Name: a triple primary cell culture model of the human blood-brain barrier for studying ischemic stroke in vitro
Uploaded: 2022-10-06T14:05:00
Description: Watch this Scientific Journal Video about A Triple Primary Cell Culture Model of the Human Blood-Brain Barrier for Studying Ischemic Stroke In Vitro at JoVE.com

这项工作得到了美国国立卫生研究院（NIH）资助MH128022，MH122235，MH072567，MH122235，HL126559，DA044579，DA039576，DA040537，DA050528和DA047157的支持。

注意：有关此协议中使用的所有电池，材料，设备和溶液的详细信息，请参阅 材料表 。
1. 三细胞培养BBB模型设置
<ol><li>接种周细胞<ol><li>在具有活化表面的T75培养瓶中培养HBVP，以便在37°C的5%CO2 培养箱中粘附细胞直至汇合。一旦达到汇合，吸出旧的周细胞培养基，并用5mL温热的Dulbecco磷酸盐缓冲盐水（DPBS）洗涤细胞。吸出DPBS并使用4mL温热?...

<ol>
	<li>Mozaffarian, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+disease+and+stroke+statistics-2016+update:+a+report+from+the+American+Heart+Association.">Heart disease and stroke statistics-2016 update: a report from the American Heart Association.</a> Circulation. 133 (94), 38 (2016).</li><li>Yousufuddin, M., Young, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+and+ischemic+stroke.">Aging and ischemic stroke.</a> Aging. 11 (9), 2542-2544 (2019).</li><li>Donkor, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stroke+in+the+21st+century:+a+snapshot+of+the+burden,+epidemiology,+and+quality+of+life.">Stroke in the 21st century: a snapshot of the burden, epidemiology, and quality of life.</a> Stroke Research and Treatment. , 3238165 (2018).</li><li>Kadry, H., Noorani, B., 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=A+blood-brain+barrier+overview+on+structure,+function,+impairment,+and+biomarkers+of+integrity.">A blood-brain barrier overview on structure, function, impairment, and biomarkers of integrity.</a> Fluids and Barriers of the CNS. 17 (1), 69 (2020).</li><li>Brown, L. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericytes+and+neurovascular+function+in+the+healthy+and+diseased+brain.">Pericytes and neurovascular function in the healthy and diseased brain.</a> Frontiers in Cellular Neuroscience. 13, 282 (2019).</li><li>Cabezas, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytic+modulation+of+blood+brain+barrier:+perspectives+on+Parkinson's+disease.">Astrocytic modulation of blood brain barrier: perspectives on Parkinson's disease.</a> Frontiers in Cellular Neuroscience. 8, 211 (2014).</li><li>Abdullahi, W., Tripathi, D., Ronaldson, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-brain+barrier+dysfunction+in+ischemic+stroke:+targeting+tight+junctions+and+transporters+for+vascular+protection.">Blood-brain barrier dysfunction in ischemic stroke: targeting tight junctions and transporters for vascular protection.</a> American Journal of Physiology-Cell Physiology. 315 (3), 343-356 (2018).</li><li>Candelario-Jalil, E., Dijkhuizen, R. M., Magnus, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroinflammation,+stroke,+blood-brain+barrier+dysfunction,+and+imaging+modalities.">Neuroinflammation, stroke, blood-brain barrier dysfunction, and imaging modalities.</a> Stroke. 53 (5), 1473-1486 (2022).</li><li>He, Y., Yao, Y., Tsirka, S. E., Cao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-culture+models+of+the+blood-brain+barrier.">Cell-culture models of the blood-brain barrier.</a> Stroke. 45 (8), 2514-2526 (2014).</li><li>Thomsen, L. B., Burkhart, A., Moos, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+triple+culture+model+of+the+blood-brain+barrier+using+porcine+brain+endothelial+cells,+astrocytes+and+pericytes.">A triple culture model of the blood-brain barrier using porcine brain endothelial cells, astrocytes and pericytes.</a> PLoS One. 10 (8), 0134765 (2015).</li><li>Song, Y., Cai, X., Du, D., Dutta, P., Lin, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+blood-brain+barrier+models+for+in+vitro+biological+analysis:+one+cell+type+vs+three+cell+types.">Comparison of blood-brain barrier models for in vitro biological analysis: one cell type vs three cell types.</a> ACS Applied Bio Materials. 2 (3), 1050-1055 (2019).</li><li>Xu, L., 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=Silver+nanoparticles+induce+tight+junction+disruption+and+astrocyte+neurotoxicity+in+a+rat+blood-brain+barrier+primary+triple+coculture+model.">Silver nanoparticles induce tight junction disruption and astrocyte neurotoxicity in a rat blood-brain barrier primary triple coculture model.</a> International Journal of Nanomedicine. 10, 6105-6118 (2015).</li><li>Appelt-Menzel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+human+blood-brain+barrier+co-culture+model+mimicking+the+neurovascular+unit+using+induced+pluri-+and+multipotent+stem+cells.">Establishment of a human blood-brain barrier co-culture model mimicking the neurovascular unit using induced pluri- and multipotent stem cells.</a> Stem Cell Reports. 8 (4), 894-906 (2017).</li><li>Zhang, Y., 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=Rational+construction+of+a+reversible+arylazo-based+NIR+probe+for+cycling+hypoxia+imaging+in+vivo.">Rational construction of a reversible arylazo-based NIR probe for cycling hypoxia imaging in vivo.</a> Nature Communications. 12 (1), 2772 (2021).</li><li>Palacio-Casta&#241;eda, V., Kooijman, L., Venzac, B., Verdurmen, W. 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R., Le Gac, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+switching+of+tumor+cells+under+hypoxic+conditions+in+a+tumor-on-a-chip+model.">Metabolic switching of tumor cells under hypoxic conditions in a tumor-on-a-chip model.</a> Micromachines. 11 (4), 382 (2020).</li><li>Ramsauer, M., Krause, D., Dermietzel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+of+the+blood-brain+barrier+in+vitro+and+the+function+of+cerebral+pericytes.">Angiogenesis of the blood-brain barrier in vitro and the function of cerebral pericytes.</a> FASEB Journal. 16 (10), 1274-1276 (2002).</li><li>Lyck, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ALCAM+(CD166)+is+involved+in+extravasation+of+monocytes+rather+than+T+cells+across+the+blood-brain+barrier.">ALCAM (CD166) is involved in extravasation of monocytes rather than T cells across the blood-brain barrier.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 37 (8), 2894-2909 (2017).</li><li>Rizzi, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+triple+culture+cell+system+modeling+the+human+blood-brain+barrier.">A triple culture cell system modeling the human blood-brain barrier.</a> Journal of Visualized Experiments. (177), (2021).</li><li>Kumar, S., Shaw, L., Lawrence, C., Lea, R., Alder, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P50:+Developing+a+physiologically+relevant+blood+brain+barrier+model+for+the+study+of+drug+disposition+in+glioma.">P50: Developing a physiologically relevant blood brain barrier model for the study of drug disposition in glioma.</a> Neuro-Oncology. 16 (6), (2014).</li><li>Stone, N. L., England, T. J., O'Sullivan, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+transwell+blood+brain+barrier+model+using+primary+human+cells.">A novel transwell blood brain barrier model using primary human cells.</a> Frontiers in Cellular Neuroscience. 13, 230 (2019).</li><li>Al Ahmad, A., Taboada, C. B., Gassmann, M., Ogunshola, O. 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在该协议中，我们描述了一种建立可靠的三重内皮细胞 - 周细胞 - 星形胶质细胞培养BBB模型的方法，用于研究 体外缺血性中风情况下的BBB功能障碍。考虑到周细胞是 体内 内皮细胞的最近邻，HBVP在该模型16中接种在孔插入物的下侧。虽然这种配置缺乏星形胶质细胞和内皮细胞之间的直接细胞间通讯，但这种排列允许通过分泌的可溶性因子 在 细胞类型之间进?...

中风是全球死亡和长期残疾的主要原因之一1。卒中的发病率随着年龄的增长而迅速增加，55岁以后每10年翻一番2.缺血性卒中是由于血栓和栓塞事件引起的脑血流中断而发生的，占所有卒中病例的80%以上3。即使是现在，可用于减少缺血性中风后组织死亡的治疗选择也相对较少。确实存在的治疗方法对时间敏感，因此并不总是导致良好的临床结果。因此，迫切需要研究缺血性脑卒中影响脑卒中后恢复的复杂细胞机制。
BBB是血液和脑实质之间分子交换的动态界面。在结构上，BBB由脑微血管内皮细胞组成，这些细胞由基底膜包围的连接复合物，周细胞和星形胶质细胞尾足4相互连接。周细胞和星形胶质细胞通过分泌形成强而紧密的连接所必需的各种因子在维持BBB完整性方面起着至关重要的作用5，6。BBB的分解是缺血性中风的标志之一。与脑缺血相关的急性炎症反应和氧化应激导致紧密连接蛋白复合物的破坏和星形胶质细胞、周细胞和内皮细胞之间的串扰失调，从而导致 BBB7 的细胞旁溶质通透性增加。BBB功能障碍进一步促进脑水肿的形成，增加出血转化的风险8。考虑到上述所有因素，人们对了解缺血性中风期间和之后BBB水平发生的分子和细胞变化非常感兴趣。
尽管近几十年来已经开发了许多体外BBB模型并用于各种研究，但它们都不能完全复制体内条件9。虽然一些模型基于在插入良好的渗透性载体上单独培养或与周细胞或星形胶质细胞组合培养的内皮细胞单层，但只有最近的研究引入了三重细胞培养模型设计。几乎所有现有的三重培养BBB模型都包含原代脑内皮细胞以及从动物物种中分离的星形胶质细胞和周细胞或来自人类多能干细胞的细胞10，11，12，13。
认识到需要在体外 更好地概括人BBB，我们建立了由人脑微血管内皮细胞（HBMEC），原代人星形胶质细胞（HA）和原代人脑血管周细胞（HBVP）组成的三细胞培养 体外 BBB模型。该三重培养BBB模型设置在孔径为0.4μm的6孔板聚酯膜插入物上。这些孔插入物为细胞附着提供了最佳环境，并可轻松进入顶端（血液）和基底外侧（脑）隔室，以进行培养基采样或化合物应用。通过测量模拟缺血性中风的体 外OGD后的TEER和细胞旁通量来评估该三细胞培养BBB模型的特征，通过使用加湿的密封室实现氧气（<1%O2）和营养物质（通过使用无葡萄糖培养基）。此外，该模型中诱导的缺血样条件通过缺氧细胞的直接可视化得到准确验证。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24 mm Transwell with 0.4 &#181;m Pore Polyester Membrane Insert</td>
			<td>Corning</td>
			<td>3450</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm Glass Bottom Dishes</td>
			<td>MatTek Life Sciences (FISHERSCI)</td>
			<td>P35GC-1.5-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Astrocyte Medium</td>
			<td>Science Cell</td>
			<td>1801</td>
			<td></td>
		</tr>
		<tr>
			<td>Attachment Factor</td>
			<td>Cell Systems (Fisher Scientific)</td>
			<td>4Z0-201</td>
			<td></td>
		</tr>
		<tr>
			<td>BD 60 mL Syringe</td>
			<td>BD</td>
			<td>309653</td>
			<td></td>
		</tr>
		<tr>
			<td>BrainPhys Imaging Optimized Medium</td>
			<td>STEMCELL Technologies</td>
			<td>5791</td>
			<td></td>
		</tr>
		<tr>
			<td>Complete Classic Medium With Serum and CultureBoost</td>
			<td>4Z0-500</td>
			<td>Cell Systems</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 50 mL PP Centrifuge Tubes (Conical Bottom with CentriStar Cap</td>
			<td>VWR</td>
			<td>430829</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 75cm&#178; U-Shaped Canted Neck Not Treated Cell Culture Flask&#160;</td>
			<td>Corning</td>
			<td>431464U</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning CellBIND 96-well Flat Clear Bottom Black Polystyrene Microplates</td>
			<td>Corning</td>
			<td>3340</td>
			<td></td>
		</tr>
		<tr>
			<td>Countes Cell Counting Chamber Slides</td>
			<td>Thermo Fisher Scientific</td>
			<td>C10228</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess II FL Automated Cell Counter</td>
			<td>Thermo Fisher Scientific</td>
			<td>ZGEXSCCOUNTESS2FL</td>
			<td></td>
		</tr>
		<tr>
			<td>Decon CiDehol 70 Isopropyl Alcohol Solution&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>04-355-71</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Petri Dishes</td>
			<td>VWR</td>
			<td>25384-088</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM Medium (No glucose, No glutamine, No phenol red)</td>
			<td>ThermoFisher</td>
			<td>A14430-01</td>
			<td>Glucose-free medium</td>
		</tr>
		<tr>
			<td>DPBS (No Calcium, No Magnesium)</td>
			<td>ThermoFisher</td>
			<td>14190250</td>
			<td></td>
		</tr>
		<tr>
			<td>EBM Endothelial Cell Growth Basal Medium, Phenol Red Free, 500 mL</td>
			<td>Lonza</td>
			<td>CC-3129</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOM2 Epithelial Volt/Ohm (TEER) Meter with STX2 electrodes</td>
			<td>World Precison Instruments</td>
			<td>NC9792051</td>
			<td>Epithelial voltohmmeter&#160;</td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate&#8211;dextran (wt 20,000)</td>
			<td>Millipore Sigma</td>
			<td>FD20-250MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate&#8211;dextran (wt 70,000)</td>
			<td>Millipore Sigma</td>
			<td>FD70S-250MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorview FV3000 Confocal Microscope</td>
			<td>Olympus</td>
			<td>FV3000</td>
			<td></td>
		</tr>
		<tr>
			<td>Gas Tank (95% N2, 5% CO2)</td>
			<td>Airgas</td>
			<td>X02NI95C2003071</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS (No calcium, No magnesium, no phenol red)</td>
			<td>Thermofisher</td>
			<td>14025092</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342, Trihydrochloride, Trihydrate - 10 mg/mL Solution in Water</td>
			<td>ThermoFisher</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Astrocytes</td>
			<td>Science Cell</td>
			<td>1800</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Brain Vascular Pericytes</td>
			<td>Science Cell</td>
			<td>1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypoxia Incubator Chamber</td>
			<td>STEMCELL Technologies</td>
			<td>27310</td>
			<td></td>
		</tr>
		<tr>
			<td>Image-iT Green Hypoxia Reagent</td>
			<td>ThermoFisher</td>
			<td>I14834</td>
			<td></td>
		</tr>
		<tr>
			<td>Pericyte Medium</td>
			<td>Science Cell</td>
			<td>1201</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary Human Brain Microvascular Endothelial Cells</td>
			<td>ACBRI 376</td>
			<td>Cell Systems</td>
			<td></td>
		</tr>
		<tr>
			<td>Rocking Platform Shaker, Double</td>
			<td>VWR</td>
			<td>10860-658</td>
			<td></td>
		</tr>
		<tr>
			<td>Single Flow Meter</td>
			<td>STEMCELL Technologies</td>
			<td>27311</td>
			<td></td>
		</tr>
		<tr>
			<td>SpectraMax iD3 Microplate Reader</td>
			<td>Molecular Devices</td>
			<td>75886-128</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Filter, 25 mm, 0.22 &#956;m, PVDF, Sterile</td>
			<td>NEST Scientific</td>
			<td>380121</td>
			<td></td>
		</tr>
		<tr>
			<td>TPP Mutli-well Plates (6 wells)</td>
			<td>MidSci</td>
			<td>TP92406</td>
			<td></td>
		</tr>
		<tr>
			<td>TPP Tissue Culture Flasks T-75 Flasks</td>
			<td>MidSci</td>
			<td>TP90075</td>
			<td>Flasks with activated surface for cell adhesion</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>ThermoFisher</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure Distilled Water</td>
			<td>Invitrogen (Life Technologies)</td>
			<td>10977-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Uno Stage Top Incubator-</td>
			<td>Oko Lab</td>
			<td>UNO-T-H-CO2-TTL</td>
			<td></td>
		</tr>
	</tbody>
</table>

a triple primary cell culture model of the human blood-brain barrier for studying ischemic stroke in vitro

缺血性中风是全世界死亡和残疾的主要原因，治疗选择有限。缺血性中风的神经病理学特征是大脑血液供应中断，导致细胞死亡和认知功能障碍。在缺血性卒中期间和之后，血脑屏障（BBB）功能障碍会促进损伤进展，并导致患者恢复不良。目前的BBB模型主要包括内皮单培养和与星形胶质细胞或周细胞的双重共培养。
这种模型缺乏完全模仿动态大脑微环境的能力，这对于细胞间通信至关重要。此外，常用的BBB模型通常包含永生化的人内皮细胞或动物源性（啮齿动物，猪或牛）细胞培养物，这些细胞培养物具有翻译局限性。本文描述了一种基于插入良好的新型BBB模型，该模型仅包含原代人细胞（脑微血管内皮细胞，星形胶质细胞和脑血管周细胞），能够在 体外研究缺血性脑损伤。 
通过被动通透性、经内皮电阻（TEER）测量和缺氧细胞直接可视化评估氧-葡萄糖剥夺（OGD）对屏障完整性的影响。所提出的方案在 模拟体内BBB的细胞间环境方面具有明显的优势，可作为更现实的 体外 BBB模型，用于在缺血性脑损伤的情况下开发新的治疗策略。

为了研究星形胶质细胞和周细胞对HBMEC屏障功能的影响，我们在细胞培养插入物上构建了三重细胞培养BBB模型（图1A），以及HBMEC单培养和两个双共培养模型作为对照（图1B）。双重共培养对照包括HBMEC与HA的非接触共培养以及HBMEC与HBVP的接触共培养。共培养6天后，所有实验装置均接受OGD4小时。通过在OGD前后以及复氧24小时后测定TEER，评估所述BBB配置中?...

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A Triple Primary Cell Culture Model of the Human Blood-Brain Barrier for Studying Ischemic Stroke In Vitro

在这里，我们描述了基于原代人脑微血管内皮细胞，星形胶质细胞和周细胞建立血脑屏障三重细胞培养模型的方法。该多细胞模型适用于 体外缺 血性卒中期间神经血管单元功能障碍的研究或候选药物的筛选。

用于体外研究缺血性中风的人血脑屏障的三重原代细胞培养模型

Ischemic stroke is a major cause of death and disability worldwide with limited therapeutic options. The neuropathology of ischemic stroke is ...

我们的方案认识到需要在体外更好地概括人类血脑屏障，以研究影响中风后恢复的复杂细胞机制。我们的三重模型表现出强大的完整性，这部分与油井插入物的大隔室有关，使我们能够降低变化频率并避免种群中断。我们的模型是研究缺血性中风期间和之后血脑屏障发生的分子和细胞变化的绝佳工具。我们的方案允许发现新的靶点和治疗方法，以改善中风后的恢复。目前缺血性卒中的治疗方法对时间敏感，因此并不总是有效的。首先在T75培养瓶中培养HBVP，该培养瓶具有活化表面，用于细胞粘附在5%二氧化碳培养箱中，温度为37摄氏度，直至汇合。一旦达到汇合，吸出旧的周细胞培养基，并用五毫升温暖的Dulbecco的PBS洗涤细胞。吸出Dulbecco的PBS，并使用四毫升温热的胰蛋白酶EDTA溶液和一毫升Dulbecco的PBS的组合将细胞从烧瓶中分离出来。将烧瓶在 37 摄氏度下在二氧化碳培养箱中孵育五分钟。在显微镜下观察以确认细胞是否与烧瓶分离。向烧瓶中加入5毫升含有2%FBS的温热周细胞培养基，并将分离的细胞转移到50毫升离心管中。以200g离心细胞悬液三分钟，使细胞在管底部形成沉淀。从管中吸出培养基，确保细胞沉淀保持完整。将细胞沉淀重新悬浮在周细胞培养基中。根据细胞的汇合度和所需的孔插入物数量计算培养基量。取10微升重新悬浮的细胞，将它们放入细胞计数载玻片中，并计数细胞数。确定细胞密度，并在一毫升周细胞培养基中每个插入物接种300，000个细胞到孔插入物的光位侧。如文本手稿中所述，使用含有2%FBS的星形胶质细胞培养原代人星形胶质细胞。测定细胞密度并将每孔接种300， 000个细胞到组织培养六孔板的底部。盖上板以防止蒸发并孵育过夜。同样，如文本手稿中所述，使用含有10%FBS的完整经典培养基在组织培养皿中培养HBMEC。从培养箱中取出含有星形胶质细胞的组织培养六孔板和含有周细胞的孔插入物。从组织培养六孔板中吸出星形胶质细胞培养基，并向每个孔中加入一毫升周细胞培养基和一毫升星形胶质细胞培养基。从孔插入物中吸出周细胞培养基，并将其放入含有接种星形胶质细胞的组织培养六孔板中。在两毫升完整的经典培养基中以每孔300，000个细胞的密度接种HBMEC，到同一孔插入物的顶端侧。用Dulbecco的PBS清洗细胞三次。对于接受氧-葡萄糖剥夺的三重细胞培养物，向顶端和基底外侧区室添加无葡萄糖培养基。用新鲜培养基和常氧对照细胞培养物替换培养基。将对照三重培养物置于37摄氏度的5%二氧化碳培养箱中。将含有20毫升无菌水的培养皿放入缺氧培养箱室中，以提供足够的培养物加湿。通过松开环夹打开腔室，然后将细胞培养物排列在架子上。通过固定环夹密封腔室。打开腔室的入口和出口，并从腔室流量计的顶部连接管道。通过空气过滤器将管道从流量计底部连接到气罐。逆时针转动罐体流量控制阀以允许最小的气体流量。顺时针转动，缓慢打开调压阀。用气体混合物以每分钟 20 升的流速冲洗腔室五分钟。断开腔室与气源的连接，并牢固地关闭两个白色塑料夹。顺时针转动关闭储罐流量控制阀。逆时针转动关闭压力调节阀。将缺氧室置于37摄氏度的常规培养箱中四个小时。将灭菌的TEER仪器放入生物安全柜中，并将电极插入上皮伏特欧姆表。在 30 毫升 70% 异丙醇溶液中对电极进行至少 30 分钟的灭菌。打开TEER仪器并将功能设置为欧姆。从 70% 异丙醇溶液中取出电极，并将它们放入 20 毫升 Dulbecco 的 PBS 中至少 30 分钟，直到 TEER 设备上的数字读数读数为零欧姆。将电极的长插脚插入空白井插入控件的井插入机库中的三个开口之一，将其降低，直到它接触孔的底部。确保短插脚位于孔插入物底部的顶端培养物上方。等到 TEER 仪器上的数字读数值水平关闭后再记录该值。将电极放回Dulbecco的PBS中，以便在测量之间清洗它们。继续收集另外两个空白孔插入对照的所有TEER测量值。如前所述收集样品板的TEER测量值。完成所有测量后，将电极放回70%异丙醇溶液中30分钟。然后将电极与TEER仪器断开连接并使其风干。计算 TEER 值。从基底外侧室吸出培养基，并在三重细胞培养血脑屏障模型中用两毫升酚红游离内皮细胞生长培养基替换它。用HBSS洗涤顶端隔室中的细胞两次。在顶端隔室中加入一毫升FITC葡聚糖溶液，并用铝箔覆盖板，然后将板放入37摄氏度的培养箱中，用5%二氧化碳放置一小时。从基底外侧隔室中取出 100 微升培养基，并将其转移到黑色 96 孔板中。使用酶标仪测量荧光，激发和发射波长分别设置为 480 和 530 纳米。在聚-D-赖氨酸包被的35毫米玻璃底培养皿的中心接种200微升HBMEC，原代星形胶质细胞和HBVP，密度为每培养皿150， 000个细胞。在播种HBMEC之前，在培养皿的底部涂上附着因子。让细胞附着在玻璃表面上，将它们在二氧化碳培养箱中在 37 摄氏度下放置过夜。一旦达到汇合，丢弃培养基并加入两毫升预热的无葡萄糖培养基用于氧-葡萄糖剥夺或正常培养基用于对照。氧-葡萄糖剥夺处理后，在所有培养皿中用成像优化培养基替换培养基，然后按照文本手稿中所述使用GFP滤光片和顶级共聚焦显微镜培养箱进行荧光活细胞成像。通过测定氧-葡萄糖剥夺前后以及再氧合 24 小时后的 TEER，评估所示血脑屏障模型配置中内皮单层的屏障完整性。氧-葡萄糖剥夺持续4小时，仅在HBMEC单一栽培以及与HBMEC和HBVP共培养模型中导致TEER值显着降低。这些降低的水平在重新氧合24小时后达到基线水平。氧-葡萄糖剥夺后，三重共培养模型中的TEER值显著高于双共培养对照或单培养对照。与常氧对照相比，内皮单层对20千道尔顿分子量FITC dextran的细胞旁通透性在HBMEC单培养中急剧增加，在与HBMEC和HBVP共培养模型中的渗透性较小。此外，在常氧和氧-葡萄糖剥夺条件下，20千道尔顿FITC葡聚糖通透性水平在所有模型中是所有模型中最低的。在任何模型中，在内皮单层的70千道尔顿FITC葡聚糖通量中均未观察到变化。暴露于氧 - 葡萄糖剥夺的所有主要人类类型都表现出强烈的绿色荧光，证明成像的活细胞是缺氧的。将人脑血管周细胞接种在孔插入物的光位侧后，用翻转板覆盖它们以防止蒸发非常重要。在我们的模型中，我们使用相对较小的0.4微米孔径的聚酯孔插入物，使其适用于筛选任何需要穿过血脑屏障进行治疗的疾病的候选药物。如果需要，该技术为可视化蛋白质和转运蛋白的修饰提供了绝佳的机会。

Research

JoVE Journal

Biochemistry

A Triple Primary Cell Culture Model of the Human Blood-Brain Barrier for Studying Ischemic Stroke In Vitro

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

热稳定，表达，纯化，和人类血清素转运蛋白结晶绑定到<em&gt;取值</em&gt; -citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

科研

生物化学

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

便于操作的体系结构基于蛋白质的凝胶中为单元格文化应用程序

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

A Colorimetric Method for Measuring Iron Content in Plants

测定植物铁含量的比色法

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

利用商用仪器和自由开源亮度分析软件对蛋白质的聚齐聚进行无标定的体外定量化

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

哺乳动物细胞周期内蛋白质激酶 rna 激活的 rna 相互作用研究

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral ...

A Strategy to Identify Compounds that Affect Cell Growth and Survival in Cultured Mammalian Cells at Low-to-Moderate Throughput

识别影响培养哺乳动物细胞在低到中度通量下细胞生长和生存的化合物的策略

Cytotoxicity is a critical parameter that needs to be quantified when studying drugs that may have therapeutic benefits. Because of this, many drug ...

A Semi-Quantitative Drug Affinity Responsive Target Stability (DARTS) assay for studying Rapamycin/mTOR interaction

A Semi-Quantitative Drug Affinity Responsive Target Stability DARTS assay for studying Rapamycin/mTOR interaction

用于研究拉帕霉素/mTOR相互作用的半定量药物亲和反应目标稳定性 （DARTS） 测定

Drug Affinity Responsive Target Stability (DARTS) is a robust method for detection of novel small molecule protein targets. It can be used to verify known ...

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription

生物层干涉测量(BLI)在转录中研究蛋白质-蛋白质相互作用的应用

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA ...