Name: methods to study epithelial transport protein function and expression in native intestine and caco-2 cells grown in 3d
Uploaded: 2017-03-16T13:00:00.000+00:00
Duration: 11 min 27 s
Description: Watch this Scientific Journal Video about Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D at JoVE.com

We thank Mr. Christopher Manzella, MD/PhD student in the RKG laboratory, for helping to edit and proofread our manuscript.

1.的Caco-2的三维培养系统肠道转运的研究:成长3D的Caco-2细胞上一种胶状蛋白质混合物<ol><li>解冻生长因子减少胶状蛋白质混合物在冰上8小时（或O / N）在4℃下。一旦解冻，使1毫升或500微升等分试样在-20℃，供以后使用使用或存储。 </li><li>在培养当天，预冷在冰上培养板（用于RNA和蛋白质的提取）或室载玻片（用于免疫染色）。添加胶状蛋白质混合物的30μL到八孔腔-载玻片（或每一个6孔板的孔120微升）的每个孔中，并均匀地分布。请注意，不要在此过程中产生的气泡。 </li><li>放置细胞培养孵化器内的载玻片/板在37℃，15 - 30分钟，并允许胶状蛋白质混合物固化。 </li><li>而胶状蛋白质的混合物固化，trypsinize Caco-2细胞的汇合烧瓶中。 </li><li>数数细胞和自旋它们在500 XG在台式离心机5分钟。根据需要重悬在三维的Caco-2培养基的细胞团块。 </li><li>种子，每孔4,000个细胞的玻璃玻片（中厚板20,000每孔）。使细胞在5％CO 2的加湿培养箱中在37℃下生长。更改每中等3 - 4天。 </li></ol> 2.免疫荧光染色上皮转运体在3D Caco-2细胞:1天<ol><li>吸出培养基并用400μL的2％多聚甲醛（PFA）（在PBS中以调节到8.5，用NaOH将pH值），用于在室温30分钟固定细胞。 注:PFA溶液不应在4℃存放1周以上，并且可以使用新鲜制备的溶液。 注意:PFA有剧毒，一个潜在的致癌物质。总是与化学罩谨慎使用个人防护装备处理。 </li><li>用1×PBS漂洗孔两次，载玻片在4℃下储存于PBS（400微升/好）。一旦固定，在4℃将它们存储长达一周。 </li><li>预热室幻灯片RT（这会变硬的胶状蛋白质混合物床，可以很容易地吸在洗涤步骤）。 </li><li>透用PBS中0.5％的Triton细胞为不超过15分钟。 </li><li>制备的PBS-甘氨酸缓冲液（130毫摩尔NaCl，7毫摩尔的 Na 2 HPO 4，3.5毫的NaH 2 PO 4，和100mM甘氨酸）。冲洗3次用1×PBS甘氨酸在RT每次10分钟。 </li><li>制备免疫缓冲液（IF）:130毫摩尔NaCl，7毫摩尔的 Na 2 HPO 4，3.5毫的NaH 2 PO 4，100mM的甘氨酸，7.7毫NaN 3的，0.1％的BSA，0.2％的TritonX-100，和0.05％Tween-20的。 </li><li>洗3次中的IF缓冲液在RT每次10分钟。在5％阻止正常山羊血清（NGS）的中频缓冲器。用200微升在IF + 1％山羊血清稀释一抗，孵育/孔，1 - 在室温2小时。与中频缓冲器3×洗涤，每次10分钟。 </li><li>继发抗孵育体（1:200）稀释在IF + 1％NGS，200微升/孔，在RT下1小时。 </li><li>带有IF缓冲液洗三次，每次10分钟。保持在黑暗中的幻灯片从上这一步。通过将腔室滑动黑色保持器及滑动白色挺杆穿过保持器，直到其边缘的接触孔的边缘抬起从载玻片的塑料腔室。轻轻拉起室（持有者和推杆应该与文化幻灯片）。 </li><li>慢防褪色中安装并允许它干在室温下10分钟。 </li><li>一旦完全干燥，密封指甲油和共聚焦显微镜图像他们的幻灯片。储存在4℃下将载玻片两周或在-20℃下几个月。 </li></ol> 3.从三维的Caco-2细胞RNA和蛋白提取<ol><li>治疗上胶状蛋白质混合物培养12细胞 - 14天与测试作用剂，如TGF-β1（10毫微克/毫升，1小时）。 </li><li>处理后，冲洗细胞1点¯xPBS。保持在冰上的细胞30分钟，以液化胶状蛋白质混合物。 </li><li>用冷PBS洗井和收集各个离心管中的细胞。使用低速离心，在500 xg离心4℃10分钟收集细胞。使用标准程序提取RNA，并使用实时定量RT-PCR。 </li><li>该蛋白质的提取，溶解用补充有1×蛋白酶抑制剂鸡尾酒1×细胞裂解缓冲液的细胞。加入细胞裂解缓冲液，以沉淀后，通过超声处理裂解细胞，并通过离心7500 xg离心4℃10分钟除去细胞碎片。 </li></ol> 4，5-HT摄取鼠标回肠利用Ussing室中的测量:肠道准备和浆肌层剥离<ol><li>继安乐死，解剖小鼠，取出小肠（胃后盲肠之前）。划分小肠分成三部分。丢弃中间的三分之一，使用近三分之一如空肠，并使用远侧三分之一回肠。避免其肠系膜附着拉肠道以防止上皮损伤。通过用冰冷的Krebs-Ringer碳酸氢盐（KBR）缓冲液覆盖它保持所述组织部分冷。 </li><li>打开纵向用剪刀肠道。孵育新鲜切除肠切片在冰冷，放气含1μM的吲哚美辛10分钟的浆肌汽提之前KBR。 </li><li>针肠节（约1厘米长）粘膜面朝下含7％琼脂糖或固化有机硅弹性体（0.5厘米厚）板。 </li><li>胶条底部照明解剖立体显微镜下的浆肌层层。切用羽毛手术刀片的浆肌层。用细镊子沿着取得肠道的纵轴层的边缘。 注:在剥离过程中，体视显微镜的底部照明有助于淋巴集结识别和避免地区上课。 </li><li>所述浆肌剥离完成之后，小心地安装在一个尤斯灌流半腔的销的粘膜。在整个过程中，照顾的边缘以保持剥离粘膜组织，以免撕破。 </li></ol> 5.平衡和组织治疗<ol><li>制备的Kreb氏溶液:115 mM氯化钠，的25mM的NaHCO 3，2.4毫K 2 HPO 4，1.2毫氯化钙 ，1.2mM的MgCl 2的，和0.4毫摩尔KH 2 PO 4在pH 7.4，用95％ 的 O 2/5％充气的 CO 2在37℃。添加10mM葡萄糖到浆膜浴以用作能量基板和10mM甘露糖醇对粘膜浴以保持渗透平衡。 注:的Kreb氏溶液补充有L-抗坏血酸（100μM）和帕吉林（100μM，单胺氧化酶抑制剂），以防止细胞内的5-HT的降解。 </li><li>插入滑块进入腔室以暴露既心尖（管腔）SI德和组织到的Kreb氏溶液的基底外侧（浆膜）侧。 </li><li> 10分钟的平衡期后，预处理氟西汀（10μM），用于在顶面上面向J 毫秒 （粘膜到浆膜磁通）30分钟的回肠组织。 </li><li>治疗与TGF-β1（10毫微克/毫升）的组织为上基底外侧面1小时，然后用3 [H] -5-HT（20 nm）的孵育它为在顶面上30分钟。 </li></ol> 6.粘膜量化到浆膜通量（J 毫秒 ）和3 [H]在组织-5-HT积累<ol><li>收集从浆膜贮存器（总5毫升）0.75毫升等分试样以测量放射活性在液体闪烁计数器以及计算粘膜到浆膜（J 毫秒 ）通量率;氟西汀敏感磁通表示SERT介导的3 [H] -5-HT摄取。 </li><li>为了避免在焊剂期跨粘膜静水压力差，采取样品从浆膜侧和再把他们带浴缸中的相同卷。 注:通量计算校正结束样品（4.25毫升/ 5毫升= 0.85）稀释。 </li><li>为3 [H] -5-HT中的组织中积累的定量，从滑块卸除粘膜，用冰冷的KRB缓冲液洗一次它，并将其放置在玻璃培养管中。 </li><li>孵育在0.5毫升10％的KOH的黏膜，在37℃过夜。测量用液体闪烁计数器在裂解物（一式三份）的150微升等分试样的放射性。 </li><li>用等分 - 裂解液（3 5μL）用Bradford法测定蛋白浓度。 注:10毫升含放射性血清素孵育培养基中被用作标准。 5-HT的保留在组织中被表达为皮摩尔/毫克蛋白质/分钟。 </li></ol>

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The authors have nothing to disclose.

作为极化上皮单层以研究肠运输10，11，12，13，14，15的完全分化的Caco-2细胞单层已被广泛使用。然而，以模仿肠上皮细胞的生理组织，出现了在3D一个的Caco-2文化的发展相当大的兴趣。若干三维细胞培养模型为肠上皮细胞的近来已经开发出模拟天然细胞-细胞和细胞-细胞外基质（ECM）配比2D系统16较大的生理相关性的相互作用。因此，被广泛地用于三维细胞培养物1的生成含有共同的部件（如层粘连蛋白，胶原蛋白IV和硫酸肝素蛋白聚糖）基于ECM的天然水凝胶6。 这里描述的方案展示了在胶状蛋白质混合物生长3D Caco-2细胞的适用性（从Engelbreth-Holm的-群（EHS）小鼠肿瘤的提取的基于ECM的天然水凝胶）上皮细胞的转运研究。 10天之内，这些细胞开始形成由细胞和显示极性的层包围，具有鲜明的心尖和基底域的中空腔内。在上一胶状蛋白质混合物播种时的细胞密度为具有中央腔定期形成囊肿的关键。对于染色实验中，我们加入4000个细胞/孔的8室滑板（以及表面积:0.7 cm 2）的。种子细胞在不规则的球体更高密度的结果。三维的Caco-2囊肿在10 - 19天秀极性和更分化比上生长的transwell 17（未发表的观测值）2D Caco-2细胞。我们已经表明在SERT mRNA的表达和显着增加的蛋白水平如排版ARED到完全分化的2D单层。另外，在使用这些方法，我们已经表明，TGF-TGF-β1增加SERT的表面的水平，如由免疫荧光染色15。 有趣的是，最近的研究已经表明，3D的Caco-2细胞反应更灵敏，因为重要的信号分量，诸如TNF受体2和MyD88的存在的病理生理的刺激，并代表比常规的2D系统17更好的模型。然而，这限制了在上皮运输研究中使用3D的Caco-2的一个问题是缺乏的不同细胞类型和其在天然肠中发现的复杂性。在这方面，人或小鼠起源的肠组织体（也称为enteroids / colonoids）代表包含所有不同的细胞类型的原生上皮研究离子，营养物，或药物的运输的状态的最先进的模型。相比于类器官，良好区别3D的Caco-2囊肿代表一种类型的上皮细胞（ 即吸收性肠）和肯定适于研究专注于管理肠转运的细胞和分子机制。这是因为，在3D Caco-2细胞是方便的处理，是具有成本效益的，并且易于操纵，诸如质粒DNA和siRNA的转染。在上皮运输研究3D Caco-2细胞的另一个限制是在访问管腔的难度。结合于管腔膜受体或对从腔侧访问上皮传染原剂可以是具有挑战性的在该系统中学习。这种限制可以通过在管腔显微注射的剂来克服，如已经在艰难梭菌感染18人肠类器官培养的情况下，先前完成的。然而，重要的是， 在体外细胞培养模型研究在天然i的验证ntestine 使用体内或离体的方法。 在过去的几十年中，尤斯室，大大的认可许多上皮转运19的功能或监管性能做出了贡献。这里，我们显示了尤斯灌流室的方法的适用性来测量小鼠肠粘膜全无浆肌层15的5-HT的磁通及5-HT的摄取。上基底外侧面（10毫微克/毫升，1小时）显著增加SERT功能，通过增加的Na证明回肠黏膜的TGF-β1的治疗+敏感的粘膜到浆膜通量（J 毫秒 ）和3 H-5- HT 摄取 。回肠组织的氟西汀处理抑制5-HT的摄取的TGF-β1介导的刺激。 这种技术的关键步骤包括安装在所述滑动孔的一个完整的层的组织（没有任何撕裂），以便achie<html>已经准确的测量。合适的手术工具，例如球剪刀，薄尖镊子和羽毛外科刀片，对于适当的处理重要和组织的剥离。 这种方法适用于研究其他上皮转运，如电解质转运（氯化物转运的Na + / H +交换，营养运输等 ）的功能。尤斯灌流的腔室等专门用途的pH计技术，测量跨上皮碳酸氢盐分泌，而同位素熔剂法，来衡量净分泌或吸收基板，已经由克拉克博士等人全面审查。 8。他们广泛的审查尤斯灌流室来输送养分19的研究应用。赠送尤斯灌流室也已经在临床上相关的情况下使用，如用于测量活检样品在运输功能裁判"&gt; 20。在外翻囊方法的尤斯灌流室技术的主要优点是能够同时测量完好，偏振光肠上皮的电和运输参数的能力。 与尤斯室方法的一个问题是有限的生存能力和体外肠道准备的最佳功能。在从动物取出后， 离体肠制剂可以仅持续在Ussing室中的19至3小时。因此，这种方法可能在测试的药剂的效果更长的时间段的限制。在这种情况下，治疗可以在体内进行，然后分离从对照和治疗组肠可用于测量与所述尤斯灌流方法传输参数。在所有情况下， 克 叔 （或R t）的大小可以是存活的实时指示和所用的区分是有用测试试剂的特异性和非特异性的效果。 总之，这里描述的方法可以方便我们理事在健康和患病的条件下，如肠道炎症，感染性腹泻，吸收障碍或代谢紊乱的功能和上皮转运调节的细胞和分子机制的理解。

A correction was made to the Authors section in: <a href="https://www-jove-com.bdigitaluss.remotexs.co/video/55304">Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D</a>.
One of the authors&#39; names was corrected from:
Arivarasu N. Anabazhagan
to:
Arivarasu N. Anbazhagan

A correction was made to the Authors section in: <a href="https://www-jove-com.bdigitaluss.remotexs.co/video/55304">Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D</a>.

Erratum: Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D

肠上皮配有各种转运蛋白执行许多功能，从营养物质，电解质的吸收（频道，ATP酶，共转运和交换器），和药物的流体分泌和离子在管腔。转运蛋白产生电化学梯度允许在矢量方式的离子或分子的运动。这是通过在极化上皮细胞的顶和底外侧膜的运输系统的非对称分布来实现的。此外，紧密连接，该系绳相邻上皮细胞，通过作为一个屏障心尖和基底外侧膜结构域之间的组件的膜内扩散在这一过程中起重要作用。模拟天然肠的这些特征合适的模型系统（ 即极性，分化和紧密连接的完整性）都为函数的的研究临界上皮运输系统nality。 对于模型，在肠上皮运输研究目前使用的典型细胞系的Caco-2，完全分化的模式，吸收小肠上皮细胞;和T84细胞或HT-29亚克隆，隐窝衍生大肠上皮细胞1的模型。传统上，这些细胞系生长在塑料表面的单层或在涂覆的transwell插入物。反孔细胞培养插入到一定程度，允许偏振细胞基底外侧喂类似于体内环境。然而，在传统的二维培养系统中的限制在于将细胞被迫适应的人工，平坦，刚性表面。因此，天然上皮的生理复杂不准确地反映在二维系统。这一限制已经克服的方法在一个特定的微环境生长的细胞在3D，如胶状蛋白质mixtur即，含有多种细胞外基质成分2,3。尽管如此， 在体外培养物不能模拟的肠上皮细胞，其具有多种细胞类型和在肠道内的特定区域的体系结构的复杂性。因此，使用细胞培养模型的研究需要在本机肠道进一步验证。使用体外三维细胞培养和小鼠肠道黏膜，我们在这里描述的简单方法来研究肠道羟色胺转运（SLC6A4，SERT）的监管。 的SERT转运调节的重要激素和神经递质5-羟色胺（5-HT）的胞外情况，通过经由的 Na + /氯迅速运送它-依赖性过程。 SERT是抗抑郁的已知靶，并在最近成为胃肠道疾病，如腹泻和肠道炎症的一种新的治疗靶标。方法来调查在肠上皮细胞的5-HT摄取之前已经描述了。例如，生长在塑料支撑物或可渗透插入Caco-2细胞已经显示在两个顶端和基底外侧结构域4呈现氟西汀敏感的3 H-5-HT的摄取。 SERT功能的分离的刷状缘膜囊（BBMVs）从人体器官捐献者小肠中制得的测量也得到了我们5描述。 3人BBVMs H-5-HT的摄取显示出是氟西汀敏感和Na + / Cl -的依赖性，并且呈现出饱和动力学具有300 K m值纳米5。利用类似的方法，我们也预先测定SERT功能如在小鼠肠BBMVs 6 的3 H-5-HT的摄取。然而，纯质膜囊泡的制备需要大量的组织黏膜。其它方法，如radiog的3 H-5-HT的摄取部位raphic可视化，也已预先在豚鼠和大鼠小肠7所示。 在尤斯灌流室提供了一个更生理系统来测量离子，营养物，和在各种上皮组织的药物的输送。在尤斯灌流室技术的主要优点是，它使完整的，偏振肠上皮的电气和运输参数的精确测量。另外，为了尽量减少固有神经肌肉系统的影响，可以进行肠粘膜浆肌剥离调查转运的调节中的上皮8。 我们表明，在三维上生长凝胶状蛋白Caco-2细胞形成中空内腔表达不同心尖和基底外侧标记。这些细胞显示的SERT比2D Caco-2细胞的高表达。方法来种植3D细胞和PErform免疫染色或RNA和蛋白质的提取进行说明。此外，我们描述的方法由TGF-β1，一种多效性细胞因子，利用Ussing室中的技术，研究SERT功能和调节小肠粘膜。

<table><tbody><tr><td>10x PBS</td><td>ATCC</td><td>ATCC&amp;reg; HTB&amp;shy;37&amp;trade;</td><td>Cells</td></tr><tr><td>10x PBS</td><td>Carl Zeiss</td><td></td><td>Microsope for confocal Imaging</td></tr><tr><td>&lt;sup&gt;3&lt;/sup&gt;[H]-5-HT&amp;nbsp;</td><td>LabtekII</td><td>152453</td><td>Culturing cells for microscopy</td></tr><tr><td>5-HT&amp;nbsp;</td><td>Gibco</td><td>70013-032</td><td>Not a hazardous substance</td></tr><tr><td>95% O&lt;sub&gt;2&lt;/sub&gt;- 5% CO&lt;sub&gt;2&lt;/sub&gt; cylinder</td><td>KPL</td><td>71-00-27</td><td>Not a hazardous substance</td></tr><tr><td>Agar (for Agar plates)</td><td>Fischer</td><td>BP151-100</td><td>Acute toxicity, Ora</td></tr><tr><td>Agar (for electrode)</td><td>Sigma</td><td>G7126-1KG</td><td>Not a hazardous substance</td></tr><tr><td>Alexa Fluor Phalloidin</td><td>Sigma</td><td>C3867-1VL</td><td>Not a hazardous substance</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma</td><td>P6148-500G</td><td>Harmful if swallowed or if inhaled</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>LifeTechnologies</td><td>A12380</td><td>Not a hazardous substance</td></tr><tr><td>Caco-2 Cells</td><td>Sigma</td><td>A8806-5G</td><td>Not a hazardous substance</td></tr><tr><td>Cell lysis Buffer</td><td>Fischer</td><td>BP337-100</td><td>Not a hazardous substance</td></tr><tr><td>Chabered slides</td><td>Sigma</td><td>S5886-1KG</td><td>Not a hazardous substance</td></tr><tr><td>Collagen Type-1 Solution</td><td>Sigma</td><td>S8045-500G</td><td></td></tr><tr><td>Electrodes</td><td>Sigma</td><td>S-0876</td><td></td></tr><tr><td>EMEM</td><td>Gibco by Life Technologies</td><td>25200-056</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Corning</td><td>356234</td><td>Used as Extracellular matrix</td></tr><tr><td>Fluoxetine</td><td>Qiagen</td><td>74104</td><td></td></tr><tr><td>Gentamicin</td><td>ATCC</td><td>30-2003</td><td>Cell culture Media</td></tr><tr><td>Glycin</td><td>Cell Signaling, Danvers, MA</td><td></td><td></td></tr><tr><td>Hepes&amp;nbsp;</td><td>Roche</td><td>11836145001</td><td></td></tr><tr><td>Indomethacin</td><td>Gibco by Life Technologies</td><td>15070-063</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Gibco by Life Technologies</td><td>15710-064</td><td></td></tr><tr><td>KOH</td><td>Gibco by Life Technologies</td><td>15630-080</td><td></td></tr><tr><td>L-ascorbic acid&amp;nbsp;</td><td>Gibco by Life Technologies</td><td>10082147</td><td></td></tr><tr><td>Liquid scintillation counter&amp;nbsp;</td><td>Gibco by Life Technologies</td><td>70013--032</td><td></td></tr><tr><td>LSM710 Meta</td><td>Physiologic Instruments, San Diego, CA&amp;nbsp;</td><td>EM-CSYS-8</td><td></td></tr><tr><td>Mannitol</td><td>Physiologic Instruments, San Diego, CA&amp;nbsp;</td><td>P2304</td><td></td></tr><tr><td>Matrigel</td><td>Physiologic Instruments, San Diego, CA&amp;nbsp;</td><td>VCC MC8</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Physiologic Instruments, San Diego, CA&amp;nbsp;</td><td>DM MC6</td><td></td></tr><tr><td>Multichannel Voltage/current Clamp</td><td>Physiologic Instruments, San Diego, CA&amp;nbsp;</td><td>P2300</td><td></td></tr><tr><td>NaCl</td><td>Physiologic Instruments, San Diego, CA&amp;nbsp;</td><td>P2023-100</td><td></td></tr><tr><td>NaCl</td><td>Fisher Scientific, Hampton, NH</td><td>BP1423</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich, St Louis, MO</td><td>S5886</td><td></td></tr><tr><td>Normal Goat Serum (NGS, 10%)</td><td>Fisher Scientific, Hampton, NH</td><td>S233</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma-Aldrich, St Louis, MO</td><td>P288</td><td></td></tr><tr><td>Pen-Strp</td><td>Sigma-Aldrich, St Louis, MO</td><td>C3881</td><td></td></tr><tr><td>Protein assay reagent</td><td>Sigma-Aldrich, St Louis, MO</td><td>10420</td><td></td></tr><tr><td>Proteinase Inhibitor Cocktail</td><td>MEDOX, Chicago, IL&amp;nbsp;</td><td>style G- 12300</td><td></td></tr><tr><td>RNA easy Mini kit</td><td>Sigma-Aldrich, St Louis, MO</td><td>M4125</td><td></td></tr><tr><td>Single Channel Electrode Input Module&amp;nbsp;</td><td>Sigma-Aldrich, St Louis, MO</td><td>A92902</td><td></td></tr><tr><td>Sliders for snapwell Chambers</td><td>Sigma-Aldrich, St Louis, MO</td><td>H9523</td><td></td></tr><tr><td>Sodium Phosphate (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich, St Louis, MO</td><td>F132</td><td></td></tr><tr><td>Sodium-Hydroxide (NaOH)</td><td>Sigma-Aldrich, St Louis, MO</td><td>T7039</td><td></td></tr><tr><td>TGF-&amp;beta;1</td><td>Perkin-Elmer,Waltham, MA</td><td>NFT1167250UC</td><td></td></tr><tr><td>Triton X-100</td><td>Packard Instruments, Downers Grove, IL</td><td>B1600</td><td></td></tr><tr><td>Trypsin EDTA</td><td>Sigma-Aldrich, St Louis, MO</td><td>221473</td><td></td></tr><tr><td>Tween-20</td><td>Bio Rad Laboratories, Hercules, CA</td><td>5000006</td><td></td></tr><tr><td>Ussing Chamber (chamber only)</td><td>Sigma-Aldrich, St Louis, MO</td><td>I7378</td><td></td></tr><tr><td>Ussing Chamber Systems</td><td>Sigma-Aldrich, St Louis, MO</td><td>A1296</td><td></td></tr></tbody></table>

methods to study epithelial transport protein function and expression in native intestine and caco-2 cells grown in 3d

肠上皮具有重要的运输和屏障功能，在身体的正常生理功能中起关键作用，同时提供一个障碍异物。受损的上皮细胞的转运（离子，营养物，或药物）已经与许多疾病相关联，并且可以具有通过影响上皮完整性和肠道微生物以外的转运，如正常生理功能延伸的后果。了解功能和转运蛋白的调控是改进治疗干预的发展至关重要。在上皮运输研究的最大挑战是开发概括了原生肠上皮细胞的重要特征一个合适的模型系统。几个体外细胞培养模型，如的Caco-2，T-84，和HT-29-Cl.19A细胞在上皮转运研究通常使用。这些细胞系代表了还原的方法来电子建模pithelium并已在许多机理研究，包括其上皮 - 微生物相互作用的检查中使用。然而，细胞单层没有准确地反映细胞-细胞相互作用和体内微环境。在三维生长的细胞都显示出有希望用于药物渗透性的研究模型。我们表明，Caco-2细胞在3D可用于研究上皮转运。同样重要的是，在Caco-2细胞的研究与其它型号的补充，以排除细胞特异性的影响，并考虑到天然肠的复杂性。几种方法先前已被用来评估转运，例如在分离的上皮细胞或分离的质膜囊泡外翻囊和吸收的功能。考虑到相对于模型和转运功能的测量在该领域的挑战，我们在这里展示一个协议到生长在3D Caco-2细胞并描述了使用Ussing室中的作为一个有效的方法来测量血清素转运，如在完整偏振光肠上皮。

免疫染色在三维的Caco-2囊肿示于图1。的XY平面内的表示与鬼笔环肽的肌动蛋白染色的三维囊肿以及各别管腔的代表性图像中的图1A中描绘。面向内腔侧表示顶侧。上皮细胞在3D环境中的结果在一个坚固的上皮表型进行培养。在第12天，当肌动蛋白和SERT共染色不同的Caco-2囊肿，示于图1B。在XY平面（从图1A中所示的平面不同）的这种表示，SERT染色是可见的，主要是在管腔膜，用亚根尖染色沿。 图2显示了SERT蛋白水平是较高的三维囊肿相比生长为在第 12天postplating单层Caco-2细胞。这些数据表明，Caco-2细胞的三维培养可以触发模仿呐信号事件略去上皮细胞和导致增加的SERT表达。 图3A描绘Ussing室中的系统的一般概述和操作，并显示在孔的销安装小肠。这种技术允许在天然肠上皮转运的活性的评估。在尤斯室方法的优点是评估管腔（黏膜）或基底（浆膜）方面表示转运的功能的能力。 图4显示增加的5-HTĴ 毫秒通量（A）和响应于TGF-β在回肠黏膜（B）中增加5-HT的累积。因为TGF-β1升高放射性标记的5-羟色胺的运动从粘膜到浆膜侧，如通过歼毫秒表示，这些数据是指示从回肠上皮细胞的管腔膜中5-HT的摄取增加。这从在细胞的增加在放射性标记的5-HT的累积，是进一步明显这是氟西汀抑制敏感，反映SERT的活动上腔膜中表示。 <img alt="图1" src="/files/ftp_upload/55304/55304fig1.jpg" /> 图1: 的Caco-2在12天显示鲜明流明生长在胶质蛋白质混合物。红色:肌动蛋白。对的Caco-2囊肿在三维生长的免疫染色，细胞在8孔腔滑动培养，并用鬼笔环肽（A）的染色，如由Debnath说等如前所述。 9。对于免疫染色三维的Caco-2培养物（B）中 ，囊肿被固定在2％的PFA 30分钟，用PBS-甘氨酸处理，并用0.5％的Triton-X-100透化，在室温15分钟。在4℃下过夜:封闭后，将囊肿在SERT抗体（100 1）温育。然后将它们洗涤并用荧光标记的二抗（1:200）温育并鬼笔环肽（1:200）在室温45分钟。最后，他们被装在抗淬灭含DAPI。图像用共焦显微镜拍摄。比例尺:20微米。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55304/55304fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55304/55304fig2.jpg" /> 图2: 在Caco-2细胞在二维和三维生长SERT蛋白表达。从Caco-2细胞裂解物上的塑料支撑培养的Caco-2三维球体通过SDS-PAGE得到解决印迹到硝酸纤维素膜上并与SERT抗体和GAPDH进行分析。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55304/55304fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55304/55304fig3.jpg"/&gt; 图3:（A）的尤斯室和"滑盖"与销和光圈显示已安装的鼠标小肠。滑块开口面积= 0.30 平方厘米。滑块装配到尤斯灌流室的两半，这是条块。电气参数，如跨上皮电压电位（V t）的 ，是由一个电位器和电极（通常为甘汞半电池或银-氯化银通过盐桥连接到每个腔室的一半）测定。盐桥是由用于洗澡粘膜，以避免在溶液中的离子组成的变化3％琼脂中的生理缓冲液熔化（ 例如，KBR）的。的尤斯室的原则（B）示意图。汽提过的肠粘膜被安装在尤斯灌流室分隔的粘膜和浆膜室的两半。放射性示踪剂加至粘膜的sidË;摄取测定由组织掺入的放射活性的量，归一化的总蛋白含量。磁通通过测量在浆膜室中的放射性在一段时间进行评价。在示意图所示的电极允许电参数，包括电流，电压和电阻的同时监测。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55304/55304fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/55304/55304fig4.jpg" /> 图4:SERT 函数在回肠黏膜测量剥去浆肌层的。短期TGF-β1处理对小鼠回肠SERT功能体外效果，通过3 [H] -5-HT摄取和3 [H] -5-HT通量使用测量表明尤斯室。 TGF-β1处理（10毫微克/毫升，1小时）上的基底外侧面显著增加SERT功能，由增加的Na证明+敏感的粘膜到浆膜通量（J 毫秒 ）（A）和3 [H] - 5-HT的摄取 （B）。回肠组织的氟西汀处理抑制TGF-β1刺激的5-HT的摄取。氟西汀敏感摄取由TGF-β1相比对照增加了2.5倍。结果表示为平均值±SEM。单向ANOVA Tukey的试验被用于统计分析。 Ĵ 毫秒流量的计算:[（CPMë - CPM S）×稀释因子] / [（:V STD点¯x浓底物）的CPM STD / xtxa。 CPM E:每在光通量测量结束分钟数; CPM 小号 :每在流量测量开始计数分钟; CPM 标准 :按标准的分钟数（从粘膜水库采取的）; V 标准 :容积ØF中的每千次展示的std; T:在H通量的时间;答:光圈（0.4 平方厘米）的面积。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55304/55304fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D at JoVE.com

Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D

我们描述了简单的方法来研究肠血清素转运蛋白（SERT）的功能和表达的使用体外细胞培养的Caco-2细胞在三维生长模型和小鼠肠的离体模型的调节。这些方法适用于其他上皮转运的研究。

方法来研究上皮转运蛋白功能和表达在纯肠和Caco-2细胞生长在3D

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing ...

methods-to-study-epithelial-transport-protein-function-expression

Universidad San Sebastian

Research

JoVE Journal

Biology

15.5K 次观看.  University of Illinois at Chicago. 我们描述了简单的方法来研究肠血清素转运蛋白（SERT）的功能和表达的使用体外细胞培养的Caco-2细胞在三维生长模型和小鼠肠的离体模型的调节。这些方法适用于其他上皮转运的研究。 

视频: Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D

Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. Additionally, traversing cellular barriers in the body is crucial for both oral delivery of therapeutic NCs into the circulation and transport from the blood into tissues, where intervention is needed. NC transport across cellular barriers is achieved by: (i) the paracellular route, via transient disruption of the junctions that interlock adjacent cells, or (ii) the transcellular route, where materials are internalized by endocytosis, transported across the cell body, and secreted at the opposite cell surface (transyctosis). Delivery across cellular barriers can be facilitated by coupling therapeutics or their carriers with targeting agents that bind specifically to cell-surface markers involved in transport. Here, we provide methods to measure the extent and mechanism of NC transport across a model cell barrier, which consists of a monolayer of gastrointestinal (GI) epithelial cells grown on a porous membrane located in a transwell insert. Formation of a permeability barrier is confirmed by measuring transepithelial electrical resistance (TEER), transepithelial transport of a control substance, and immunostaining of tight junctions. As an example, ~200&#160;nm polymer NCs are used, which carry a therapeutic cargo and are coated with an antibody that targets a cell-surface determinant. The antibody or therapeutic cargo is labeled with 125I for radioisotope tracing and labeled NCs are added to the upper chamber over the cell monolayer for varying periods of time. NCs associated to the cells and/or transported to the underlying chamber can be detected. Measurement of free 125I allows subtraction of the degraded fraction. The paracellular route is assessed by determining potential changes caused by NC transport to the barrier parameters described above. Transcellular transport is determined by addressing the effect of modulating endocytosis and transcytosis pathways.

Many therapeutic applications require safe and efficient transport of drug carriers and their cargoes across cellular barriers in the body. This article describes an adaptation of established methods to evaluate the rate and mechanism of transport of drug nanocarriers (NCs) across cellular barriers, such as the gastrointestinal (GI) epithelium.

模型和方法来评价药物输送系统在整个细胞屏障的运输

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. ...

科研

生物工程

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological questions in cell and developmental biology. In addition, organoids together with recent technical advances in gene editing and viral gene delivery promises to advance medical research and development of new drugs for treatment of diseases. Organoids grown in vitro in basement matrix provide powerful model systems for studying the behavior and function of various proteins and are well suited for live-imaging of fluorescent-tagged proteins. However, establishing the expression and localization of the endogenous proteins in ex vivo tissue and in in vitro organoids is important to verify the behavior of the tagged proteins. To this end we have developed and modified tissue isolation, fixation, and immuno-labeling protocols for localization of microtubules, centrosomal, and associated proteins in ex vivo intestinal tissue and in in vitro intestinal organoids. The aim was for the fixative to preserve the 3D architecture of the organoids/tissue while also preserving antibody antigenicity and enabling good penetration and clearance of fixative and antibodies. Exposure to cold depolymerizes all but stable microtubules and this was a key factor when modifying the various protocols. We found that increasing the ethylenediaminetetraacetic acid (EDTA) concentration from 3 mM to 30 mM gave efficient detachment of villi and crypts in the small intestine while 3 mM EDTA was sufficient for colonic crypts. The developed formaldehyde/methanol fixation protocol gave very good structural preservation while also preserving antigenicity for effective labeling of microtubules, actin, and the end-binding (EB) proteins. It also worked for the centrosomal protein ninein although the methanol protocol worked more consistently. We further established that fixation and immuno-labeling of microtubules and associated proteins could be achieved with organoids isolated from or remaining within the basement matrix.

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

We present protocols for isolation of intestinal 3D structures from in vivo tissue and in vitro basement matrix embedded organoids, and detail different fixation and staining protocols optimized for immuno-labeling of microtubule, centrosomal, and junctional proteins as well as cell markers including the stem cell protein Lgr5.

免疫荧光标记的微管和中心蛋白在体内肠道组织和 3D体外肠道 Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological ...

发育生物学

Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted in sophisticated in vitro models of the intestinal mucosa. Leveraging these emerging model systems will require adaptation of tools and techniques developed for 2D culture systems and animals. Here, we describe a technique for measuring epithelial barrier permeability in human intestinal organoids in real-time. This is accomplished by microinjection of fluorescently-labeled dextran and imaging on an inverted microscope fitted with epifluorescent filters. Real-time measurement of the barrier permeability in intestinal organoids facilitates the generation of high-resolution temporal data in human intestinal epithelial tissue, although this technique can also be applied to fixed timepoint imaging approaches. This protocol is readily adaptable for the measurement of epithelial barrier permeability following exposure to pharmacologic agents, bacterial products or toxins, or live microorganisms. With minor modifications, this protocol can also serve as a general primer on microinjection of intestinal organoids and users may choose to supplement this protocol with additional or alternative downstream applications following microinjection.

This protocol describes the measurement of epithelial barrier permeability in real-time following pharmacologic treatment in human intestinal organoids using fluorescent microscopy and live cell microscopy.

人肠道 Organoids 上皮屏障通透性的实时测量

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

人类舱内单聚物研究病原体、共脉和宿主肠道上皮细胞之间的相互作用

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...

免疫与感染

Investigating Intestinal Barrier Breakdown in Living Organoids

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was not possible in classical cell culture monolayers. Moreover, monolayer cell cultures are good in vitro model systems but do not represent the complex cellular differentiation processes and functions that rely on 3D structure. This has so far only been possible in animal experiments, which are laborious, time consuming, and hard to assess by optical techniques. Here we describe an assay to quantitatively determine the barrier integrity over time in living small intestinal mouse organoids. To validate our model, we applied interferon gamma (IFN-&#947;) as a positive control for barrier destruction and organoids derived from IFN-&#947; receptor 2 knock out mice as a negative control. The assay allowed us to determine the impact of IFN-&#947; on the intestinal barrier integrity and the IFN-&#947; induced degradation of the tight junction proteins claudin-2, -7, and -15. This assay could also be used to investigate the impact of chemical compounds, proteins, toxins, bacteria, or patient-derived probes on the intestinal barrier integrity.

Here we describe a technique to quantify the barrier integrity of small intestinal organoids. The fact that the method is based on living organoids enables the sequential investigation of different barrier integrity modulating substances or combinations thereof in a time-resolved manner.

研究活体器官的肠道屏障分解

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was ...

医学

Using Caco-2 Cells to Study Lipid Transport by the Intestine

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely accepted as the in vivo model of dietary fat absorption, the surgical techniques involved are challenging and expensive. Genetic manipulation of the animal model is also costly and time consuming. The alternative in vitro model is arguably more affordable, timesaving, and less challenging. Importantly, the in vitro model allows investigators to examine the enterocytes as an isolated system, reducing the complexity inherent in the whole organism model. This paper describes how human colon carcinoma cells (Caco-2) can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs and vitamins. It explains the proper maintenance of Caco-2 cells and the preparation of their lipid mixture; and it further discusses the valuable option of using the permeable membrane system. Since differentiated Caco-2 cells are polarized, the main advantage of using the permeable membrane system is that it separates the apical from the basolateral compartment. Consequently, the lipid mixture can be added to the apical compartment while the lipoproteins can be collected from the basolateral compartment. In addition, the effectiveness of the lentivirus expression system in upregulating gene expression in Caco-2 cells is discussed. Lastly, this paper describes how to confirm the successful isolation of intestinal lipoproteins by transmission electron microscopy (TEM).

Caco-2 cells can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs/vitamins. The permeable membrane system separates the apical from the basolateral compartment, while the lentivirus expression system offers an effective gene overexpression method. The isolation of lipoproteins is confirmed by TEM.

采用Caco-2细胞研究脂质运输由肠

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely ...

生物学

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.

肠上皮细胞通透性的体外及体内测定方法

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and ...

Organoid-Derived Epithelial Monolayer: A Clinically Relevant In Vitro Model for Intestinal Barrier Function

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations as the former do not faithfully represent original tissue physiology, and the availability of the latter is limited. Hence, their application hampers fundamental and drug development research. Adult stem-cell-based organoids (henceforth referred to as organoids) are miniatures of normal or diseased epithelial tissue from which they are derived. They can be established very efficiently from different gastrointestinal (GI) tract regions, have long-term expandability, and simulate tissue- and patient-specific responses to treatments in vitro. Here, the establishment of intestinal organoid-derived epithelial monolayers has been demonstrated along with methods to measure epithelial barrier integrity, permeability and transport,&#160;antimicrobial protein secretion, as well as histology. Moreover, intestinal organoid-derived monolayers can be enriched with proliferating stem and transit-amplifying cells as well as with key differentiated epithelial cells. Therefore, they represent a model system that can be tailored to study the effects of compounds on target cells and their mode of action. Although organoid cultures are technically more demanding than cell lines, once established, they can reduce failures in the later stages of drug development as they truly represent in vivo epithelium complexity and interpatient heterogeneity.

Here, we describe the preparation of human organoid-derived intestinal epithelial monolayers for studying intestinal barrier function, permeability, and transport. As organoids represent original epithelial tissue response to external stimuli, these models combine the advantages of expandability of cell lines and the relevance and complexity of primary tissue.

类器官来源的上皮单层:临床相关的肠道屏障功能体外模型

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations ...

Culture Methods to Study Apical-Specific Interactions using Intestinal Organoid Models

The lining of the gut epithelium is made up of a simple layer of specialized epithelial cells that expose their apical side to the lumen and respond to external cues. Recent optimization of in vitro culture conditions allows for the re-creation of the intestinal stem cell niche and the development of advanced 3-dimensional (3D) culture systems that recapitulate the cell composition and the organization of the epithelium. Intestinal organoids embedded in an extracellular matrix (ECM) can be maintained for long-term and self-organize to generate a well-defined, polarized epithelium that encompasses an internal lumen and an external exposed basal side. This restrictive nature of the intestinal organoids presents challenges in accessing the apical surface of the epithelium in vitro and limits the investigation of biological mechanisms such as nutrient uptake and host-microbiota/host-pathogen interactions. Here, we describe two methods that facilitate access to the apical side of the organoid epithelium and support the differentiation of specific intestinal cell types. First, we show how ECM removal induces an inversion of the epithelial cell polarity and allows for the generation of apical-out 3D organoids. Second, we describe how to generate 2-dimensional (2D) monolayers from single cell suspensions derived from intestinal organoids, comprised of&#160;mature and differentiated cell types. These techniques provide novel tools to study apical-specific interactions of the epithelium with external cues in vitro and promote the use of organoids as a platform to facilitate precision medicine.

Here we present two protocols that allow the modeling of intestinal apical-specific interactions. Organoid-derived intestinal monolayers and Air-Liquid Interface (ALI) cultures facilitate the generation of well-differentiated epithelia accessible from both luminal and basolateral sides, whereas polarity-inverted intestinal organoids expose their apical side and are amenable to high throughput assays.

使用肠道器官模型研究猿特异性相互作用的文化方法

The lining of the gut epithelium is made up of a simple layer of specialized epithelial cells that expose their apical side to the lumen and respond to ...

石蜡包埋的 3D 肠粘膜模型的构建

Basic Three-Dimensional (3D) Intestinal Model System with an Immune Component

There has been an increase in the use of in vivo and in vitro intestinal models to study the pathophysiology of inflammatory intestinal diseases, for the pharmacological screening of potentially beneficial substances, and for toxicity studies on potentially harmful food components. Of relevance, there is a current demand for the development of cell-based in vitro models to substitute animal models. Here, a protocol for a basic, &#8220;healthy tissue&#8221; three-dimensional (3D) intestinal equivalent model using cell lines is presented with the dual benefit of providing both experimental simplicity (standardized and easily repeatable system) and physiological complexity (Caco-2 enterocytes with a supporting immune component of U937 monocytes and L929 fibroblasts). The protocol also includes paraffin embedding for light microscopic evaluation of fixed intestinal equivalents, thereby providing the advantage of analyzing multiple visual parameters from a single experiment. Hematoxylin and eosin (H&#38;E) stained sections showing the Caco-2 columnar cells forming a tight and regular monolayer in control treatments are used to verify the efficacy of the model as an experimental system. Using gluten as a pro-inflammatory food component, parameters analyzed from sections include reduced monolayer thickness, as well as disruption and detachment from the underlying matrix (H&#38;E), decreased tight junction protein expression as shown from occludin staining (quantifiable statistically), and immune-activation of migrating U937 cells as evidenced from the cluster of differentiation 14 (CD14) staining and CD11b-related differentiation into macrophages. As shown by using lipopolysaccharide to simulate intestinal inflammation, additional parameters that can be measured are increased mucus staining and cytokine expression (such as midkine) that can be extracted from the medium prior to fixation. The basic three-dimensional (3D) intestinal mucosa model and fixed sections can be recommended for inflammatory status and barrier integrity studies with the possibility of analyzing multiple visual quantifiable parameters.

作者聚焦：使用 3D 人类细胞系模型研究化合物对肠道组织的影响 

Here we describe constructing a basic three-dimensional (3D) intestinal cell line model system and a paraffin embedding protocol for light microscopic evaluation of fixed intestinal equivalents. Staining of selected proteins permits the analysis of multiple visual parameters from a single experiment for potential use in preclinical drug screening studies.

具有免疫成分的基本三维 （3D） 肠道模型系统

There has been an increase in the use of in vivo and in vitro intestinal models to study the pathophysiology of inflammatory intestinal diseases, for the ...

方法来研究上皮转运蛋白功能和表达在纯肠和Caco-2细胞生长在3D

摘要

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模型和方法来评价药物输送系统在整个细胞屏障的运输

免疫荧光标记的微管和中心蛋白在体内肠道组织和 3D体外肠道 Organoids

人肠道 Organoids 上皮屏障通透性的实时测量

人类舱内单聚物研究病原体、共脉和宿主肠道上皮细胞之间的相互作用

研究活体器官的肠道屏障分解

采用Caco-2细胞研究脂质运输由肠

肠上皮细胞通透性的体外及体内测定方法

类器官来源的上皮单层:临床相关的肠道屏障功能体外模型

使用肠道器官模型研究猿特异性相互作用的文化方法

具有免疫成分的基本三维（3D）肠道模型系统

方法来研究上皮转运蛋白功能和表达在纯肠和Caco-2细胞生长在3D

摘要

探索更多视频

此视频中的章节

模型和方法来评价药物输送系统在整个细胞屏障的运输

免疫荧光标记的微管和中心蛋白在体内肠道组织和 3D体外肠道 Organoids

人肠道 Organoids 上皮屏障通透性的实时测量

人类舱内单聚物研究病原体、共脉和宿主肠道上皮细胞之间的相互作用

研究活体器官的肠道屏障分解

采用Caco-2细胞研究脂质运输由肠

肠上皮细胞通透性的体外及体内测定方法

类器官来源的上皮单层:临床相关的肠道屏障功能体外模型

使用肠道器官模型研究猿特异性相互作用的文化方法

具有免疫成分的基本三维 （3D） 肠道模型系统

具有免疫成分的基本三维（3D）肠道模型系统