感谢国家自然科学基金（82274207 82104533）、宁夏重点研发计划（2023BEG02012）、成都中医药大学星林学者研究推进项目（XKTD2022013）的资助。

注：将小鼠脑源性内皮细胞.3 （bEnd.3） 接种到 24 孔板的腔室中，以在特定培养基条件下构建简单的 BBB 体外 模型。通过TEER仪测量正常细胞和缺氧细胞的TEER（图1 和 图2）。
1. 溶液制备
<ol><li>制备含有FBS（10%，v / v），100U / mL青霉素和10mg / mL链霉素的DMEM细胞培养基（参见 材料表）。</li>
	<li>通过向 100 μL DMSO 溶液中加入 1.30 mg CoCl2 来制备 100 mM CoCl2 储备溶液。 注：上述所有溶液均在4°C条件下储存，使用前根据所需浓度稀释储备溶液。</li>
	<li>通过将 2 mL 次氯酸钠溶液加入 38 mL 双蒸水中来制备 5% 次氯酸钠溶液 （v/v）。</li>
</ol>2. 细胞培养和细胞活力
<ol><li>在含有密度为1×10 6个细胞/mL的DMEM培养基的培养皿（100mm）中接种1mL bEnd.3细胞，并在37°C下在5%CO2的湿化气氛中培养。每 2 至 3 天更换培养基，每周 2 次对细胞进行传代培养。</li>
	<li>bEnd.3细胞生长至80%汇合后，用0.25%胰蛋白酶消化细胞30秒。</li>
	<li>使用DMEM培养基通过细胞计数器制备密度为7 x 104 个细胞/mL的bEnd.3细胞悬浮液。然后，在 96 孔板中接种 100 μL bEnd.3 细胞悬液。</li>
	<li>细胞粘附后，用PBS清洁细胞，并在相同条件下用100μL培养基或含药物培养基（100μM，200μM，300μM，400μM，500μM CoCl2）培养细胞24小时。除去孔板内的培养基并用PBS清洗后，加入100μLCCK-8溶液。 注意： 不要在孔中产生气泡，这会影响光密度 （OD） 的值。</li>
	<li>将96孔板置于37°C环境中，孵育1小时。使用酶标仪测量96孔板在450nm处的吸光度。 注意：为避免组内差异，在检测前将孔板冷却至室温。</li>
	<li>根据公式[OD药物 - OD空白]/[OD对照 - OD空白]×100%，计算不同浓度的CoCl2诱导的细胞活力。选择与对照组相比，细胞活力降低有显着差异的CoCl2浓度进行下一次实验。</li>
</ol>3.模型组装
<ol><li>用PBS冲洗24孔板的上腔室。 注意：细胞在24孔板上腔室的底部生长和融合，细胞之间逐渐形成紧密的连接以发挥屏障作用。如果某些研究中使用的内皮细胞不能独立地在PET膜上形成完全屏障，则需要在细胞接种前用胶原IV溶液涂覆膜。</li>
	<li>使用涡旋混合器将 bEnd.3 细胞与 DMEM 培养基混合，以 5 x 105 个细胞/mL 的密度制成悬浮液。然后，在 24 孔板（0.33 cm 2,0.4 μm 膜孔径）的上腔室中的 PET 膜上接种 200 μL bEnd.3 细胞悬浮液。 注：该方案的初步研究发现，当使用5至10次bEnd.3细胞进行实验时，结果没有差异。为确保建模成功的概率，请参考本协议的细胞数间隔。</li>
	<li>向板的下腔室中加入 1200 μL 完全培养基，以确保上下腔室的渗透压趋于稳定。根据类型，添加的培养基体积存在差异;确保上腔和下腔室的液位齐平。</li>
	<li>每天固定时间更换上腔室和下腔室的介质，同时监测电阻值。<ol><li>更换培养基时，细胞层的完整性会因人工触摸或过度运动而受到破坏。为避免上述情况，用负压移液器从一侧缓慢去除旧介质，并在流体交换过程中沿壁缓慢添加新介质。</li>
	</ol></li>
</ol>4. TEER的测量
<ol><li>工作开始前，将电阻器、5% 次氯酸钠溶液、75% 乙醇和双蒸水溶液放入超净工作台中。打开紫外线照射 30 分钟以消除残留的细菌和病原体。</li>
	<li>将电极置于5%次氯酸钠溶液中，缓慢摇动3秒至5秒，然后浸入75%乙醇中15分钟。最后，转移到PBS或双蒸水溶液中直至使用。</li>
	<li>打开电池电阻表背面的开关，根据表1中的参数点击选择板，选择24孔板。</li>
	<li>根据操作需要，选择合适的检测顺序。我们在这项研究中选择了 A1 程序。</li>
	<li>将 kΩ 电阻插入右侧插头以校准仪器。如果校准结果为 1000 ± 5 Ω，则认为仪器的精度正常。<ol><li>如果校准结果不是1000 Ω，点击主界面的 模式单位 选择 欧姆，再点击主界面的 校准 重新校准仪器。</li>
	</ol></li>
	<li>拉出右侧的kΩ电阻，用连接线替换测量电极。</li>
	<li>将电极放入 24 孔板中，不垂直接种细胞，然后单击仪器主屏幕上的 空白处理 。没有接种细胞的板抗性的背景值约为 134.4 Ω。</li>
	<li>将两个电极插入带有电池的播种板的上下腔室中，使电池层位于它们之间，然后轻轻踩 踏踏板记录电阻值。<ol><li>确保电极不接触上腔室和下腔室底部的电池。电极在溶液中的浸泡时间对电阻值没有影响，只需要确保电极处于正确的位置即可。</li>
	</ol></li>
	<li>通过将电阻值（欧姆）乘以上腔室的底部面积（cm2）获得TEER值（Ωcm2），如等式所示： TEER （Ωcm2） = 电阻 （Ω） x S插件 （cm2）</li>
	<li>绘制 TEER-Time 的折线图（以天为单位）。当电阻值没有随时间进一步增加（以天为单位测量）时，考虑细胞形成屏障。 注意：bEnd.3 细胞的单层 BBB 模型将通过培养具有本研究中参数的 bEND.3 细胞约 6 天来成功构建。bEnd.3细胞单层模型的TEER范围为16.49±2.12 Ωcm2 至27.59±1.50 Ωcm2,6 天内（n=8，平均值± SD）。</li>
</ol>5. 屏障破坏和统计分析
<ol><li>根据TEER-时间曲线，选择具有屏障功能的孔，并将其分为对照组和CoCl2 组（n=4）。</li>
	<li>将培养基和含有300μM CoCl2 （200μL）的培养基分别加入对照组和CoCl2 组上腔。</li>
	<li>使用步骤4中描述的程序在37°C孵育的12小时和24小时检测对照组和CoCl2 组的电阻值。</li>
	<li>使用商业软件绘制用或不用 300 μM CoCl2 培养的细胞屏障的 TEER 值趋势。</li>
	<li>使用统计分析软件分析CoCl2处理的细胞与正常细胞之间的抗性值差异（n=4，*p<0.05，**p<0.01，***p<0.001）。</li>
</ol>

跨内皮抗性测量以评估细胞屏障完整性

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X., 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=Endothelial+Cdk5+deficit+leads+to+the+development+of+spontaneous+epilepsy+through+CXCL1/CXCR2-mediated+reactive+astrogliosis.">Endothelial Cdk5 deficit leads to the development of spontaneous epilepsy through CXCL1/CXCR2-mediated reactive astrogliosis.</a> J Exp Med. 217 (1), 20180992 (2020).</li><li>Chen, X., 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=Modeling+Sporadic+Alzheimer's+Disease+in+Human+Brain+Organoids+under+Serum+Exposure.">Modeling Sporadic Alzheimer's Disease in Human Brain Organoids under Serum Exposure.</a> Adv Sci (Weinh). 8 (18), 2101462 (2021).</li><li>Qi, D., Lin, H., Hu, B., Wei, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+on+in+vitro+model+of+the+blood-brain+barrier+(BBB)+based+on+hCMEC/D3+cells.">A review on in vitro model of the blood-brain barrier (BBB) based on hCMEC/D3 cells.</a> J Control Release. 358, 78-97 (2023).</li><li>Artus, C., 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=The+Wnt/planar+cell+polarity+signaling+pathway+contributes+to+the+integrity+of+tight+junctions+in+brain+endothelial+cells.">The Wnt/planar cell polarity signaling pathway contributes to the integrity of tight junctions in brain endothelial cells.</a> J Cereb Blood Flow Metab. 34 (3), 433-440 (2014).</li><li>Sivandzade, F., Cucullo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-vitro+blood-brain+barrier+modeling:+A+review+of+modern+and+fast-advancing+technologies.">In-vitro blood-brain barrier modeling: A review of modern and fast-advancing technologies.</a> J Cereb Blood Flow Metab. 38 (10), 1667-1681 (2018).</li><li>Galla, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocultures+of+primary+porcine+brain+capillary+endothelial+cells:+Still+a+functional+in+vitro+model+for+the+blood-brain-barrier.">Monocultures of primary porcine brain capillary endothelial cells: Still a functional in vitro model for the blood-brain-barrier.</a> J Control Release. 285, 172-177 (2018).</li><li>Srinivasan, B., Kolli, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transepithelial/transendothelial+electrical+resistance+(TEER)+to+measure+the+integrity+of+blood-brain+barrier.">Transepithelial/transendothelial electrical resistance (TEER) to measure the integrity of blood-brain barrier.</a> Blood-Brain Barrier. 142, 99-114 (2019).</li><li>Liang, Y., Yoon, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+sensors+for+blood-brain+barrier+(BBB)+on+a+chip.">In situ sensors for blood-brain barrier (BBB) on a chip.</a> Sens Actuators Rep. 3, 100031 (2021).</li><li>Ozg&#252;r, B., Helms, H. C. C., Tornabene, E., Brodin, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+increases+expression+of+selected+blood-brain+barrier+transporters+GLUT-1,+P-gp,+SLC7A5+and+TFRC,+while+maintaining+barrier+integrity,+in+brain+capillary+endothelial+monolayers.">Hypoxia increases expression of selected blood-brain barrier transporters GLUT-1, P-gp, SLC7A5 and TFRC, while maintaining barrier integrity, in brain capillary endothelial monolayers.</a> Fluids Barriers CNS. 19 (1), (2022).</li></ol>

作者没有什么可透露的。

大脑是最发达的身体器官之一，控制着各种复杂的生理过程，包括记忆、认知、听觉、嗅觉和运动27.大脑是人体最复杂、最患病的器官之一。由于空气污染、饮食习惯不规律和其他因素，许多中枢神经系统疾病的发生呈逐年增长趋势 27,28,29。有趣的是，一些中枢神经系统疾病的开始和进展，如中风、癫痫和阿...

血脑屏障是血液循环和神经组织之间独特的生物界面，由血管内皮细胞、周细胞、星形胶质细胞、神经元和其他细胞结构组成1。离子、化学物质和细胞在血液和大脑之间的流动受到该屏障的严格调节。这种稳态可以保护神经组织免受毒素和病原体的侵害，同时也使大脑神经能够正常运作2,3。维持血脑屏障的完整性可以有效预防影响中枢神经系统的疾病的发展和进展，如神经元功能障碍、水肿和神经炎症4.然而，BBB独特的生理特性阻止了超过98%的小分子药物和100%的大分子药物进入中枢神经系统5。因此，在中枢神经系统药物开发过程中增加药物通过血脑屏障的渗透对于实现治疗效果至关重要 6,7。尽管底物的计算机模拟筛选大大提高了候选药物穿越 BBB 的概率，但仍需要可靠且负担得起的体外/体内 BBB 模型来满足科学研究的需要8。
一种快速且经济实惠的高通量药物筛选技术是体外模型9。为了阐明药物对血脑屏障功能影响的基本过程及其在疾病发展和进展中的作用，已经创建了一系列简化的体外血脑屏障模型。目前，常见的体外BBB模型有单层、共培养、动态和微流控模型10,11,12，由血管内皮细胞和星形胶质细胞、周细胞或小胶质细胞13,14构建。尽管 3D 细胞培养物更符合 BBB15 的生理结构，但它们作为 BBB 药物筛选手段的应用仍然受到其复杂设计和低于标准的可重复性的限制。相比之下，单层体外模型是最常用于研究BBB的模型，适用于确定特定细胞中膜转运蛋白和紧密连接蛋白的表达。
跨膜电阻 （TEER） 测量是一种评估和监测电阻层的细胞层并评估屏障的细胞完整性和渗透性的技术。通过同时将两个电极插入单层两侧的生长培养基或缓冲溶液中，可以测量通过电池致密层16,17的交流电或电阻抗。为了确定体外BBB模型是否已正确创建，TEER的测量通常将采用作为金标准18。另一方面，通过测量药物受累后细胞层电阻的变化，可以准确预测药物作用对BBB通透性的趋势19。例如，Feng等人发现，地黄科的主要活性单体楸波尔（地黄科的主要活性单体）可以有效逆转脂多糖诱导的血脑屏障中紧密连接蛋白的下调，提高小鼠脑内皮细胞层20的TEER值。
神经炎症反应通常是血脑屏障稳态失衡的主要原因21.低氧治疗诱发神经炎症损伤是破坏血脑屏障的主要方法，主要包括物理方法和化学试剂方法。前者主要利用三气体培养箱来改变细胞生长环境中的氧含量以模拟缺氧条件22，而后者是通过人为地将脱氧试剂（如CoCl2）引入细胞培养基23来实现的。如果 Fe2+ 被血红素中的 Co2+ 取代，细胞将保持脱氧状态。如果催化基团中的Fe2+被Co2+取代，则脯氨酸羟化酶和天冬氨酸羟化酶活性将受到抑制，导致缺氧诱导因子-1α（HIF-1α）24的积累。在持续缺氧下，细胞质中HIF-1α的去磷酸化触发细胞死亡并激活血管内皮生长因子，最终提高血管通透性。在先前的研究 25,26 中，已经充分证明缺氧可以显着降低内皮紧密连接蛋白的表达，从而增加 BBB 的通透性。在这项研究中，测量了接种在 24 孔板中的 bEnd.3 细胞的时间电阻曲线，以创建一个简单的 BBB 模型。使用该模型，我们表征了 CoCl2 干预后细胞 TEER 的变化，以构建可用于筛选 BBB 保护药物的细胞模型。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24-well transwell plate</td>
			<td>Corning (Corning 3470, 0.33 cm2, 0.4 &#181;m)</td>
			<td>10522023</td>
			<td></td>
		</tr>
		<tr>
			<td>75 % ethanol</td>
			<td>ChengDu Chron Chemicals Co,.Ltd</td>
			<td>2023052901</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Guangzhou Jet Bio-Filtration Co., Ltd</td>
			<td>220412-078-B</td>
			<td></td>
		</tr>
		<tr>
			<td>bEnd.3 cells</td>
			<td>Hunan Fenghui Biotechnology Co., Ltd</td>
			<td>CL0049</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell counting kit-8 (CCK-8)</td>
			<td>Boster Biological Technology Co., Ltd</td>
			<td>BG0025</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture dish (100mm)</td>
			<td>Zhejiang Sorfa Life Science Research Co., Ltd</td>
			<td>1192022</td>
			<td></td>
		</tr>
		<tr>
			<td>Cobalt Chloride (CoCl2)</td>
			<td>Sigma</td>
			<td>15862</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Boster Biological Technology Co., Ltd</td>
			<td>PYG0040</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified eagle medium (1x)</td>
			<td>Gibco ThermoFisher Scientific</td>
			<td>8121587</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco ThermoFisher Scientific</td>
			<td>2166090RP</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism software</td>
			<td>GraphPad Software</td>
			<td>9.0.0(121)</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel (Contains collagen IV)</td>
			<td>MedChemexpress</td>
			<td>HY-K6002</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Molecular Devices</td>
			<td>SpectraMax iD5</td>
			<td></td>
		</tr>
		<tr>
			<td>OriginPro 8 software</td>
			<td>OriginLab Corporation</td>
			<td>v8.0724(B724)</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (100x)</td>
			<td>Boster Biological Technology Co., Ltd</td>
			<td>17C18B16</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS, 1x)</td>
			<td>Gibco ThermoFisher Scientific</td>
			<td>8120485</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hypochlorite</td>
			<td>ChengDu Chron Chemicals Co,.Ltd</td>
			<td>2022091501</td>
			<td></td>
		</tr>
		<tr>
			<td>Transmembrane resistance meter</td>
			<td>World Precision Instruments LLC</td>
			<td>VOM3 (verison 1.6)</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin 0.25% (1x)</td>
			<td>HyClone</td>
			<td>J210045</td>
			<td></td>
		</tr>
	</tbody>
</table>

barrier functional integrity recording on bend.3 vascular endothelial cells via transendothelial electrical resistance detection

血脑屏障（BBB）是由微血管内皮细胞、星形胶质细胞和周细胞组成的动态生理结构。通过协调有害物质的受限转运、营养吸收和大脑中代谢物清除之间的相互作用，血脑屏障对于维持中枢神经系统稳态至关重要。建立血脑屏障的 体外 模型是探索神经系统疾病的病理生理学和创建药物治疗的宝贵工具。本研究描述了一种通过将 bEnd.3 细胞接种到 24 孔板的上腔室中来创建 体外 单层 BBB 细胞模型的过程。为了评估细胞屏障功能的完整性，采用常规的上皮细胞电压表实时记录正常细胞和CoCl2诱导的缺氧细胞的跨膜电阻。我们预计上述实验将为创建BBB的 体外 模型和治疗中枢神经系统疾病的药物提供有效的思路。

BBB is a unique biological interface between blood circulation and nerve tissue, which is composed of vascular endothelial cells, pericytes, astrocytes, neurons, and other cellular structures1. The flow of ions, chemicals, and cells between the blood and the brain is strictly regulated by this barrier. This homeostasis safeguards the nervous tissues against toxins and pathogens while also enabling the appropriate operation of the brain&#39;s nerves2,3. Maintaining the integrity of the BBB can effectively prevent the development and progression of disorders affecting the central nervous system, such as neuronal dysfunction, edema, and neuroinflammation4. However, the unique physiological properties of the BBB prevent more than 98% of small molecule medications and 100% of macromolecular pharmaceuticals from entering the central nervous system5. Therefore, increasing the penetration of medications through the BBB during the development of drugs for the central nervous system is essential for achieving therapeutic efficacy6,7. Even though computer simulation screening of substrates has significantly raised the probability of drug candidates crossing the BBB, reliable and affordable in vitro/in vivo BBB models are still needed to meet the needs of scientific research8.
A quick and affordable technique for high-throughput drug screening is the in vitro model9. To shed light on the fundamental processes of medicines&#39; effects on BBB function and their part in the development and progression of disease, a series of simplified in vitro BBB models has been created. At present, the common in vitro BBB models are the monolayer, co-culture, dynamic, and microfluidic models10,11,12, constructed by vascular endothelial cells and astrocytes, pericytes, or microglia13,14. Although 3D cell cultures are morein line with the physiological structure of BBB15, their application as a means of drug screening for BBB is still constrained by their intricate design and subpar reproducibility. In contrast, the monolayer in vitro model is the one most frequently used to research the BBB and is applicable for determining the expression of membrane transporters and tight junction proteins in particular cells.
Transmembrane electrical resistance (TEER) measurement is a technique to evaluate and monitor the layer of cells across the resistance and evaluate the cell integrity and permeability of the barrier. By simultaneously inserting two electrodes into the growth medium or buffer solution on either side of the monolayer, it is possible to measure the alternating current or electrical impedance through the cell&#39;s compact layer16,17. In order to determine whether the in vitro BBB model has been properly created, the measurement of TEER will usually be employed as the gold standard18. On the other hand, the trend of medication action on BBB permeability can be accurately predicted by measuring the change in electrical resistance of the cell layer after drug involvement19. For example, Feng et al. discovered that catalpol (the primary active monomer of rehmanniae) could effectively reverse the lipopolysaccharide-induced down-regulation of tight junction proteins in the BBB and raise the TEER value of the mouse brain endothelial cell layer20.
The neuroinflammatory response is usually the main cause of BBB homeostasis imbalance21. Hypoxic treatment to induce neuroinflammatory injury is the main method to destroy the blood-brain barrier, mainly including physical methods and chemical reagent methods. The former primarily utilizes a three-gas incubator to vary the oxygen content in the cell growth environment to simulate hypoxic conditions22,while the latter is achieved by artificially introducing deoxy reagents such as CoCl2 to the cell culture medium23. The cells will remain in a deoxygenated condition if Fe2+ is substituted for Co2+ in the heme. If Fe2+ is substituted for Co2+ in the catalytic group, proline hydroxylase and aspartate hydroxylase activity will be inhibited, resulting in an accumulation of hypoxia-inducible factor-1&#945; (HIF-1&#945;)24. Under persistent hypoxia, the dephosphorylation of HIF-1&#945; in the cytoplasm triggers cell death and activates vascular endothelial growth factor, which ultimately raises vascular permeability. In previous studies25,26, it has been well demonstrated that hypoxia can significantly reduce the expression of endothelial tight junction proteins to increase the permeability of BBB. In this study, the time-resistance curve of bEnd.3 cells seeded in 24-well plates were measured in order to create a straightforward BBB model. Using this model, we characterized the changes in cell TEER after CoCl2 intervention in order to construct a cell model that can be used to screen drugs for BBB protection.

作者聚焦：TEER 检测在评估细胞屏障完整性中的应用 

通过跨内皮电阻检测对bEnd.3血管内皮细胞进行屏障功能完整性记录

Current studies typically use permeability tests and associated protein expression to verify the accuracy of TEER detection. The BBB model of monolayer cells in virtue is less effective for drug screening, and the use of more cells makes it more difficult to construct the model. The protocol of this study is relatively simple, as the non-invasive procedure allows the cell samples to be used in other experiments after TEER detection.The detection of TEER to assess the permeability of cell models can accelerate the screening of drugs for treatment and prevention of the central nervous system to increase the success rate of new drug development. In the future, we will detect the expression of tight junction protein in upper compartment cells of chest wall to verify that TEER test results can effectively represent the integrity of BBB barrier function.

该协议允许根据跨内皮电阻仪中设置的参数记录细胞电阻值的变化。采用CCK-8法筛选不同浓度CoCl2处理的bEnd.3细胞（活细胞数）。CoCl2 产生的细胞损伤越大，细胞活力越低。我们发现 300 μM 的 CoCl2 在体外具有显着的细胞毒性，该浓度用于后续实验（图 3A）。通过监测bEnd.3的生长抗性变化，我们发现细胞TEER值在第5天开始稳定。在第6 ...

Watch this Scientific Journal Video about Applications of TEER Detection to Assess Cell Barrier Integrity at JoVE.com

该协议描述了一种可靠且有效的脑血屏障 体外 模型。该方法使用小鼠脑血管内皮细胞bEnd.3并测量跨膜电阻。

The blood-brain barrier (BBB) is a dynamic physiological structure composed of microvascular endothelial cells, astrocytes, and pericytes. By coordinating ...

目前的研究通常使用渗透性测试和相关的蛋白质表达来验证TEER检测的准确性。单层细胞的BBB模型对药物筛选效果较差，使用较多的细胞使得模型的构建更加困难。本研究的方案相对简单，因为非侵入性程序允许细胞样品在TEER检测后用于其他实验。检测TEER以评估细胞模型的渗透性，可以加速中枢神经系统治疗和预防药物的筛选，从而提高新药开发的成功率。未来，我们将检测胸壁上区室细胞中紧密连接蛋白的表达，以验证TEER测试结果可以有效代表BBB屏障功能的完整性。

barrier-functional-integrity-recording-on-bend3-vascular-endothelial

Research

JoVE Journal

Neuroscience

Barrier Functional Integrity Recording on bEnd.3 Vascular Endothelial Cells via Transendothelial Electrical Resistance Detection

2.0K 次观看.  Chengdu University of Traditional Chinese Medicine. 目前的研究通常使用渗透性测试和相关的蛋白质表达来验证TEER检测的准确性。单层细胞的BBB模型对药物筛选效果较差，使用较多的细胞使得模型的构建更加困难。本研究的方案相对简单，因为非侵入性程序允许细胞样品在TEER检测后用于其他实验。检测TEER以评估细胞模型的渗透性，可以加速中枢神经系统治疗和预防药物的筛选，从而提高新药开发的成功率。未来，我们?...

视频: 作者聚焦：TEER 检测在评估细胞屏障完整性中的应用 

The blood-brain barrier (BBB) is a dynamic physiological structure composed of microvascular endothelial cells, astrocytes, and pericytes. By coordinating the interaction between restricted transit of harmful substances, nutrient absorption, and metabolite clearance in the brain, the BBB is essential in preserving central nervous system homeostasis. Building in vitro models of the BBB is a valuable tool for exploring the pathophysiology of neurological disorders and creating pharmacological treatments. This study describes a procedure for creating an in vitro monolayer BBB cell model by seeding bEnd.3 cells into the upper chamber of a 24-well plate. To assess the integrity of cell barrier function, the conventional epithelial cell voltmeter was used to record the transmembrane electrical resistance of normal cells and CoCl2-induced hypoxic cells in real-time. We anticipate that the above experiments will provide effective ideas for the creation of in vitro models of BBB and drugs to treat disorders of central nervous system diseases.

This protocol describes a dependable and efficient in vitro model of the brain blood barrier. The method uses mouse cerebral vascular endothelial cells bEnd.3 and measures transmembrane electrical resistance.

科研

神经科学

Culture of bEnd.3 Cells and Cell Viability After CoCl2 Treatment

Culture of bEnd.3 Cells and Cell Viability After CoCl2 Treatment  

To begin, prepare DMEM cell culture medium containing FBS, penicillin, and streptomycin. Also, prepare a 100 millimolar cobalt chloride stock solution by adding 1.30 milligrams of cobalt chloride to 100 microliters of DMSO. Next, seed one milliliter of BEND3 cells in a 100-milliliter culture dish containing DMEM medium and culture at 37 degrees Celsius in a humidified atmosphere of 5%carbon dioxide.Change the medium every two to three days and subculture the cells two times a week. After BEND3 cells grow to 80%confluence, digest the cells with 0.25%trypsin for 30 seconds. Count the cells using a cell counter and make a suspension of density seven times 10 to the fourth cells per milliliter.Then, seed 100 microliters of this cell suspension in a 96-well plate for cell adhesion. Then wash the cells with PBS and add 100 microliters of culture medium containing cobalt chloride. Culture the cells for 24 hours.After incubation, remove the medium and wash with PBS. Next, add 100 microliters of CCK-8 solution. Incubate the plates at 37 degrees Celsius for one hour, then measure the absorbance at 450 nanometers using a microplate reader.Calculate the cell viability induced by cobalt chloride according to this formula. Select the concentration with a significant difference in cell viability reduction compared with the control group. The viability of BEND3 cells treated with different concentrations of cobalt chloride was analyzed by CoCl2.The results indicated that 300 micromolar of cobalt chloride showed significant in vitro cytotoxicity.

bEnd.3 细胞的培养和 CoCl2 处理后的细胞活力

In Vitro Blood-Brain Barrier Model Assembly for Measurement of TEER

To begin, rinse the upper chamber of 24 well plates with PBS. Mix the BEND3 cells with DMEM medium using a vortex mixer to make a suspension. Then, seed 200 microliters of BEND3 cell suspension on the PET membrane in the upper chamber of a 24 well plate.Add 1, 200 microliters of complete medium to the lower chamber of the plate to stabilize the osmotic pressure of the upper and lower chambers. Change the medium by slowly removing the old medium from one side with a negative-pressure pipette and adding a new medium along the wall during fluid exchange. Before measuring TEER, place the resistor, 5%sodium hypochlorite solution, 75%ethanol, and double distilled water on an ultra clean table.Turn on the UV irradiation for 30 minutes to eliminate residual bacteria and pathogens. Place the electrodes in 5%sodium hypochlorite solution with slow shaking for three to five seconds. Then immerse in 75%ethanol for 15 minutes.Finally, transfer to PBS or double distilled water until use. Next, turn on the switch at the back of the cell resistor meter. Click Select Plate and Select 24 well plate.According to the operation, select the appropriate detection sequence. Insert a kilo ohm resistor into the right plug to calibrate the instrument. If the calibration result is 1000 plus minus 5 ohms, consider the instrument accurate.If not, click on Mode Units on the main interface to select Ohms and then click Calibrate. Then, remove the kilo ohm resistor and replace it with the measuring electrode using a connecting wire. Place the electrode vertically into the 24 well plate containing only medium and click Blank Handling on the instrument.The background value of the plate resistance without seated cells should be about 134.4 ohm. Next, insert the two electrodes into the upper and lower chambers of the seated plate, such that the cell layer is between them. Record the resistance value by gently stepping on the pedal.Make sure that the electrode does not touch the cells in the upper chamber and the bottom of the lower chamber. Obtain TEER values by multiplying electrical resistance values with the bottom area of the upper chamber, as shown in the equation. Draw a line plot of TEER versus time in days.When the resistance value does not increase with time, the cells have formed a barrier. The resistance values plotted versus time for BEND3 cells showed that the cell TEER values began to stabilize on the fifth day.

用于测量TEER的体外血脑屏障模型组装

Barrier Destruction and Statistical Analysis After TEER Measurement in an In Vitro Blood-Brain Barrier Model

Begin by culturing the BEND3 cells, then divide the wells into the control group and cobalt chloride group. Add 200 microliters of culture medium into the upper chamber of the control group and culture medium containing 300 micromolar cobalt chloride into the cobalt chloride group. Incubate the plates at 37 degrees Celsius and detect the resistance values at 12 and 24 hours of incubation using TEER method.Use commercial software to map the trend of TEER values of the cell barrier. Use statistical analysis software to analyze the difference in resistance values between cobalt chloride treated cells and control cells. Addition of 300 micromolar cobalt chloride resulted in a significant decrease in TEER values compared to the control at both the 12-hour and 24-hour time points.After 24 hours, the TEER value of 300 micromolar cobalt chloride group reduced compared to the control group, indicating breakdown of the cell barrier that was induced by hypoxic conditions due to cobalt chloride.