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In vitro Assays for Measuring Endothelial Permeability by Transwells and Electrical Impedance Systems
利用Transwell试验和电阻抗系统体外测量内皮通透性

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Abstract

Vascular leakage is an important feature in several diseases, such as septic shock, viral hemorrhagic fever, cancer metastasis and ischemia-reperfusion injuries. Thus establishing assays for measuring endothelial permeability will provide insight into the establishment or progression of such diseases. Here, we provide transwell permeability assay and electrical impedance sensing assay for studying endothelial permeability in vitro. With these methods, the effect of a molecule on endothelial permeability could be defined.

Keywords: Endothelial permeability(内皮通透性), Vascular leakage(血管渗漏), Transwell(Transwell)

Background

The endothelial barrier is a well-regulated structure, which maintains a minimal and selective permeability to fluid and molecules under normal physiological conditions (Komarova and Malik, 2010). The disruption of the endothelial barrier occurs during exposure to inflammatory cytokines, pathogen infection, or cancer metastasis, which induces the disruption of cytoskeleton, cell-cell junction, or cell-to-matrix attachments. The increase in vascular permeability is an important feature in many diseases, including ischemia-reperfusion injury, sepsis, viral hemorrhagic fevers and cancers. To screen which molecules modulate vascular permeability, it is necessary to establish in vitro systems to test endothelial permeability before expanding to animal studies. There are two available systems to test endothelial permeability in vitro, transwell permeability assay and electrical impedance sensing devices (Bischoff et al., 2016). The transwell permeability assays directly detect the penetration of macromolecules and the electrical impedance sensing devices measure the cell layer’s tightness for ion flow. Basically, molecules which can be detected by a spectrometer-based absorbance reader can be used in the transwell permeability assay. As a result, the materials required for this assay are relatively easy to prepare. For the electrical impedance sensing assay, we used the xCELLigence Real-Time Cell Analysis (RTCA) systems to measuring endothelial permeability in a 96-well microplate. Compared to transwell permeability assay, electrical impedance sensing device is more sensitive, and is suitable for time-lapse tracking. However, it is also more expensive, and it may not accurately reflect the penetration of molecules through cell-cell junction. As a result, it is more accurate to apply both systems in parallel. Here, we show the protocol for using these two methods to measure the dengue virus nonstructural protein 1-induced endothelial hyperpermeability in vitro (Chen et al., 2016).

Materials and Reagents

  1. General materials and reagents
    1. Pipet tips  
    2. Human microvascular endothelial cells (HMEC-1) (ATCC, catalog number: CRL-3243 )
    3. Medium 200 (Thermo Fisher Scientific, GibcoTM, catalog number: M-200-500 )
    4. 10% fetal bovine serum (FBS) (GE Healthcare, HyCloneTM, catalog number: SH30071.03HI )
    5. Penicillin-streptomycin solution at a concentration of 100 U/ml (Caisson Laboratories, catalog number: PSL01-100ML )
    6. Endothelial cell growth medium (see Recipes)

  2. Materials and reagents for transwell permeability assay 
    1. Corning 6.5 mm Transwell inserts with 0.4 µm polycarbonate membranes in a 24-well plate (Corning, catalog number: 3413 )
    2. 96-well plate (clear polystyrene wells, flat bottom) (ExtraGene, catalog number: EL-1190-F )
    3. Streptavidin-horseradish peroxidase (HRP) (R&D Systems, catalog number: DY998 )
    4. 3,3’,5,5’-tetramethylbenzidine (TMB) substrate (Sigma-Aldrich, catalog number: T0440 )
    5. Stop solution (2 N H2SO4 water solution) (Sigma-Aldrich, catalog number: 30743 )

  3. Materials and reagents for RTCA 
    1. 96-well E-plate (ACEA BIO, catalog number: 05232368001 )

Equipment

  1. Forceps
  2. General equipment: 37 °C cell incubator supplied with 5% CO2 atmosphere
  3. Transwell permeability assay: VersaMax microplate reader (Molecular Devices, model: VersaMax ELISA Microplate Reader )
  4. RTCA: xCELLigence RTCA System (ACEA BIO, model: xCELLigence RTCA SP System , catalog number: 00380601030)

Software

  1. GraphPad Prism software

Procedure

  1. Transwell permeability assay
    1. Grow 2 x 105 HMEC-1 cells in 300 µl endothelial growth medium on the membrane of each 6.5 mm transwell insert (Figure 1).


      Figure 1. A schematic outline procedure and typical results of transwell permeability assay

    2. Fill the 24-well plate with 1 ml endothelial growth medium.
    3. Transfer the inserts to the wells with medium by using forceps, and then cover the plate with the lid. The sterilization of the forceps is not necessary, although treating with 80% ethanol is recommended.
    4. Incubate the plate in a 37 °C incubator for two days. 
    5. Aspirate the medium from the top and bottom chamber, then replace with the same volume of fresh Medium 200 medium as described above.
      Note: Every movement in this step should be done carefully and slowly as to not activate the endothelial cells. Pipet tips should not touch the membrane. Refill the chambers with fresh medium in a dropwise manner to avoid shear flow.
    6. Put the plate back into the 37 °C incubator for another two days.
    7. Move the inserts to empty wells with forceps, and wait for 5 min to observe whether medium from the upper chambers leaks into the lower chamber. If there is no leak, then proceed to the next steps. If there is a leak repeat steps A6-A7 and wait till the cells are confluent. The endothelial cells must be fully confluent to ensure the formation of cell-cell junctions and a good endothelial barrier. In general, the cells form confluent monolayer after 5 days of incubation.
    8. Repeat step A5 and add the desired stimulation to the medium in the top chambers. The stimulation can be cytokines (TNF-α), molecules which induce inflammatory responses (lipopolysaccharide, histamine), or recombinant proteins (thrombin, dengue virus nonstructural protein 1).
    9. Incubate the plate in the 37 °C incubator for desired time period. This assay works best when the cells are stimulated between 10 min to 24 h. Here we show the result of stimulating the endothelial cells with thrombin for 30 min (Figure 1).
    10. Before measuring the permeability of the endothelial monolayer, prepare medium containing streptavidin-HRP (15 µl streptavidin-HRP per 1 ml of serum-free medium).
    11. Aspirate medium in top chambers and refill them with streptavidin-HRP-containing medium.
      Note: As mentioned in step A6, every movement should be gentle and careful, and pipet tips should not touch the membrane.   
    12. Add 1 ml serum-free medium in each well of a new 24-well plate. And then put the inserts in.
    13. Cover the plate, and place the plate in the 37 °C incubator.
    14. After incubation for 5 min, the inserts are removed, and 20 µl of media from the lower chamber is transferred to a new 96-well plate. Every condition should be aliquoted in triplicate. 
    15. Add 50 µl TMB substrate into each well of the 96-well plate, and wait 5 min at room temperature for the reaction to stabilize. After incubation, TMB substrate should turn blue. If there is no change in the color, extend the incubation time. However, the incubation time should not exceed 20 min.
    16. Add 25 µl stop solution into each well, and acquire the absorption at 450 nm with an ELISA reader.
      A schematic outline procedure and typical results are shown in Figure 1.

  2. RTCA
    1. Grow 1 x 104 HMEC-1 cells in each well of a 96-well E-plate, and fill the well with 100 µl of endothelial growth medium.
    2. Place the plate in the xCELLigence RTCA System, which is set up in a 37 °C incubator.
    3. Set the program to detect the resistance every hour and monitor when the cells have grown to confluency. In general, the cells grow to confluency after incubating for 2 days, so we suggest checking the cell index curve after 36 h of incubation.
    4. After the cell index curve stabilizes, refresh the medium, and wait 2 h for the cell index curve to stabilize again.
    5. Add the desired stimulation into each well. Every condition should be done in triplicate.
    6. Put the plate back into the xCELLigence RTCA System in the 37 °C incubator.
    7. Set up the desired detection period and time interval, and start the recording.
      The typical results of RTCA are shown in Figure 2.


      Figure 2. Thrombin-induced endothelial permeability increase as detected by RTCA

Data analysis

  1. For the transwell permeability assay, the resultant absorption intensity values should be normalized over control (e.g., time zero, vehicle control). In order to determine the relative permeability. Each experiment should be repeated more than 3 times, then statistics can be performed using GraphPad Prism software and relevant statistical protocols. Here we show the result which is analyzed with one-way ANOVA and Bonferroni’s multiple comparison test as post-test (Figure 1).
  2. For RTCA, the cell index of every time point should be normalized over the time zero to determine the relative cell index. The average cell index is calculated and plotted verse time.

Notes

  1. Other types of endothelial cells, such as HUVECs or hCMEC/D3, can also be used for either assays; however, pre-coating the membranes or plates with substrates such as fibronectin, collagen, gelatin, and/or laminin may be required to promote cell adhesion.
  2. The integrity of the endothelial monolayer is crucial for the accuracy of either method, so shear flow should always be avoided when handling the cells. For RTCA, as the electrode of 96-well E-plate is very fragile and sensitive, it should always be handled with care. Pipet tips should not touch the electrode on the plate thus avoiding any disruption of the endothelial monolayer. Hands should not touch the electrode beneath the plate as well, as it might disrupt the detection of the resistance. After trypsinizing and washing, the E-plate could be reused (Stefanowicz-Hajduk et al., 2016). An E-plate can be reused at least 3 times without affecting data reproducibility in previous literature and in our experiences (Stefanowicz-Hajduk et al., 2016).
    Run a pretest program before use. If it shows errors in detecting electrical resistance, the E-plate is unsuitable for further assays.

Recipes

  1. Endothelial cell growth medium
    Medium 200 supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin solution at a final concentration of 100 U/ml

Acknowledgments

This study is supported by grants from the Ministry of Science and Technology of Taiwan (102-2320-B-006-025-MY3) (105-2321-B-006-023 and the Center of Infectious Disease and Signaling Research of NCKU, Tainan, Taiwan). This protocol is a modification of those published by Chuang et al. (2011) and Bao Dang et al. (2013).

References

  1. Bao Dang, Q., Lapergue, B., Tran-Dinh, A., Diallo, D., Moreno, J. A., Mazighi, M., Romero, I. A., Weksler, B., Michel, J. B., Amarenco, P. and Meilhac, O. (2013). High-density lipoproteins limit neutrophil-induced damage to the blood-brain barrier in vitro. J Cereb Blood Flow Metab 33(4): 575-582.
  2. Bischoff, I., Hornburger, M. C., Mayer, B. A., Beyerle, A., Wegener, J. and Furst, R. (2016). Pitfalls in assessing microvascular endothelial barrier function: impedance-based devices versus the classic macromolecular tracer assay. Sci Rep 6: 23671.
  3. Chen, H. R., Chuang, Y. C., Lin, Y. S., Liu, H. S., Liu, C. C., Perng, G. C. and Yeh, T. M. (2016). Dengue virus nonstructural protein 1 induces vascular leakage through macrophage migration inhibitory factor and autophagy. PLoS Negl Trop Dis 10(7): e0004828.
  4. Chuang, Y. C., Lei, H. Y., Liu, H. S., Lin, Y. S., Fu, T. F. and Yeh, T. M. (2011). Macrophage migration inhibitory factor induced by dengue virus infection increases vascular permeability. Cytokine 54(2): 222-231.
  5. Komarova, Y. and Malik, A. B. (2010). Regulation of endothelial permeability via paracellular and transcellular transport pathways. Annu Rev Physiol 72: 463-493.
  6. Stefanowicz-Hajduk, J., Adamska, A., Bartoszewski, R. and Ochocka, J. R. (2016). Reuse of E-plate cell sensor arrays in the xCELLigence Real-Time Cell Analyzer. Biotechniques 61(3): 117-122.

简介

血管渗漏是脓毒性休克,病毒性出血热,癌症转移和缺血再灌注损伤等几种疾病的重要特征。因此,建立测定内皮通透性的测定将提供对这些疾病的建立或进展的洞察。在这里,我们提供transwell渗透性测定和用于研究体内内皮通透性的电阻抗感测测定。通过这些方法,可以定义分子对内皮通透性的影响。

背景 内皮屏障是一个良好调节的结构,其在正常生理条件下保持对流体和分子的最小和选择性渗透性(Komarova和Malik,2010)。内皮屏障的破坏在暴露于炎性细胞因子,病原体感染或癌症转移中发生,其诱导细胞骨架,细胞 - 细胞连接或细胞与基质附着的破坏。血管通透性的增加是许多疾病的重要特征,包括缺血再灌注损伤,败血症,病毒性出血热和癌症。为了筛选哪个分子调节血管通透性,有必要建立体外的体外试验以在扩张到动物研究之前测试内皮通透性。有两种可用的体外试验体外渗透性试验,transwell渗透性测定法和电阻抗检测装置(Bischoff等,2016)。 transwell渗透性测定法直接检测大分子的渗透性,电阻抗感测装置测量细胞层对离子流的紧密度。基本上,可以通过基于光谱仪的吸光度读数器检测的分子可用于渗透渗透性测定。因此,该测定所需的材料相对容易制备。对于电阻抗感测测定,我们使用xCELLigence实时细胞分析(RTCA)系统测量96孔微量培养板中的内皮通透性。与transwell渗透性测定相比,电阻抗感应器更灵敏,适合延时跟踪。然而,它也更昂贵,并且可能不能准确地反映分子穿过细胞 - 细胞连接的渗透。因此,并行应用两个系统更为准确。在这里,我们展示了使用这两种方法来测量登革热病毒非结构蛋白1诱导的内皮超高渗透性体外的方案(Chen等人,2016)。

关键字:内皮通透性, 血管渗漏, Transwell

材料和试剂

  1. 一般材料和试剂
    1. 吸管提示
    2. 人微血管内皮细胞(HMEC-1)(ATCC,目录号:CRL-3243)
    3. 培养基200(Thermo Fisher Scientific,Gibco TM,目录号:M-200-500)
    4. 10%胎牛血清(FBS)(GE Healthcare,HyClone TM,目录号:SH30071.03HI)
    5. 浓度为100U/ml的青霉素 - 链霉素溶液(Caisson Laboratories,目录号:PSL01-100ML)
    6. 内皮细胞生长培养基(参见食谱)

  2. 用于transwell渗透性测定的材料和试剂
    1. Corning 6.5 mm Transwell刀片,带有0.4μm聚碳酸酯膜的24孔板(Corning,目录号:3413)
    2. 96孔板(透明聚苯乙烯孔,平底)(ExtraGene,目录号:EL-1190-F)
    3. 链霉亲和素 - 辣根过氧化物酶(HRP)(R& D Systems,目录号:DY998)
    4. 3,3',5,5'-四甲基联苯胺(TMB)底物(Sigma-Aldrich,目录号:T0440)
    5. 停止溶液(2 N H 2 SO 4水溶液)(Sigma-Aldrich,目录号:30743)

  3. RTCA的材料和试剂
    1. 96孔E板(ACEA BIO,目录号:05232368001)

设备

  1. 镊子
  2. 一般设备:37℃的细胞培养箱,供应5%CO 2 气氛
  3. 透孔渗透性测定法:VersaMax酶标仪(Molecular Devices,VersaMax ELISA Microplate Reader)
  4. RTCA:xCELLigence RTCA系统(ACEA BIO,型号:xCELLigence RTCA SP系统,目录号:00380601030)

软件

  1. GraphPad Prism软件

程序

  1. 透孔渗透性测定法
    1. 在每个6.5mm transwell插入物膜上的300μl内皮生长培养基中生长2×10 5个HMEC-1细胞(图1)。


      图1. transwell渗透性测定的示意图概述程序和典型结果

    2. 向24孔板中加入1ml内皮生长培养基
    3. 使用镊子将介质用介质转移到孔中,然后用盖子盖住板。镊子的灭菌是不必要的,尽管用80%乙醇处理是推荐的。
    4. 将板在37℃的培养箱中孵育两天。
    5. 从顶部和底部腔室吸出培养基,然后用如上所述的相同体积的新鲜培养基200培养基替换。
      注意:此步骤中的每一个运动都应该仔细而缓慢地进行,以免激活内皮细胞。吸头不应接触膜。用新鲜的介质以液体方式填充室以避免剪切流动。
    6. 将板放回37°C的孵化器两天。
    7. 用镊子将插入物移入空井,等待5分钟观察上腔室介质是否泄漏到下腔室。如果没有泄漏,请继续下一步。如果有泄漏重复步骤A6-A7,并等待细胞汇合。内皮细胞必须完全融合,以确保细胞细胞连接的形成和良好的内皮屏障。通常,孵育5天后,细胞形成汇合单层。
    8. 重复步骤A5,并将所需的刺激添加到顶部腔室中的培养基。刺激可以是细胞因子(TNF-α),诱导炎症反应的分子(脂多糖,组胺)或重组蛋白(凝血酶,登革热病毒非结构蛋白1)。
    9. 在37℃的培养箱中孵育平板所需的时间。当细胞在10分钟至24小时之间被刺激时,该测定最有效。这里我们显示用凝血酶刺激内皮细胞30分钟的结果(图1)
    10. 在测量内皮单层的渗透性之前,制备含有链霉抗生物素蛋白-HRP的培养基(每1ml无血清培养基为15μl链霉抗生物素蛋白-HRP)。
    11. 在顶部腔室中吸入培养基,并用含链霉亲和素-HRP的培养基补充它们。
      注意:如步骤A6所述,每次运动都应该温和谨慎,移液管吸头不要接触膜。   
    12. 在一个新的24孔板的每个孔中加入1毫升无血清培养基。然后将插入物放入。
    13. 覆盖板,并将板放在37℃的培养箱中。
    14. 孵育5分钟后,除去插入物,将来自下室的20μl培养基转移到新的96孔板中。每一个条件都应该一式三份。
    15. 在96孔板的每个孔中加入50μlTMB底物,并在室温下等待5分钟使反应稳定。孵育后,TMB底物应变蓝。如果颜色没有变化,请延长孵化时间。但是,孵化时间不应超过20分钟
    16. 在每个孔中加入25μl终止溶液,并用ELISA读数器获得450nm的吸收。
      示意图概要程序和典型结果如图1所示。

  2. RTCA
    1. 在96孔E板的每个孔中生长1×10 4个HMEC-1细胞,并用100μl内皮生长培养基填充孔。
    2. 将板放在xCELLigence RTCA系统中,该系统设置在37℃的孵化器中。
    3. 设置程序以每小时检测电阻,并监测细胞何时增长到融合。一般来说,孵化2天后,细胞生长至融合,因此建议在孵育36小时后检查细胞指数曲线。
    4. 细胞指数曲线稳定后,刷新培养基,再等待2小时,使细胞指数曲线再次稳定
    5. 将所需的刺激物添加到每个孔中。每一个条件都应该一式三份。
    6. 将板放回37℃培养箱中的xCELLigence RTCA系统。
    7. 设置所需的检测周期和时间间隔,并开始录制。
      RTCA的典型结果如图2所示

      图2.由RTCA检测到的凝血酶诱导的内皮通透性增加

数据分析

  1. 对于transwell渗透性测定,所得到的吸收强度值应该通过控制(例如,时间零,载体对照)进行归一化。以确定相对渗透率。每个实验应重复3次以上,然后可以使用GraphPad Prism软件和相关统计方案进行统计。在这里,我们显示了使用单因素方差分析和Bonferroni的多重比较检验作为检验后的结果(图1)。
  2. 对于RTCA,每个时间点的细胞指数应在零时间内归一化以确定相对细胞指数。计算平均细胞指数并绘制时间。

笔记

  1. 其他类型的内皮细胞,如HUVECs或hCMEC/D3也可以用于任一种测定;然而,可能需要用诸如纤连蛋白,胶原,明胶和/或层粘连蛋白之类的底物预涂膜或板以促进细胞粘附。
  2. 内皮单层的完整性对于任一方法的准确度至关重要,因此处理细胞时应始终避免剪切流动。对于RTCA,由于96孔E板的电极非常脆弱和敏感,因此应始终小心处理。吸管尖端不应接触板上的电极,从而避免内皮单层的任何破坏。手也不应该接触板下面的电极,因为它可能会破坏电阻的检测。胰蛋白酶消化和洗涤后,E板可以重复使用(Stefanowicz-Hajduk等人,2016)。 E板可以重复使用至少3次,而不影响先前文献和我们的经验中的数据再现性(Stefanowicz-Hajduk等人,2016)。
    使用前运行预测试程序。如果在检测电阻中显示错误,则E板不适合进一步测定。

食谱

  1. 内皮细胞生长培养基
    补充有10%胎牛血清(FBS)和青霉素 - 链霉素溶液的培养基200,终浓度为100U/ml

致谢

本研究得到台湾科技部(102-2320-B-006-025-MY3)(105-2321-B-006-023)和NCKU传染病与信号研究中心的资助,台南,台湾)。该协议是由Chuang等人发表的那些修改。 (2011)和宝钢等人。 (2013年)。

参考

  1. Bao Dang,Q.,Lapergue,B.,Tran-Dinh,A.,Diallo,D.,Moreno,JA,Mazighi,M.,Romero,IA,Weksler,B.,Michel,JB,Amarenco, Meilhac,O.(2013)。高密度脂蛋白限制嗜中性粒细胞对体外血脑屏障的损伤。 .J Cereb Blood Flow Metab 33(4):575-582。
  2. Bischoff,I.,Hornburger,MC,Mayer,BA,Beyerle,A.,Wegener,J.and Furst,R。(2016)。  评估微血管内皮屏障功能的缺陷:基于阻抗的装置与经典的大分子示踪剂测定。 > 6:23671.
  3. Chen,HR,Chuang,YC,Lin,YS,Liu,HS,Liu,CC,Perng,GC和Yeh,TM(2016)。  登革热病毒非结构蛋白1通过巨噬细胞迁移抑制因子和自噬诱导血管渗漏。 PLoS Negl Trop Dis 10(7):e0004828。
  4. (2011)。登革热病毒感染引起的巨噬细胞迁移抑制因子增加血管通透性。细胞因子 54(2):222-231。 br />
  5. Komarova,Y。和Malik,AB(2010)。  通过旁分泌细胞和跨细胞转运途径调节内皮细胞通透性。 Annu Rev Physiol 72:463-493。
  6. Stefanowicz-Hajduk,J.,Adamska,A.,Bartoszewski,R.and Ochocka,JR(2016)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih。 gov/pubmed/27625205"target ="_ blank">在xCELLigence实时细胞分析仪中重复使用E板细胞传感器阵列。生物技术 61(3):117-122。
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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Chen, H. and Yeh, T. (2017). In vitro Assays for Measuring Endothelial Permeability by Transwells and Electrical Impedance Systems. Bio-protocol 7(9): e2273. DOI: 10.21769/BioProtoc.2273.
  2. Chen, H. R., Chuang, Y. C., Lin, Y. S., Liu, H. S., Liu, C. C., Perng, G. C. and Yeh, T. M. (2016). Dengue virus nonstructural protein 1 induces vascular leakage through macrophage migration inhibitory factor and autophagy. PLoS Negl Trop Dis 10(7): e0004828.
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