (面板G) 和/或无序的细胞团簇 (面板H)。缩放条 = 100 µm.<a href="/files/ftp_upload/57492/57492fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57492/57492fig2.jpg"> 图 2: 他球体的功能特性.wtCFTR 球体的代表性功能反应从一个捐赠者, 当刺激与佛司可林/IBMX 与/不存在 CFTR 抑制剂 Inh172 显示在面板 (a) 中, 每个点表示在单个球体中的响应。在面板 (B) 中, 用佛司可林/IBMX 与/不存在 VX809 和 VX770 来刺激 F508del 纯合球体的代表性功能反应。误差条 = SEM < 0.01;p < 0.001。<a href="/files/ftp_upload/57492/57492fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Generation of Human Nasal Epithelial Cell Spheroids for Individualized Cystic Fibrosis Transmembrane Conductance Regulator Study at JoVE.com

Generation of Human Nasal Epithelial Cell Spheroids for Individualized Cystic Fibrosis Transmembrane Conductance Regulator Study

这里我们描述了一种生成三维的人鼻上皮细胞的球形培养方法。球体被刺激以驱动囊性纤维化跨膜电导调节剂 (CFTR) 依赖的离子和液体分泌物, 将球体腔内大小的变化量化为 CFTR 功能的代表。

人鼻上皮细胞球体的生成与个体化囊性纤维膜电导调节的研究

While the introduction of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) modulator drugs has revolutionized care in Cystic Fibrosis (CF), the ...

generation-human-nasal-epithelial-cell-spheroids-for-individualized

Nanyang Technological University

Research

JoVE Journal

Medicine

10.6K 次观看.  Cincinnati Children's Hospital Medical Center. 这里我们描述了一种生成三维的人鼻上皮细胞的球形培养方法。球体被刺激以驱动囊性纤维化跨膜电导调节剂 (CFTR) 依赖的离子和液体分泌物, 将球体腔内大小的变化量化为 CFTR 功能的代表。

视频: Generation of Human Nasal Epithelial Cell Spheroids for Individualized Cystic Fibrosis Transmembrane Conductance Regulator Study

Cultivate Primary Nasal Epithelial Cells from Children and Reprogram into Induced Pluripotent Stem Cells

Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They are also involved in many airway diseases through their innate immune response and interaction with immune and airway stromal cells. NECs are of particular interest for studies in children due to their accessibility during clinical visits. Human induced pluripotent stem cells (iPSCs) have been generated from multiple cell types and are a powerful tool for modeling human development and disease, as well as for their potential applications in regenerative medicine. This is the first protocol to lay out methods for successful generation of iPSCs from NECs derived from pediatric participants for research purposes. It describes how to obtain nasal epithelial cells from children, how to generate primary NEC cultures from these samples, and how to reprogram primary NECs into well-characterized iPSCs. Nasal mucosa samples are useful in epidemiological studies related to the effects of air pollution in children, and provide an important tool for studying airway disease. Primary nasal cells and iPSCs derived from them can be a tool for providing unlimited material for patient-specific research in diverse areas of airway epithelial biology, including asthma and COPD research.

This publication demonstrates methods for successful sampling and culture of nasal epithelial mucosa from children, and reprogramming these cells to induced Pluripotent Stem Cells (iPSCs).

从培养儿童原发性鼻腔上皮细胞和重新编程为诱导多能干细胞

Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They ...

科研

发育生物学

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

人类神经内分泌肿瘤细胞株为研究人类神经内分泌肿瘤治疗的三维模型

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

医学

Using Continuous Data Tracking Technology to Study Exercise Adherence in Pulmonary Rehabilitation

Pulmonary rehabilitation (PR) is an important component in the management of respiratory diseases. The effectiveness of PR is dependent upon adherence to exercise training recommendations. The study of exercise adherence is thus a key step towards the optimization of PR programs. To date, mostly indirect measures, such as rates of participation, completion, and attendance, have been used to determine adherence to PR. The purpose of the present protocol is to describe how continuous data tracking technology can be used to measure adherence to a prescribed aerobic training intensity on a second-by-second basis.
In our investigations, adherence has been defined as the percent time spent within a specified target heart rate range. As such, using a combination of hardware and software, heart rate is measured, tracked, and recorded during cycling second-by-second for each participant, for each exercise session. Using statistical software, the data is subsequently extracted and analyzed. The same protocol can be applied to determine adherence to other measures of exercise intensity, such as time spent at a specified wattage, level, or speed on the cycle ergometer. Furthermore, the hardware and software is also available to measure adherence to other modes of training, such as the treadmill, elliptical, stepper, and arm ergometer. The present protocol, therefore, has a vast applicability to directly measure adherence to aerobic exercise.

Pulmonary rehabilitation is widely recognized in the management of respiratory diseases. A key component to successful pulmonary rehabilitation is adherence to the recommended exercise training. The purpose of the present protocol is to describe how continuous data tracking technology can be used to precisely measure adherence to a prescribed aerobic training intensity.

使用连续数据跟踪技术在肺康复研究锻炼坚持性

Pulmonary rehabilitation (PR) is an important component in the management of respiratory diseases. The effectiveness of PR is dependent upon adherence to ...

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 ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

毛喉素诱导在肠组织体溶胀:一个<em&gt;体外</em&gt;检测方法的评估囊性纤维化患者对药物的反应

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

人鼻粘膜上皮细胞在气液界面培养

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

生物学

Culture and Imaging of Human Nasal Epithelial Organoids

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis Transmembrane conductance Regulator (CFTR) activity and restoration from small molecule compounds. Our group recently focused on establishing a well-differentiated organoid model directly derived from primary human nasal epithelial cells (HNE). Histology of sectioned organoids, whole-mount immunofluorescent staining, and imaging (using confocal microscopy, immunofluorescent microscopy, and bright field) are essential to characterize organoids and confirm epithelial differentiation in preparation for functional assays. Furthermore, HNE organoids produce lumens of varying sizes that correlate with CFTR activity, distinguishing between CF and non-CF organoids. In this manuscript, the methodology for culturing HNE organoids are described in detail, focusing on the assessment of differentiation using the imaging modalities, including the measurement of baseline lumen area (a method of CFTR activity measurement in organoids that any laboratory with a microscope can employ) as well as the developed automated approach to a functional assay (which requires more specialized equipment).

A detailed protocol is presented here to describe an in vitro organoid model from human nasal epithelial cells. The protocol has options for measurements requiring standard laboratory equipment, with additional possibilities for specialized equipment and software.

人鼻上皮类器官的培养和成像

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis ...

Collection, Expansion, and Differentiation of Primary Human Nasal Epithelial Cell Models for Quantification of Cilia Beat Frequency

Measurements of cilia function (beat frequency, pattern) have been established as diagnostic tools for respiratory diseases such as primary ciliary dyskinesia. However, the wider application of these techniques is limited by the extreme susceptibility of ciliary function to changes in environmental factors e.g., temperature, humidity, and pH. In the airway of patients with Cystic Fibrosis (CF), mucus accumulation impedes cilia beating. Cilia function has been investigated in primary airway cell models as an indicator of CF Transmembrane conductance Regulator (CFTR) channel activity. However, considerable patient-to-patient variability in cilia beating frequency has been found in response to CFTR-modulating drugs, even for patients with the same CFTR mutations. Furthermore, the impact of dysfunctional CFTR-regulated chloride secretion on ciliary function is poorly understood. There is currently no comprehensive protocol demonstrating sample preparation of in vitro airway models, image acquisition, and analysis of Cilia Beat Frequency (CBF). Standardized culture conditions and image acquisition performed in an environmentally controlled condition would enable consistent, reproducible quantification of CBF between individuals and in response to CFTR-modulating drugs. This protocol describes the quantification of CBF in three different airway epithelial cell model systems: 1) native epithelial sheets, 2) air-liquid interface models imaged on permeable support inserts, and 3) extracellular matrix-embedded three-dimensional organoids. The latter two replicate in vivo lung physiology, with beating cilia and production of mucus. The ciliary function is captured using a high-speed video camera in an environment-controlled chamber. Custom-built scripts are used for the analysis of CBF. Translation of CBF measurements to the clinic is envisioned to be an important clinical tool for predicting response to CFTR-modulating drugs on a per-patient basis.

This protocol describes nasal epithelial cell collection, expansion, and differentiation to organotypic airway epithelial cell models and quantification of cilia beat frequency via live-cell imaging and custom-built scripts.

原代人鼻上皮细胞模型的收集、扩增和分化，用于定量纤毛搏动频率

Measurements of cilia function (beat frequency, pattern) have been established as diagnostic tools for respiratory diseases such as primary ciliary ...

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 ...

Primary Human Nasal Epithelial Cells: Biobanking in the Context of Precision Medicine

Human nasal epithelial (HNE) cells are easy to collect by simple, non-invasive nasal brushing. Patient-derived primary HNE cells can be amplified and differentiated into a pseudo-stratified epithelium in air-liquid interface conditions to quantify cyclic AMP-mediated Chloride (Cl-) transport as an index of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) function. If critical steps such as quality of nasal brushing and cell density upon cryopreservation are performed efficiently, HNE cells can be successfully biobanked. Moreover, short-circuit current studies demonstrate that freeze-thawing does not significantly modify HNE cells&#39; electrophysiological properties and response to CFTR modulators. In the culture conditions used in this study, when less than 2 x 106 cells are frozen per cryovial, the failure rate is very high. We recommend freezing at least 3 x 106 cells per cryovial. We show that dual therapies combining a CFTR corrector with a CFTR potentiator have a comparable correction efficacy for CFTR activity in F508del-homozygous HNE cells. Triple therapy VX-445 + VX-661 + VX-770 significantly increased correction of CFTR activity compared to dual therapy VX-809 + VX-770. The measure of CFTR activity in HNE cells is a promising pre-clinical biomarker useful to guide CFTR modulator therapy.

Here we describe the isolation, amplification, and differentiation of primary human nasal epithelial (HNE) cells at the air-liquid interface and a biobanking protocol allowing to successfully freeze and then thaw amplified HNE. The protocol analyzes electrophysiological properties of differentiated HNE cells and CFTR-related chloride secretion correction upon different modulator treatments.

原代人鼻上皮细胞:精准医学背景下的生物样本库

Human nasal epithelial (HNE) cells are easy to collect by simple, non-invasive nasal brushing. Patient-derived primary HNE cells can be amplified and ...