意大利核物理研究所 (INFN) 通过 INFN-子午线项目部分资助了这项工作。作者承认 Prof. 伯父 Milotti (意大利的里雅斯特大学物理系) 为 INFN-子午线项目协调;Dr. 罗伯特 Chignola (生物技术系, 意大利维罗纳大学) 提供 Caco-2 细胞, 欧姆计, 并为他的宝贵的帮助和培训。我们还感谢 Agnese 索拉里的技术援助。

下面的协议涉及人体血液从健康的志愿者中撤出。捐助者在入学前提供书面知情同意。这一程序符合《赫尔辛基宣言》的规定, 并由一名专业保健助理进行采血。
1. 细胞培养与共文化的建立
<ol><li>照射前一周, 在新鲜的 RPMI1640 培养基中, 用10% 胎牛血清 (FBS)、2毫米 l-谷氨酰胺、100 U/毫升青霉素和100微克/毫升链霉素, 制备含有 2.5 x 105细胞/毫升的 Caco-2 细胞悬液。</li>
	<li>种子2毫升的细胞悬浮在无菌1µm 孔直径细胞培养插入 6-井板, 并把插入到一个6井板。 注意: 细胞培养插入可能需要激活的孵化与无菌完整培养基之前, 细胞播种。在这种情况下, 应丢弃区域性介质, 并将其替换为单元悬浮介质。</li>
	<li>添加3毫升新鲜 RPMI1640 培养基补充 10% FBS, 2 毫米 l-谷氨酰胺, 100 毫升青霉素, 和100µg/毫升链霉素在每个底部车厢, 并培养细胞在37° c, 在一个孵化器与湿化气氛含有 5% CO2。</li>
	<li>在同一天的 Caco-2 细胞照射, 收集人的全血在商用锂肝素涂层6毫升管 (管大小:13 x 100 mm)。</li>
	<li>随后, 利用聚梯度分离外周血单个核细胞 (PBMC)。要分离 PBMC, 把25毫升的聚在50毫升圆锥离心管和一层等量的全血稀释1:1 与 RPMI1640 到聚表面。 注意: 正常的健康捐赠者通常有大约 4-10 x 106 PBMC/毫升。</li>
	<li>离心50毫升管在 400 x g 在室温下30分钟。</li>
	<li>用巴斯德吸管轻轻地收集聚和血浆界面的 PBMC, 并将其放入15毫升锥形管中。</li>
	<li>洗 PBMC 两次, 加入10毫升磷酸盐缓冲盐水 (PBS) 和离心 PBMC 在 250 x g 10 分钟。</li>
	<li>文化 PBMC 在 T25 cm2烧瓶中的最大值为 3-5 h, 在完整的 RPMI1640 介质中, 如前所述, 在37° c 中含有 5% CO2的湿化气氛中。 注: 在实验当日收集 PBMC, 种子 2 x 106细胞/井, 悬浮在3毫升的完整 RPMI1640 培养基中, 在共培养的底部隔间。带 Caco-2 细胞的插入物在其辐照后30分钟在含 PBMC 的井中转移。如果不用e. g进行刺激, 从全血中收集的 PBMC 在文化中不能维持超过约72小时。phytohaemagglutinin (临医局), 之后细胞的生存能力丧失。 注意: 所有血液和血液制品的质量和安全必须在整个过程中得到保证。</li>
</ol>2. 辐照设置
注意: Caco-2 细胞的照射是在依的放疗部进行的, Ricovero 库拉索 Carattere Scientifico (IRCCS) s Maugeri (意大利帕维亚), 通常使用线性加速器来治疗不同类型的癌症。
<ol><li>将光子 X 射线能量设置为 6 MV 峰值。线性加速器操作在一个搏动的政权 (时间在两个连续的脉冲之间大约4毫秒; 一个单一的脉冲的期间大约5µs 以药量率由 1 x 10-3 ./p)。</li>
	<li>在1.4 厘米厚的有机玻璃板上放置带有 Caco-2 的6井板, 在 X 射线的轨道上 (相对于所用辐射的建造距离稍大一些), 从辐射源的100厘米处。</li>
	<li>在辐射发生前, 在每个样品上放置一个0.5 厘米厚的丸, 以保证散射辐射分量和带电粒子的平衡。</li>
	<li>照射细胞 (剂量在 2-10 的范围内) 与一个单位和对称 (± 2%)20 x 20 厘米2辐射场和剂量率为3的/分钟。 注意: 控制 "假"-辐照细胞经历了相同的程序条件照射的, 没有进入辐照室 (接受剂量: 0)。 警告: 电离辐射产生装置必须由专门人员使用。</li>
</ol>3. 细胞活力测定法 (MTT 法)
注: Caco-2 细胞代谢活性通过 3-(45-dimethylthiazol-2-基)-25-diphenyltetrazolium 溴 (MTT) 法测定14。
<ol><li>种子 2 x 105 Caco-2 在 24-井板 24 h 前辐照1.25 毫升的完全介质。</li>
	<li>照射在步骤 2.1-2.4 中所描述的细胞, 然后孵育在37° c 在一个含 5% CO2的湿润气氛中的细胞。</li>
	<li>21小时辐照后, 加入100µL 5 毫克/毫升 MTT 溶液, 并保持在37° c 的湿化气氛中含有 5% CO2为3小时的细胞。</li>
	<li>丢弃上清液, 用 PBS 1 毫升冲洗细胞。</li>
	<li>通过在每个井中加入500µL 的二甲基亚砜 (亚砜) 来溶解甲晶体, 并在λ = 570 nm 上用多井板读写器评估吸光度。</li>
	<li>在辐照后, 重复步骤 3.3-3.4-3.5-3.6 在45小时。 注: 结果显示为一个扰动从相应的假条件, 这是正常化到100%。 注意: 亚砜是易燃和刺激性的皮肤, 眼睛和呼吸系统 (GHS07, GHS08) 的代理。如果接触眼睛, 立即用大量的水冲洗, 并寻求医疗建议。MTT 是皮肤, 眼睛和呼吸系统的刺激性剂 (GHS07, GHS08)。如果接触眼睛, 立即用大量的水冲洗, 并寻求医疗建议。</li>
</ol>4. 活细胞测定的百分比 (台盼蓝染料排除法)
注意: 用台盼蓝染料排除法评估活细胞百分率。
<ol><li>种子 2 x 105 Caco-2 在 24-井板 24 h 前辐照1.25 毫升完全介质。</li>
	<li>照射细胞与剂量在范围 0-10, (参见步骤 2.1-2.4) 然后孵育细胞在37° c 在一个湿的大气包含 5% CO2为 24-48 h。</li>
	<li>经过两个选择的时间点, 用 PBS 冲洗细胞, 丢弃它, 然后加入100µL 的胰蛋白酶-乙酸酸 (EDTA) 溶液。将细胞置于37° c 的湿润气氛中, 含 5% CO2为2分钟, 然后添加完整培养基以阻止酶反应。</li>
	<li>收集在1.5 毫升管的细胞和离心管在 500 x g 5 分钟, 丢弃上清和重新悬浮在50µL 的 PBS 细胞。</li>
	<li>将细胞悬浮液与50µL 的台盼蓝染料溶液混合, 在室温下孵育3分钟。</li>
	<li>计数的染色 (非可行) 和不 (可行) 细胞与 Bürker 室。</li>
</ol>5. 反式上皮电阻 (TEER)
<ol><li>种子 5 x 105 Caco-2 细胞在 6-井板 co 培养插入物 (聚乙烯对苯二甲酸乙二醇酯 (PET), 2 x 106毛孔/cm2) 前一周照射。</li>
	<li>在照射前1小时, 用伏特计/欧姆计测量 TEER。</li>
	<li>照射细胞, 如步骤 2.1-2.4 所述。</li>
	<li>在含有 5% co2的湿化气氛中, 在37° c 的情况下孵育 Caco-2 细胞与 PBMC 的共培养。</li>
	<li>在头几个小时辐照后, 测量 TEER 每小时, 随后每3ĥ高达 48 h。</li>
</ol>6. Claudin-1、蛋白、Afadin、ZO-1、ZO-2、NF-kB 和 XIAP 的免疫印迹分析
<ol><li>种子 5 x 105细胞1周前 X 射线曝光在 6-井板块 co 培养插入物 (PET, 2 X 106毛孔/cm2) 和照射细胞, 如步骤 2.1-2.4 所述。</li>
	<li>在含有 5% co2的湿化气氛中, 在37° c 的情况下孵育 Caco-2 细胞与 PBMC 的共培养。</li>
	<li>48小时照射后, 用细胞裂解缓冲液和-20 ° c 储存样品, 制备 Caco-2 和 PBMC 细胞裂解。 注意: 协议可以在此处暂停。</li>
	<li>用二辛可宁酸法测定总蛋白量。 注意: 协议可以在此处暂停。</li>
	<li>将等量的总蛋白与β-基 (β-基的最终浓度为 5%) 相加, 将样品在95° c 加热5分钟, 然后在 1万 x g Laemli。</li>
	<li>在 4-20% 的预制凝胶中加载样品, 并在 polyvinildiene 氟 (PVDF) 膜上进行 120 V 的电泳, 用于 1 h. 转移蛋白。</li>
	<li>在室温下用5% 非脂肪的干奶 (NFDM) 在 PBS 中加入 0.2% Tween-20 (PBT) 来阻止非特定的结合点。冲洗膜三次, 10 毫升 0.2% PBT 5 分钟。</li>
	<li>在室温下, 在4° c 下过夜时, 用温和搅拌的方法孵育1小时的膜, 其主要抗体有: anti-claudin-1、anti-ZO-1、anti-ZO-2、抗 afadin、抗蛋白、抗 NF kB、抗 XIAP 和抗肌动蛋白。冲洗膜三次, 10 毫升 0.2% PBT 5 分钟。</li>
	<li>在室温下, 用辣根过氧化物酶 (HRP)-共轭二级抗体孵育1小时, 温和搅拌。冲洗膜三次, 10 毫升 0.2% PBT 5 分钟。</li>
	<li>在室温下, 利用增强的化学发光试剂盒来孵化膜, 来冲洗胶片。</li>
	<li>利用扫描系统获取照相胶片, 并用合适的图像分析软件对获得的波段进行量化。 注意: Claudin-1、蛋白、Afadin、ZO-1 和 ZO-2 的评价是在 Caco-2 细胞中进行的。在 PBMC 中对 NF-kB 和 XIAP 进行了评价。 注意: β-基是有毒的 (GHS05, GHS06, GHS08, GHS09)。不要吸入蒸汽, 避免释放到环境中, 并佩戴个人防护装置和呼吸道保护。如果吞下, 立即向医生咨询。</li>
</ol>7. 统计分析
<ol><li>为了确定辐射照射和共培养物是否引起统计学上的显著摄动, 对方差 (方差分析) 试验进行了两种方法的比较, 对重复测量进行了多次对比 (Bonferroni 后的测试比较复制方式)。</li>
	<li>如果没有另有说明, 计算的统计意义 (p) 由两尾学生的 t 检验。每个值均为3独立实验平均值 (SEM) 的标准误差。</li>
</ol>

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作者声明没有利益冲突。

在发达国家, 大肠癌是发病率和死亡率最常见的原因之一。它通常由外科手术, 化疗和放疗1。在放疗治疗的框架内, 必须特别注意健康组织暴露的影响4;此外, 系统地研究辐射暴露与免疫系统的关系, 是发展无线电免疫治疗方法的基础3。
我们在这项工作中采用的方法是针对 Caco-2 细胞和 PBMC 串扰的研究而量身定做的。我们关注的是 X 射线照射肿瘤细胞的效果, 但同样的协议可以适应与药理学的研究。与标准细胞培养更接近的生理条件, 这种方法的优势是有可能全面解剖复杂的系统, 考虑到分析两种不同的细胞类型和释放信号分子到联合文化的二个隔间里本身。通过这种方法, 常规应用生物方法有助于了解细胞相互作用的相关机制。
X 射线诱导的 Caco-2 细胞的初步表征是基于两个互补的测量,即MTT 比色法和台盼蓝染料排斥试验。这些明显不一致的结果可以用这两种分析的不同焦点来解释。MTT 法评价氧化酶活性, 而台盼蓝染料是基于活细胞排斥染料的机制。
对 Caco-2-PBMC 相互作用的研究需要建立一个上皮单层能够导致在两个共文化的隔间完全分离。在共培养的插入 Caco-2 细胞的可能性允许仅辐射这个细胞人口。由于共培养开始后, 辐射, 没有任何偏见, 由于任何意外暴露的 PBMC。这一设置需要小心处理, 以避免损坏 (或污染) 的蜂窝单层在移动的插入从一个6井板到另一个。当仔细地执行时, TEER 测量是一个简单和非侵入的方法研究单层渗透性。这种测定方法与共培养方法密切相关, 它不是单层渗透性研究的唯一选择。它可以很好地重现的措施, 一旦它是良好的校准与新鲜完整的介质。常见的化验重点是化学染料从上部的"顶端"隔间扩散到底部 "侧"一 (荧光素异硫氰酸酯 (FITC)-葡聚糖检测)16。然而, 由于 PBMC 在本研究的侧室, 我们决定避免在实验中引入化学试剂。
在这项工作中采用的不同技术中, TEER 测量是唯一需要共用区域性设置17,18的方法。然而, 其他常见的实验室技术提供了更多的信息的结果时, 应用于细胞的共同培养, 因为他们允许调查的设置更接近生理条件和数据具有更强的生物学意义。另一方面, 必须指出的是, 在多孔支架上生长的细胞的使用可能会在准备样品所需的操作中遇到一些困难, 如细胞裂解制备细胞提取物, 以便通过印迹来分析。
本研究采用的系统具有进一步改进的潜力,例如应用能够重现细胞外基质环境的物质。然而, 这也将导致系统的复杂性增加, 而对设置的完整剖析将更难实现。
细胞共培养的设置, 当然代表了一个强大的工具, 促进体外的研究, 以及对复杂系统的理解。这项技术有可能增加基本机制的知识, 为分子信号的基础研究提供新的投入, 并且可能的应用在免疫系统的活动的调节在框架肿瘤临床病人管理。

这项工作所采用的方法是为了研究大肠癌细胞与免疫系统之间的相互作用, 在与正常的2维细胞培养相比, 利用一个更接近生理状态的装置。
大肠癌 (CRC) 被认为是第三种最常见的癌症, 全世界有超过100万的病例 (全球癌症观察站, 国际癌症研究机构, 世界卫生组织, http://gco.iarc.fr)。CRC 的管理通常通过手术、化疗或放疗进行,1。与手术或化疗等侵入性技术相比, 放疗在很大程度上避免了从这些临床方法中产生的典型的有害系统反应, 这得益于局部放射剂量的传递。然而, 副作用可能出现在周围的健康组织, 触发炎症与直接损害健康细胞和损害介导的非靶向效应2,3,4。针对大肠癌放疗中的不良反应, 需要研究两个方面。首先, 负责肠道抗渗性的机制可以通过辐射传递来改变, 反过来, 由于细菌数量的改变和分子和溶质的细胞通道的影响, 导致了副作用的可能性。其次, 肠道相关淋巴组织的存在, 作为免疫系统的前哨, 具有控制细菌生长的功能, 并调解一般免疫应答5,6,7。为了实现这些功能, 肠道的抗渗性是由于细胞膜间连接配合物的作用而保持的。由于这些原因, 在 Caco-2 细胞单独和与 PBMC 的 co 培养中, 对不同剂量的 X 射线所引起的有害后果进行了研究。
尽管对细胞培养进行研究是生物医学研究的第一 setline, 但缺乏对细胞生物学和不同细胞类型相互作用的机制的详细了解, 可能成为关键研究在实验室中不易重现的器官、系统和器械的生理学。因此, 我们决定采用一种联合培养的方式, 允许两个细胞共同研究, 并解剖有关的方面与细胞和细胞外的机制。
共培养是研究上皮功能和不同细胞类型间相互作用的一种技术。特别是, 在我们的情况下, 这种技术的使用成为强制性的, 因为上皮细胞是由具有极性特征的单元组成的。在肠道屏障的情况下, 细胞显示一个明确的极化, 顶端和侧极点通常分离, 由于存在紧密连接-创造黏附分子。这种条块分割是需要的组织生理学, 避免细胞贩运和允许通过的坚定分子只。这一功能当然是不可能的重建与正常细胞培养设置。此外, 联合培养的采用, 在侧表面只复制免疫细胞的存在, 而顶端表面 (对应于肠腔) 不直接与其他细胞接触。
近年来, Caco-2 细胞株作为一种肠道屏障的体外模型变得越来越重要。虽然从人类结肠腺癌中获得, Caco-2 细胞保持分化能力, 并创建一个功能极化单层8, 这使得研究的细胞膜特性时, 成长在一个共同文化插入。
由于 Caco-2 在多孔膜上的培养是一种成熟的肠单层结构的体外模型, 改善了 Caco-2 与其他细胞的共培养。这种设置已经被频繁地用来测量不同单元类型之间的串扰9 , 可以用来解开 Caco-2 扰动反应的外来刺激时, 在共同文化, 就 Caco-2 单独培养。
许多研究都解决了 Caco-2 行为时, 与非病原菌和外周血单个核细胞共同培养, 以阐明特别是与免疫系统10的串扰。Pozo-卢比奥et al.11研究了双歧杆菌刺激 Caco-2 细胞 Caco-2/PBMC 共培养中几种细胞因子的表达。他们的工作强调了对细胞因子表达谱的实质性修改, 这取决于在 PBMC 的存在/缺席下进行的细菌刺激。结果表明, PBMC 使 Caco-2 对双歧杆菌存在。
不同的研究组对 Caco-2 细胞对非致病和致病细菌的不同反应进行了评价。Parlesak et al。12显示了 Caco-2 细胞对大肠杆菌刺激 PBMC 的免疫抑制作用。此外, Haller et al。13研究了致病大肠杆菌或非致病细菌的 Caco-2 细胞 (LPS) 对 i.e.E. 大肠杆菌、乳酸杆菌的反应, 加强推测 Caco-2 细胞的反应严格依赖于在共同培养中的白细胞的存在。
通过执行不同的辅助实验室化验 (例如西方印迹, 反式上皮电阻, MTT,等), 除了分析在共同培养的不同细胞类型, 整个方法论承诺结果, 可以被认为是真正发生在体内的更具代表性。此外, 这一设置允许分离不同的共培养舱室, 不仅允许研究所涉及的细胞类型, 而且还可以在上vs.下部舱内或在存在中释放的胞间信号分子, vs.缺少合作文化。

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a co-culture method to investigate the crosstalk between x-ray irradiated caco-2 cells and pbmc

这项工作所采用的议定书旨在解开 X 射线如何扰乱肠屏障的功能, 重点是大肠肿瘤细胞与免疫系统之间的相互作用。大肠癌是最常见的癌症之一, 通常通过手术、化疗和放疗来治疗。放疗在肿瘤靶向中的优势是众所周知的。然而, 即使是有限的健康组织暴露是非常令人关注的, 特别是关于肠道屏障和免疫系统的影响。所采用的设置允许研究两个细胞之间的相互作用在一个更类似于生理的条件, 当与正常细胞培养。为此, 我们采用了不同的技术, 我们使用了一个体外共培养模型, 基于 Caco-2 细胞分化为单层和 PBMC, 共享相同的培养基。本议定书的发展重点是宏观效应,即细胞活力和跨上皮电阻 (TEER), 并通过西方印迹, 分子改变,即炎症通路的活化免疫细胞和 Caco-2 细胞的紧密连接蛋白表达。通过 3-(45-dimethylthiazol-2-基)-25-diphenyltetrazolium 溴化铵 (MTT) 和台盼蓝测定对 Caco-2 细胞活力的辐射效应进行了初步评估, 而 TEER 是通过欧姆计在固定时间间隔测量的。专门为合作文化系统设计的。这样, 辐射的影响, 外周血单个核细胞 (PBMC) 的存在, 并最终它们的协同作用, 可以证明。通过这些互补的技术, 我们观察到 Caco-2 在 2-10 X 射线的范围内的高射电电阻和增加的 Caco-2 单层通透性, 当 PBMCs 增加。特别是, PBMC 存在被发现与变化紧密连接支架蛋白表达。

在实验前一周, 细胞被播种在插入物的多孔膜上, 并允许在接下来的几天内生长。汇合的水平可以用倒置显微镜或通过测量 TEER 值被检查。事实上, 在生长阶段, TEER 不断增加, 直到所有的多孔膜已被细胞覆盖, 它们形成了分化的细胞单层。如果细胞增殖速度更快/较慢, 实验可以在播种后的较早/更晚的时间点开始。当在汇合处, 细胞然后被带到辐照设施, 最小化被传授的环境重音 (温度或 pH 值), 在开始与/不 PBMC 的 co 文化之前, 在底部舱内播种 (图 1A), 或评估 Caco-2 细胞的增殖。考虑到初始播种密度, 日0细胞应达到100% 汇合, 并创建一个分化单层上皮细胞, 可以观察到的高原在 TEER 显示在图 1B。一旦细胞达到这样的状态, TEER 值在接下来的一周内保持相对恒定, 只要旧培养基被替换为新鲜培养基, 至少每周一次 (图 1B)。如图 1C所示, MTT 法不显示 Caco-2 细胞增殖状态的任何统计学意义上的改变, 无论是在24小时内, 还是在48小时, 独立于所接受的剂量 (最多 10)。
对 Caco-2 细胞的短期死亡率有不同的观察结果。在两个时间点, 台盼蓝染色显示剂量依赖性增加的细胞死亡率。这些结果显示了辐射暴露的明显影响, 虽然死亡细胞的百分比看起来很低, 特别是当考虑到最高的被提供的剂量 (10) 只产生大约20% 的细胞死亡 (图 1D)。
样品在下隔间有或没有 PBMC 的共同培养。鉴于 PBMC 没有得到任何外部刺激增殖, 48 h 实验被认为是理想的, 以避免 PBMC 引入的偏见开始死亡。因此, 从紧接照射前, TEER 被定期测量为48小时, 以跟踪可能的暂时影响的辐射协议。如图 1 e-F所示, TEER 值被显示为相对于预处理条件 (1200年-1500 Ω·cm2) 的相关变化, 以便更好地突出 X 射线辐照引起的摄动和/或在共同文化中 PBMC 的存在/缺席。在这两种情况下, 一个初始的瞬时峰值可以清楚地看到在第一时间点后, 辐射照射, 这可以归因于辐照程序。
当不与 PBMC (图 1E) 共用时, TEER 值几乎是恒定的 48 h, 而在 10 X 射线束后, 细胞显示 TEER 开始在 3 h 后照射时的长时间下降。PBMCs 的存在完全修改了 TEER 时间动态 (图 1F)。对于所有剂量, TEER 的减少从 3 h 到大约 30 h 后照射, 当 TEER 似乎以一个恒定的值 (图 1F) 结算时, 可以清楚地观察到。
Caco-2 细胞裂解通过免疫印迹法研究了致密结复合体的表达水平。Caco-2 细胞暴露于电离辐射 (剂量为0、2和 10), 随后单独生长或在共培养与 PBMCs 在底部车厢为 48 h (如图 2-f所示)。Claudin-1 和蛋白 (图 2A、2B) 均未被 X 射线和/或与 PBMC 的 co 区域性显著改变。在支架蛋白 ZO-1、ZO-2 和 Afadin (图 2C、2D、2E) 中观察到了较大的波动。特别是, ZO-2 表达水平的减少在2以后被观察了, 当在 co 文化与 PBMC, 当仅在 10, 当 Caco-2 单独增长时。Afadin 表达水平只在10束 X 射线之后才会受到影响, 当 Caco-2 与 PBMCs 共培养时, 会有额外的减少。
PBMCs 共培养与 Caco-2 分析的炎症状态, 特别是, 核转录因子 kb (NF-kb) 和 X 连锁抑制剂的凋亡蛋白 (XIAP) 水平已被调查 (图 2 G I)。NF-kB 总金额不受与 Caco-2 暴露于不同辐射剂量的联合培养 (图 2G) 的影响。相反, XIAP 水平在2和10的合作文化中都有4倍的上调, 尽管大的变化需要大量的样品分析来减少这种波动, 并获得更好的统计能力。
如图 2F和2I所示, 某些非特定的频带可能出现在感兴趣的蛋白质的预期分子量旁边。除非真正的和非特异的波段是容易区分, 不同的抗体和/或 BSA 或 NFDM 浓度应考虑。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56908/56908fig1.jpg"> 图 1.辐射照射和/或 PBMC 共培养的总体实验设置和宏观效果.A) 共同文化模式的示意图描述。B) 每天从 Caco-2 细胞的初始种子中测量 TEER 值, 以评估单层的适当生长和分化状况。C) 细胞活力和 D) Caco-2 暴露于 X 射线 (0、2、5和 10) 的死亡率。E) 在 Caco-2 细胞中, 用0、2和10光照射的 X 射线对 PBMCs 进行 TEER 测量。每个值都是3独立实验的平均值. * p-val < 0.05;** va l < 0.01;p-val < 0.001。从辛约格·莫里尼et al改编的图表。15.<a href="//cloudflare.jove.com/files/ftp_upload/56908/56908fig1large.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/56908/56908fig2.jpg"> 图 2。Caco-2 和 PBMC 裂解的印迹结果.在 Afadin 0、2、10 X 射线和 Caco-2 的存在/缺席的情况下, 在 co 培养中, 紧密连接蛋白 (Claudin-1 (A)、蛋白 (B)、ZO-1 (C)、ZO-2 (D) 和 PBMC (E)) 的表达水平。值在肌动蛋白水平上正常化。在面板 (F) 中显示了每个紧密接合蛋白和条件的说明薄膜。PBMCs 与 Caco-2 细胞共培养的 NF-nf-(G) 和 XIAP (H) 的表达水平。在面板中显示了 NF-kB、XIAP 和肌动蛋白的代表性薄膜。每个值都是3独立实验的平均值. 从辛约格·莫里尼et al改编的图表。15.<a href="//cloudflare.jove.com/files/ftp_upload/56908/56908fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about A Co-culture Method to Investigate the Crosstalk Between X-ray Irradiated Caco-2 Cells and PBMC at JoVE.com

A Co-culture Method to Investigate the Crosstalk Between X-ray Irradiated Caco-2 Cells and PBMC

我们提出了一个协议来研究 X 射线辐照 Caco-2 和外周血单个核细胞 (PBMC) 之间的串扰。该协议始于 Caco-2 辐照和与 PBMC 共文化的建立;随后, 反式上皮电阻是定期测量超过48小时和西方印迹执行在 Caco-2 和 PBMC。

X 射线辐照 Caco-2 细胞与 PBMC 间串扰的共培养方法研究

The protocol adopted in this work aims at unraveling how X-rays perturb the functioning of the intestinal barrier, focusing on the interplay between ...

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JoVE Journal

Immunology and Infection

13.0K 次观看.  Università degli Studi di Pavia. 我们提出了一个协议来研究 X 射线辐照 Caco-2 和外周血单个核细胞 (PBMC) 之间的串扰。该协议始于 Caco-2 辐照和与 PBMC 共文化的建立;随后, 反式上皮电阻是定期测量超过48小时和西方印迹执行在 Caco-2 和 PBMC。

视频: A Co-culture Method to Investigate the Crosstalk Between X-ray Irradiated Caco-2 Cells and PBMC

A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events leading to stromal activation and establishment of the tumor microenvironment (TME). Several in vitro and in vivo models of the TME have been developed; however, in general these models do not readily permit isolation of individual cell populations, under non-perturbing conditions, for further study. To circumvent this difficulty, we have employed an in vitro TME model using a cell growth substrate consisting of a permeable microporous membrane insert that permits simple generation of highly enriched cell populations grown intimately, yet separately, on either side of the insert&#39;s membrane for extended co-culture times. Through use of this model, we are capable of generating greatly enriched cancer-associated fibroblast (CAF) populations from normal diploid human fibroblasts following co-culture (120 hr) with highly metastatic human breast carcinoma cells, without the use of fluorescent tagging and/or cell sorting. Additionally, by modulating the pore-size of the insert, we can control for the mode of intercellular communication (e.g., gap-junction communication, secreted factors) between the two heterotypic cell populations, which permits investigation of the mechanisms underlying the development of the TME, including the role of gap-junction permeability. This model serves as a valuable tool in enhancing our understanding of the initial events leading to cancer-stroma initiation, the early evolution of the TME, and the modulating effect of the stroma on the responses of cancer cells to therapeutic agents.

We adapted a permeable microporous membrane insert to mimic the tumor microenvironment (TME). The model consists of a mixed cell culture, allows simplified generation of highly enriched individual cell populations without using fluorescent tagging or cell sorting, and permits studying intercellular communication within the TME under normal or stress conditions.

肿瘤微环境的模仿:产生富集的细胞群及侦查间通讯的简单方法

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events ...

科研

癌症研究

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract (ECME)-based Three-dimensional System

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, thus greatly influencing normal tissue homeostasis and disease outcome. In breast cancer, tumor-associated macrophages (TAMs) play a critical role in disease progression, metastasis, and recurrence; therefore, understanding the mechanisms of monocyte chemoattraction to the tumor microenvironment and their interactions with tumor cells is important to control the disease. Here, we provide a detailed description of a three-dimensional (3D) co-culture system of human breast cancer (BrC) cells and human monocytes. BrC cells produced high basal levels of regulated on-activation, normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1), and granulocyte-macrophage colony-stimulating factor (G-CSF), while in co-culture with monocytes, pro-inflammatory cytokines Interleukin (IL)-1 beta (IL-1&#946;) and IL-8 were enriched together with matrix metalloproteinases (MMP)-1, MMP-2, and MMP-10. This tumor stroma microenvironment promoted resistance to anoikis in MCF-10A 3D acini-like structures, chemoattraction of monocytes, and invasion of aggressive BrC cells. The protocols presented here provide an affordable alternative to study intra-tumor communication and are an example of the great potential that in vitro 3D cell systems provide to interrogate specific features of tumor biology related to tumor aggression.

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ECME-based Three-dimensional System

Here, we describe a three-dimensional culture method to analyze the morphology of primary breast cancer cells, as well as to study their direct/indirect interactions with monocytes and the outcomes such as collagen degradation, immune cell recruitment, cell invasion, and promotion of cancer-related inflammation.

基于细胞外基质提取物 (ECME) 的三维系统中单核细胞与原代乳癌细胞的通讯分析

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, ...

Dual Effects of Melanoma Cell-derived Factors on Bone Marrow Adipocytes Differentiation

The crosstalk between bone marrow adipocytes and tumor cells may play a critical role in the process of bone metastasis. A variety of methods are available for studying the significant crosstalk; however, a two-dimensional transwell system for coculture remains a classic, reliable, and easy way for this crosstalk study. Here, we present a detailed protocol that shows the coculture of bone marrow adipocytes and melanoma cells. Nevertheless, such a coculture system could not only contribute to the study of cell signal transductions of cancer cells induced by bone marrow adipocytes, but also to the future mechanistic study of bone metastasis which may reveal new therapeutic targets for bone metastasis.

Here, we present a reliable and straightforward two-dimensional (2D) coculture system for studying the interaction between tumor cells and bone marrow adipocytes, which reveals a dual effect of melanoma cell-derived factors on the bone marrow adipocytes differentiation and also poses a classic method for the mechanistic study of bone metastasis.

黑色素瘤细胞衍生因子对骨髓脂肪分化的双重影响

The crosstalk between bone marrow adipocytes and tumor cells may play a critical role in the process of bone metastasis. A variety of methods are ...

Studying the Effects of Tumor-Secreted Paracrine Ligands on Macrophage Activation using Co-Culture with Permeable Membrane Supports

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer growth and metastasis. Paracrine signals can involve cell-cell contact between different cell types, such as PD-L1 expressed on the surface of tumors interacting directly with PD-1 on the surface of T cells, or the secretion of ligands by a tumor cell to affect an immune cell. Here we describe a co-culture method to interrogate the effects of tumor-secreted ligands on immune cell (macrophage) activation. This straightforward procedure utilizes commercially available 0.4 &#181;m polycarbonate membrane permeable supports and standard tissue culture plates. In the process described, macrophages are cultured in the upper chamber and tumor cells in the lower chamber. The presence of the 0.4 &#181;m barrier allows for the study of intercellular signaling without the confounding variable of physical contact, because the two cell types share the same medium and exposure to paracrine ligands. This approach can be combined with others, such as genetic alterations of the macrophage (e.g., isolation from genetic knock-out mice) or tumor (e.g., CRISPR-mediated alterations) to study the role of specific secreted factors and receptors. The approach also lends itself to standard molecular biological analyses such as quantitative reverse transcription polymerase chain reaction (qRT-PCR) or Western blot analysis, without the need for flow sorting to separate the two cell populations. Enzyme-linked immunosorbent assays (ELISAs) can similarly be utilized to measure secreted ligands to better understand the dynamic interaction of cell signaling in the multiple cell type context. Duration of co-culture can also be varied for the study of temporally regulated events. This co-culture method is a robust tool that facilitates the study of tumor-secreted signals in the immune context.

Here, we present a method using permeable membrane supports to facilitate the study of non-contact paracrine signaling used by tumor cells to suppress the immune response. The system is amenable to studying the role of tumor-secreted factors in dampening macrophage activation.

研究肿瘤分泌性副体结扎对宏噬细胞活化的影响，使用可渗透膜支架共培养

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer ...

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells.
Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.

The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity.

监测三D培养中的癌细胞入侵和T细胞毒性

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited ...

Real-Time Detection and Capture of Invasive Cell Subpopulations from Co-Cultures

Invasion and metastatic spread of cancer cells are the major cause of death from cancer. Assays developed early on to measure the invasive potential of cancer cell populations typically generate a single endpoint measurement that does not distinguish between cancer cell subpopulations with different invasive potential. Also, the tumor microenvironment consists of different resident stromal and immune cells that alter and participate in the invasive behavior of cancer cells. Invasion into tissues also plays a role in immune cell subpopulations fending off microorganisms or eliminating diseased cells from the parenchyma and endothelial cells during tissue remodeling and angiogenesis. Real-Time Cellular Analysis (RTCA) that utilizes impedance biosensors to monitor cell invasion was a major step forward beyond endpoint measurement of invasion: this provides continuous measurements over time and thus can reveal differences in invasion rates that are lost in the endpoint assay. Using current RTCA technology, we expanded dual-chamber arrays by adding a further chamber that can contain stromal and/or immune cells and allows measuring the rate of invasion under the influence of secreted factors from co-cultured stromal or immune cells over time. Beyond this, the unique design allows for detaching chambers at any time and isolating of the most invasive cancer cell, or other cell subpopulations that are present in heterogeneous mixes of tumor isolates tested. These most invasive cancer cells and other cell subpopulations drive malignant progression to metastatic disease, and their molecular characteristics are important for in-depth mechanistic studies, the development of diagnostic probes for their detection, and the assessment of vulnerabilities. Thus, the inclusion of small- or large-molecule drugs can be used to test the potential of therapies that target cancer and/or stromal cell subpopulations with the goal of inhibiting (e.g., cancer cells) or enhancing (e.g., immune cells) invasive behavior.

We describe an approach to detect and capture invasive cell subpopulations in real-time. The experimental design uses Real-Time Cellular Analysis by monitoring changes in the electric impedance of cells. Invasive cancer, immune, endothelial or stromal cells in complex tissues can be captured, and the impact of co-cultures can be assessed.

实时检测和捕获共培养物中的侵袭细胞亚群

Invasion and metastatic spread of cancer cells are the major cause of death from cancer. Assays developed early on to measure the invasive potential of ...

基于流式细胞术的癌症免疫周期监测 

Co-Culture In Vitro Systems to Reproduce the Cancer-Immunity Cycle

Fundamental cancer research and the development of effective counterattack therapies both rely on experimental studies detailing the interactions between cancer and immune cells, the so-called cancer-immunity cycle. In vitro co-culture systems combined with multiparametric flow cytometry (mFC) and tumor-on-a-chip microfluidic devices (ToCs) enable simple, fast, and reliable monitoring and characterization of each step of the cancer-immunity cycle and lead to the identification of the mechanisms responsible for tipping the balance between cancer immunosurveillance and immunoevasion. A thorough understanding of the dynamic interplays between cancer and immune cells provides critical insights to outsmart tumors and will accelerate the pace of therapeutic personalization and optimization in patients. Specifically, here we detail a straightforward mFC- and ToC-assisted protocol for unraveling the dynamic complexities of each step of the cancer-immunity cycle in murine cancer cell lines and mouse-derived immune cells and focus on immunosurveillance. Considering the time- and cost-related features of this protocol, it is certainly feasible on a large scale. Moreover, with minor variations, this protocol can be both adapted to human cancer cell lines and human peripheral-blood-derived immune cells and combined with genetic and/or pharmacologic inhibition of specific pathways in order to identify biomarkers of immune response.

监测癌症免疫周期并探索肿瘤微环境动力学

Here, we detail a simple, fast, and reliable multiparametric flow cytometry- and tumor-on-a-chip-assisted protocol to monitor and characterize the steps constituting the cancer-immunity cycle. Indeed, a thorough understanding of the interplays between cancer and immune cells provides critical insights to outsmart tumors and guide clinical care.

体外共培养系统以重现癌症免疫循环

Fundamental cancer research and the development of effective counterattack therapies both rely on experimental studies detailing the interactions between ...

Microfluidic Co-Culture Models for Dissecting the Immune Response in in vitro Tumor Microenvironments

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise invisible processes. Advanced cell assays closely mimicking in vivo scenery are establishing themselves as novel ways to visualize and measure the bidirectional tumor-host interplay influencing the progression of cancer. Here we describe two versatile protocols to recreate highly controllable 2D and 3D co-cultures in microdevices, mimicking the complexity of the tumor microenvironment (TME), under natural and therapy-induced immunosurveillance. In section 1, an experimental setting is provided to monitor crosstalk between adherent tumor cells and floating immune populations, by bright field time-lapse microscopy. As an applicative scenario, we analyze the effects of anti-cancer treatments, such as the so-called immunogenic cancer cell death inducers on the recruitment and activation of immune cells. In section 2, 3D tumor-immune microenvironments are assembled in a competitive layout. Differential immune infiltration is monitored by fluorescence snapshots up to 72 h, to evaluate combination therapeutic strategies. In both settings, image processing steps are illustrated to extract a plethora of immune cell parameters (e.g., immune cell migration and interaction, response to therapeutic agents). These simple and powerful methods can be further tailored to simulate the complexity of the TME encompassing the heterogeneity and plasticity of cancer, stromal and immune cells subtypes, as well as their reciprocal interactions as drivers of cancer evolution. The compliance of these rapidly evolving technologies with live-cell high-content imaging can lead to the generation of large informative datasets, bringing forth new challenges. Indeed, the triangle &#39;&#39;co-cultures/microscopy/advanced data analysis&#34; sets the path towards a precise problem parametrization that may assist tailor-made therapeutic protocols. We expect that future integration of cancer-immune on-a-chip with artificial intelligence for high-throughput processing will synergize a large step forward in leveraging the capabilities as predictive and preclinical tools for precision and personalized oncology.

In the age of immunotherapy and single-cell genomic profiling, cancer biology requires novel in vitro and computational tools for investigating the tumor-immune interface in a proper spatiotemporal context. We describe protocols to exploit tumor-immune microfluidic co-cultures in 2D and 3D settings, compatible with dynamic, multiparametric monitoring of cellular functions.

用于剖析体外肿瘤微环境中免疫反应的微流控共培养模型

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise ...

X 射线辐照 Caco-2 细胞与 PBMC 间串扰的共培养方法研究

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