Name: Pleins feux sur l’auteur : Applications de la détection TEER pour évaluer l’intégrité de la barrière cellulaire
Uploaded: 2023-09-29T13:40:00
Description: Watch this Scientific Journal Video about Applications of TEER Detection to Assess Cell Barrier Integrity at JoVE.com

Nous apprécions le soutien financier de la Fondation nationale des sciences naturelles de Chine (82274207 et 82104533), du programme clé de recherche et de développement du Ningxia (2023BEG02012) et du projet de promotion de la recherche Xinglin Scholar de l’Université de Chengdu de MTC (XKTD2022013).

NOTE : Des cellules endothéliales dérivées du cerveau de souris.3 (bEnd.3) ont été inoculées dans les chambres d’une plaque à 24 puits pour construire un modèle in vitro simple de la BHE dans des conditions de milieu spécifiques. Les TEER des cellules normales et des cellules hypoxiques ont été mesurés à l’aide d’un TEER (Figure 1 et Figure 2).
1. Préparation de la solution
<ol><li>Prép...

Mesure de la résistance transendothéliale pour évaluer l’intégrité de la barrière cellulaire

<ol>
	<li>Kim, 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=CLEC14A+deficiency+exacerbates+neuronal+loss+by+increasing+blood-brain+barrier+permeability+and+inflammation.">CLEC14A deficiency exacerbates neuronal loss by increasing blood-brain barrier permeability and inflammation.</a> J Neuroinflammation. 17 (1), 48 (2020).</li><li>Bagchi, 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=In-vitro+blood-brain+barrier+models+for+drug+screening+and+permeation+studies:+an+overview.">In-vitro blood-brain barrier models for drug screening and permeation studies: an overview.</a> Drug Des Devel Ther. 13, 3591-3605 (2019).</li><li>Daneman, R., Prat, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blood-brain+barrier.">The blood-brain barrier.</a> Cold Spring Harb perspect Biol. 7 (1), 020412 (2015).</li><li>Profaci, C. P., Munji, R. N., Pulido, R. S., Daneman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blood-brain+barrier+in+health+and+disease:+Important+unanswered+questions.">The blood-brain barrier in health and disease: Important unanswered questions.</a> J Exp Med. 217 (4), 20190062 (2020).</li><li>Pardridge, W. M. <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+delivery.">Blood-brain barrier delivery.</a> Drug Discov Today. 12 (1-2), 54-56 (2007).</li><li>Eigenmann, D. 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=Comparative+study+of+four+immortalized+human+brain+capillary+endothelial+cell+lines,+hCMEC/D3,+hBMEC,+TY10,+and+BB19,+and+optimization+of+culture+conditions,+for+an+in+vitro+blood-brain+barrier+model+for+drug+permeability+studies.">Comparative study of four immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, and optimization of culture conditions, for an in vitro blood-brain barrier model for drug permeability studies.</a> Fluids Barriers CNS. 10 (1), 33 (2013).</li><li>Gajd&#225;cs, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+concept+of+an+ideal+antibiotic:+Implications+for+drug+design.">The concept of an ideal antibiotic: Implications for drug design.</a> Molecule. 24 (5), 892 (2019).</li><li>Stanimirovic, D. B., Bani-Yaghoub, M., Perkins, M., Haqqani, A. S. <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+models:+in+vitro+to+in+vivo+translation+in+preclinical+development+of+CNS-targeting+biotherapeutics.">Blood-brain barrier models: in vitro to in vivo translation in preclinical development of CNS-targeting biotherapeutics.</a> Expert Opin Drug Discov. 10 (2), 141-155 (2015).</li><li>Helms, H. 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=In+vitro+models+of+the+blood-brain+barrier:+An+overview+of+commonly+used+brain+endothelial+cell+culture+models+and+guidelines+for+their+use.">In vitro models of the blood-brain barrier: An overview of commonly used brain endothelial cell culture models and guidelines for their use.</a> J Cereb Blood Flow Metab. 36 (5), 862-890 (2016).</li><li>&#214;zyurt, M. G., Bayir, E., Do&#286;an, &. #. 3. 5. 0. ;., &#214;zt&#220;rk, &. #. 3. 5. 0. ;., &#350;endem&#304;r, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coculture+model+of+blood-brain+barrier+on+electrospun+nanofibers.">Coculture model of blood-brain barrier on electrospun nanofibers.</a> Turk J Biol. 44 (4), 121-132 (2020).</li><li>Kim, W., 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=Functional+validation+of+the+simplified+in+vitro+3D+Co-culture+based+BBB+model.">Functional validation of the simplified in vitro 3D Co-culture based BBB model.</a> Biochem Biophys Res Commun. 625, 128-133 (2022).</li><li>Aazmi, A., 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=Vascularizing+the+brain+in+vitro.">Vascularizing the brain in vitro.</a> iScience. 25 (4), 104110 (2022).</li><li>Burkhart, A., 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=Transfection+of+brain+capillary+endothelial+cells+in+primary+culture+with+defined+blood-brain+barrier+properties.">Transfection of brain capillary endothelial cells in primary culture with defined blood-brain barrier properties.</a> Fluids Barriers CNS. 12, 19 (2015).</li><li>Campisi, M., 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=3D+self-organized+microvascular+model+of+the+human+blood-brain+barrier+with+endothelial+cells,+pericytes+and+astrocytes.">3D self-organized microvascular model of the human blood-brain barrier with endothelial cells, pericytes and astrocytes.</a> Biomaterials. 180, 117-129 (2018).</li><li>Peng, 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=Neuroinflammatory+in+vitro+cell+culture+models+and+the+potential+applications+for+neurological+disorders.">Neuroinflammatory in vitro cell culture models and the potential applications for neurological disorders.</a> Front Pharmacol. 12, 671734 (2021).</li><li>Secker, P. F., Schlichenmaier, N., Beilmann, M., Deschl, U., Dietrich, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+transepithelial+transport+measurements+to+detect+nephrotoxicity+in+vitro+using+the+RPTEC/TERT1+cell+line.">Functional transepithelial transport measurements to detect nephrotoxicity in vitro using the RPTEC/TERT1 cell line.</a> Arch Toxicol. 93 (7), 1965-1978 (2019).</li><li>Nazari, H., 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=Advances+in+TEER+measurements+of+biological+barriers+in+microphysiological+systems.">Advances in TEER measurements of biological barriers in microphysiological systems.</a> Biosens Bioelectron. 234, 115355 (2023).</li><li>Nicolas, A., 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=High+throughput+transepithelial+electrical+resistance+(TEER)+measurements+on+perfused+membrane-free+epithelia.">High throughput transepithelial electrical resistance (TEER) measurements on perfused membrane-free epithelia.</a> Lab Chip. 21 (9), 1676-1685 (2021).</li><li>Yang, Z., 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=Autophagy+alleviates+hypoxia-induced+blood-brain+barrier+injury+via+regulation+of+CLDN5+(claudin+5).">Autophagy alleviates hypoxia-induced blood-brain barrier injury via regulation of CLDN5 (claudin 5).</a> Autophagy. 17 (10), 3048-3067 (2021).</li><li>Feng, 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=RhoA/ROCK-2+pathway+inhibition+and+tight+junction+protein+upregulation+by+catalpol+suppresses+lipopolysaccaride-induced+disruption+of+blood-brain+barrier+permeability.">RhoA/ROCK-2 pathway inhibition and tight junction protein upregulation by catalpol suppresses lipopolysaccaride-induced disruption of blood-brain barrier permeability.</a> Molecules. 23 (9), (2018).</li><li>Sulhan, S., Lyon, K. A., Shapiro, L. A., Huang, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroinflammation+and+blood-brain+barrier+disruption+following+traumatic+brain+injury:+Pathophysiology+and+potential+therapeutic+targets.">Neuroinflammation and blood-brain barrier disruption following traumatic brain injury: Pathophysiology and potential therapeutic targets.</a> J Neurosci Res. 98 (1), 19-28 (2020).</li><li>Liu, B., 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=Notoginsenoside+R1+intervenes+degradation+and+redistribution+of+tight+junctions+to+ameliorate+blood-brain+barrier+permeability+by+Caveolin-1/MMP2/9+pathway+after+acute+ischemic+stroke.">Notoginsenoside R1 intervenes degradation and redistribution of tight junctions to ameliorate blood-brain barrier permeability by Caveolin-1/MMP2/9 pathway after acute ischemic stroke.</a> Phytomedicine. 90, 153660 (2021).</li><li>Hou, 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=Salidroside+intensifies+mitochondrial+function+of+CoCl2-damaged+HT22+cells+by+stimulating+PI3K-AKT-MAPK+signaling+pathway.">Salidroside intensifies mitochondrial function of CoCl2-damaged HT22 cells by stimulating PI3K-AKT-MAPK signaling pathway.</a> Phytomedicine. 109, 154568 (2023).</li><li>Mu&#241;oz-S&#225;nchez, J., Ch&#225;nez-C&#225;rdenas, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+cobalt+chloride+as+a+chemical+hypoxia+model.">The use of cobalt chloride as a chemical hypoxia model.</a> J Appl Toxicol. 39 (4), 556-570 (2019).</li><li>Jiang, 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=Salidroside+attenuates+high+altitude+hypobaric+hypoxia-induced+brain+injury+in+mice+via+inhibiting+NF-&#954;B/NLRP3+pathway.">Salidroside attenuates high altitude hypobaric hypoxia-induced brain injury in mice via inhibiting NF-&#954;B/NLRP3 pathway.</a> Eur J Pharmacol. 925, 175015 (2022).</li><li>Xie, N., 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=Rhodiola+crenulate+alleviates+hypobaric+hypoxia-induced+brain+injury+via+adjusting+NF-&#954;B/NLRP3-mediated+inflammation.">Rhodiola crenulate alleviates hypobaric hypoxia-induced brain injury via adjusting NF-&#954;B/NLRP3-mediated inflammation.</a> Phytomedicine. 103, 154240 (2022).</li><li>Thiebaut de Schotten, M., Forkel, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emergent+properties+of+the+connected+brain.">The emergent properties of the connected brain.</a> Science. 378 (6619), 505-510 (2022).</li><li>Tu, W. J., 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=Estimated+burden+of+stroke+in+China+in+2020.">Estimated burden of stroke in China in 2020.</a> JAMA Netw Open. 6 (3), 231455 (2023).</li><li>Alzheimers Dement. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+disease+facts+and+figures.">Alzheimer's disease facts and figures.</a> Alzheimers Dement. 17 (3), 327-406 (2021).</li><li>Wang, 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=Neutrophil+extracellular+traps+promote+tPA-induced+brain+hemorrhage+via+cGAS+in+mice+with+stroke.">Neutrophil extracellular traps promote tPA-induced brain hemorrhage via cGAS in mice with stroke.</a> Blood. 138 (1), 91-103 (2021).</li><li>Liu, X. 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>

L’un des organes corporels les plus développés, le cerveau contrôle un large éventail de processus physiologiques complexes, notamment la mémoire, la cognition, l’ouïe, l’odorat etle mouvement. Le cerveau est à la fois l’un des organes les plus compliqués et les plus malades du corps humain. L’apparition de nombreux troubles du système nerveux central montre une tendance croissante d’année en année en raison de facteurs tels que la pollution de l’air, les habitudes aliment...

La BHE est une interface biologique unique entre la circulation sanguine et le tissu nerveux, qui est composée de cellules endothéliales vasculaires, de péricytes, d’astrocytes, de neurones et d’autres structures cellulaires1. Le flux d’ions, de produits chimiques et de cellules entre le sang et le cerveau est strictement régulé par cette barrière. Cette homéostasie protège les tissus nerveux contre les toxines et les agents pathogènes tout en permettant le bon fonctionnement des nerfs du cerveau 2,3. Le maintien de l’intégrité de la BHE peut prévenir efficacement le développement et la progression de troubles affectant le système nerveux central, tels que le dysfonctionnement neuronal, l’œdème et la neuroinflammation4. Cependant, les propriétés physiologiques uniques de la BHE empêchent plus de 98 % des médicaments à petites molécules et 100 % des produits pharmaceutiques macromoléculaires de pénétrer dans le système nerveux central5. Par conséquent, il est essentiel d’augmenter la pénétration des médicaments à travers la BHE pendant le développement des médicaments pour le système nerveux central afin d’atteindre l’efficacité thérapeutique 6,7. Même si le criblage des substrats par simulation informatique a considérablement augmenté la probabilité que les candidats-médicaments franchissent la BHE, des modèles de BHE in vitro/in vivo fiables et abordables sont encore nécessaires pour répondre aux besoins de la recherche scientifique8.
Une technique rapide et abordable pour le criblage de médicaments à haut débit est le modèle9 in vitro. Pour faire la lumière sur les processus fondamentaux des effets des médicaments sur la fonction de la BHE et leur rôle dans le développement et la progression de la maladie, une série de modèles de BHE in vitro simplifiés a été créée. À l’heure actuelle, les modèles de BHE in vitro courants sont les modèles monocouches, de coculture, dynamiques et microfluidiques 10,11,12, construits par des cellules endothéliales vasculaires et des astrocytes, des péricytes ou des microglies 13,14. Bien que les cultures cellulaires 3D soient plus conformes à la structure physiologique de la BHE15, leur application en tant que moyen de criblage de médicaments pour la BHE est encore limitée par leur conception complexe et leur reproductibilité médiocre. En revanche, le modèle in vitro monocouche est celui qui est le plus fréquemment utilisé pour la recherche sur la BHE et est applicable pour déterminer l’expression des transporteurs membranaires et des protéines à jonction serrée dans des cellules particulières.
La mesure de la résistance électrique transmembranaire (TEER) est une technique permettant d’évaluer et de surveiller la couche de cellules à travers la résistance et d’évaluer l’intégrité cellulaire et la perméabilité de la barrière. En insérant simultanément deux électrodes dans le milieu de culture ou la solution tampon de part et d’autre de la monocouche, il est possible de mesurer le courant alternatif ou l’impédance électrique à travers la couche compacte de la cellule16,17. Afin de déterminer si le modèle de la BHE in vitro a été correctement créé, la mesure de l’ETER sera généralement utilisée comme étalon-or18. D’autre part, la tendance de l’action des médicaments sur la perméabilité de la BHE peut être prédite avec précision en mesurant le changement de résistance électrique de la couche cellulaire après l’implication du médicament19. Par exemple, Feng et al. ont découvert que le catalpol (le principal monomère actif de rehmanniae) pouvait effectivement inverser la régulation négative induite par les lipopolysaccharides des protéines à jonction serrée dans la BHE et augmenter la valeur TEER de la couche20 de cellules endothéliales du cerveau de souris.
La réponse neuro-inflammatoire est généralement la principale cause du déséquilibre de l’homéostasie de la BHE21. Le traitement hypoxique pour induire des lésions neuro-inflammatoires est la principale méthode pour détruire la barrière hémato-encéphalique, comprenant principalement des méthodes physiques et des méthodes de réactifs chimiques. Le premier utilise principalement un incubateur à trois gaz pour faire varier la teneur en oxygène dans l’environnement de croissance cellulaire afin de simuler des conditions hypoxiques22, tandis que le second est obtenu en introduisant artificiellement des réactifs désoxy tels que CoCl2 dans le milieu de culture cellulaire23. Les cellules resteront dans un état désoxygéné si le Fe2+ est substitué au Co2+ dans l’hème. Si le Fe2+ est substitué au Co2+ dans le groupe catalytique, l’activité de la proline hydroxylase et de l’aspartate hydroxylase sera inhibée, ce qui entraînera une accumulation de facteur 1α inductible par l’hypoxie (HIF-1α)24. En cas d’hypoxie persistante, la déphosphorylation de HIF-1α dans le cytoplasme déclenche la mort cellulaire et active le facteur de croissance de l’endothélium vasculaire, ce qui augmente finalement la perméabilité vasculaire. Dans des études antérieures25,26, il a été bien démontré que l’hypoxie peut réduire considérablement l’expression des protéines de jonction serrée endothéliale pour augmenter la perméabilité de la BHE. Dans cette étude, la courbe de résistance temporelle des cellules bEnd.3 ensemencées dans des plaques à 24 puits a été mesurée afin de créer un modèle BHE simple. À l’aide de ce modèle, nous avons caractérisé les changements dans le TEER cellulaire après l’intervention de CoCl2 afin de construire un modèle cellulaire qui peut être utilisé pour cribler les médicaments pour la protection contre la BHE.

<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

La barrière hémato-encéphalique (BHE) est une structure physiologique dynamique composée de cellules endothéliales microvasculaires, d’astrocytes et de péricytes. En coordonnant l’interaction entre le transit restreint des substances nocives, l’absorption des nutriments et la clairance des métabolites dans le cerveau, la BHE est essentielle à la préservation de l’homéostasie du système nerveux central. La construction de modèles in vitro de la BHE est un outil précieux pour explorer la physiopathologie des troubles neurologiques et créer des traitements pharmacologiques. Cette étude décrit une procédure de création d’un modèle de cellules monocouches de BHE in vitro en ensemencant des cellules bEnd.3 dans la chambre supérieure d’une plaque à 24 puits. Pour évaluer l’intégrité de la fonction de barrière cellulaire, le voltmètre de cellules épithéliales conventionnel a été utilisé pour enregistrer en temps réel la résistance électrique transmembranaire des cellules normales et des cellules hypoxiques induites par la CoCl2. Nous prévoyons que les expériences ci-dessus fourniront des idées efficaces pour la création de modèles in vitro de BHE et de médicaments pour traiter les troubles des maladies du système nerveux central.

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.

Pleins feux sur l’auteur : Applications de la détection TEER pour évaluer l’intégrité de la barrière cellulaire 

Enregistrement de l’intégrité fonctionnelle de la barrière sur les cellules endothéliales vasculaires bEnd.3 via la détection de résistance électrique transendothéliale

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.

Ce protocole a permis d’enregistrer les changements dans les valeurs de résistance des cellules en fonction des paramètres définis dans le compteur de résistance transendothéliale. La viabilité des cellules bEnd.3 (nombre de cellules vivantes) traitées avec différentes concentrations de CoCl2 a été examinée par le test CCK-8. Des dommages cellulaires plus importants produits par CoCl2 étaient représentés par une viabilité cellulaire plus faible. Nous avons constaté que 300 μM de C...

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

Ce protocole décrit un modèle in vitro fiable et efficace de la barrière hémato-encéphalique. La méthode utilise des cellules endothéliales vasculaires cérébrales de souris bEnd.3 et mesure la résistance électrique transmembranaire.

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

Les études actuelles utilisent généralement des tests de perméabilité et l’expression des protéines associées pour vérifier l’exactitude de la détection TEER. Le modèle de la BHE des cellules monocouches en vertu est moins efficace pour le criblage de médicaments, et l’utilisation d’un plus grand nombre de cellules rend plus difficile la construction du modèle. Le protocole de cette étude est relativement simple, car la procédure non invasive permet d’utiliser les échantillons de cellules dans d’autres expériences après la détection TEER.La détection de TEER pour évaluer la perméabilité des modèles cellulaires peut accélérer le criblage de médicaments pour le traitement et la prévention du système nerveux central afin d’augmenter le taux de réussite du développement de nouveaux médicaments. À l’avenir, nous détecterons l’expression de la protéine de jonction serrée dans les cellules du compartiment supérieur de la paroi thoracique afin de vérifier que les résultats des tests TEER peuvent représenter efficacement l’intégrité de la fonction barrière de la BHE.

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

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.

Recherche

Neurosciences

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.

Culture de cellules bEnd.3 et viabilité cellulaire après traitement par CoCl2

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.

Assemblage in vitro d’un modèle de barrière hémato-encéphalique pour la mesure de l’ETER

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.