Name: Autor im Rampenlicht: Anwendungen der TEER-Detektion zur Beurteilung der Integrität der Zellbarriere
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

Wir freuen uns über die finanzielle Unterstützung der National Natural Science Foundation of China (82274207 und 82104533), des Key Research and Development Program of Ningxia (2023BEG02012) und des Xinglin Scholar Research Promotion Project der Chengdu University of TCM (XKTD2022013).

ANMERKUNG: Aus dem Gehirn von Mäusen gewonnene Endothelzellen.3 (bEnd.3) wurden in die Kammern einer 24-Well-Platte inokuliert, um ein einfaches In-vitro-Modell der BHS unter bestimmten Mediumbedingungen zu konstruieren. Die TEER von normalen Zellen und hypoxischen Zellen wurden mit einem TEER-Messgerät gemessen (Abbildung 1 und Abbildung 2).
1. Vorbereitung der Lösung
<ol><li>Bereiten Sie das DMEM-Zellk...

Messung des transendothelialen Widerstands zur Beurteilung der Integrität der Zellbarriere

<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>

Das Gehirn ist eines der am weitesten entwickelten Körperorgane und steuert eine Vielzahl komplizierter physiologischer Prozesse, darunter Gedächtnis, Kognition, Hören, Riechen und Bewegung27. Das Gehirn ist eines der kompliziertesten und zugleich am meisten erkrankten Organe des menschlichen Körpers. Das Auftreten vieler Erkrankungen des Zentralnervensystems zeigt eine wachsende Tendenz von Jahr zu Jahr, was auf Faktoren wie Luftverschmutzung, unregelmäßiges Essverhalten und andere Faktoren...

BHS ist eine einzigartige biologische Schnittstelle zwischen Blutkreislauf und Nervengewebe, die aus vaskulären Endothelzellen, Perizyten, Astrozyten, Neuronen und anderen zellulären Strukturen besteht1. Der Fluss von Ionen, Chemikalien und Zellen zwischen Blut und Gehirn wird durch diese Barriere streng reguliert. Diese Homöostase schützt das Nervengewebe vor Toxinen und Krankheitserregern und ermöglicht gleichzeitig die entsprechende Funktion der Nerven des Gehirns 2,3. Die Aufrechterhaltung der Integrität der BHS kann die Entwicklung und das Fortschreiten von Störungen, die das zentrale Nervensystem betreffen, wie z. B. neuronale Dysfunktion, Ödeme und Neuroinflammation, wirksam verhindern4. Die einzigartigen physiologischen Eigenschaften der BHS verhindern jedoch, dass mehr als 98 % der niedermolekularen Medikamente und 100 % der makromolekularen Arzneimittel in das zentrale Nervensystem gelangen5. Daher ist es für die Erzielung der therapeutischen Wirksamkeit unerlässlich, die Penetration von Medikamenten durch die BHS während der Entwicklung von Medikamenten für das zentrale Nervensystem zu erhöhen 6,7. Auch wenn das Computersimulations-Screening von Substraten die Wahrscheinlichkeit, dass Wirkstoffkandidaten die BHS überschreiten, erheblich erhöht hat, werden immer noch zuverlässige und erschwingliche In-vitro-/In-vivo-BHS-Modelle benötigt, um den Anforderungen der wissenschaftlichen Forschung gerecht zu werden8.
Eine schnelle und kostengünstige Technik für das Hochdurchsatz-Wirkstoffscreening ist das In-vitro-Modell 9. Um die grundlegenden Prozesse der Auswirkungen von Medikamenten auf die BHS-Funktion und ihre Rolle bei der Entstehung und dem Fortschreiten von Krankheiten zu beleuchten, wurde eine Reihe vereinfachter In-vitro-BHS-Modelle erstellt. Gegenwärtig sind die üblichen In-vitro-BHS-Modelle die Monolayer-, Co-Kultur-, dynamischen und mikrofluidischen Modelle 10,11,12, die aus vaskulären Endothelzellen und Astrozyten, Perizyten oder Mikroglia 13,14 aufgebaut sind. Obwohl 3D-Zellkulturen eher der physiologischen Struktur von BHS15 entsprechen, ist ihre Anwendung als Mittel zum Wirkstoffscreening auf BHS immer noch durch ihr kompliziertes Design und ihre unterdurchschnittliche Reproduzierbarkeit eingeschränkt. Im Gegensatz dazu wird das Monolayer-In-vitro-Modell am häufigsten zur Erforschung der BHS verwendet und ist für die Bestimmung der Expression von Membrantransportern und Tight-Junction-Proteinen in bestimmten Zellen geeignet.
Die Messung des elektrischen Transmembranwiderstands (TEER) ist eine Technik zur Bewertung und Überwachung der Zellschicht über dem Widerstand und zur Bewertung der Zellintegrität und Durchlässigkeit der Barriere. Durch gleichzeitiges Einführen von zwei Elektroden in das Wachstumsmedium oder die Pufferlösung auf beiden Seiten der Monoschicht ist es möglich, den Wechselstrom oder die elektrische Impedanz durch die kompakte Schicht16,17 der Zelle zu messen. Um festzustellen, ob das In-vitro-BHS-Modell ordnungsgemäß erstellt wurde, wird in der Regel die Messung der TEER als Goldstandard verwendet18. Auf der anderen Seite kann der Trend der medikamentösen Wirkung auf die BHS-Permeabilität genau vorhergesagt werden, indem die Änderung des elektrischen Widerstands der Zellschicht nach Arzneimittelbeteiligung gemessen wird19. Zum Beispiel entdeckten Feng et al., dass Catalpol (das primäre aktive Monomer von Rehmanniae) die Lipopolysaccharid-induzierte Herunterregulierung von Tight-Junction-Proteinen in der BHS effektiv umkehren und den TEER-Wert der Endothelzellschicht des Mäusegehirns20 erhöhen kann.
Die neuroinflammatorische Reaktion ist in der Regel die Hauptursache für ein Ungleichgewicht der BHS-Homöostase21. Die hypoxische Behandlung zur Induktion neuroinflammatorischer Schäden ist die Hauptmethode zur Zerstörung der Blut-Hirn-Schranke, hauptsächlich mit physikalischen Methoden und chemischen Reagenzien. Ersteres verwendet in erster Linie einen Drei-Gas-Inkubator, um den Sauerstoffgehalt in der Zellwachstumsumgebung zu variieren, um hypoxische Bedingungen zu simulieren22, während letzteres durch künstliches Einbringen von Desoxyreagenzien wie CoCl2in das Zellkulturmedium23 erreicht wird. Die Zellen bleiben in einem sauerstoffarmen Zustand, wenn Fe2+ durch Co2+ im Häm ersetzt wird. Wenn Fe2+ in der katalytischen Gruppe durch Co2+ ersetzt wird, wird die Aktivität der Prolinhydroxylase und der Aspartathydroxylase gehemmt, was zu einer Akkumulation des Hypoxie-induzierbaren Faktors-1α (HIF-1α)24 führt. Bei anhaltender Hypoxie löst die Dephosphorylierung von HIF-1α im Zytoplasma den Zelltod aus und aktiviert den vaskulären endothelialen Wachstumsfaktor, der letztendlich die Gefäßpermeabilität erhöht. In früheren Studien 25,26 wurde gut gezeigt, dass Hypoxie die Expression von endothelialen Tight-Junction-Proteinen signifikant reduzieren kann, um die Permeabilität der BHS zu erhöhen. In dieser Studie wurde die Zeit-Widerstands-Kurve von bEnd.3-Zellen gemessen, die in 24-Well-Platten ausgesät wurden, um ein einfaches BHS-Modell zu erstellen. Mit Hilfe dieses Modells haben wir die Veränderungen der Zell-TEER nach CoCl2-Intervention charakterisiert, um ein Zellmodell zu konstruieren, das für das Screening von Medikamenten auf BHS-Schutz verwendet werden kann.

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		<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

Die Blut-Hirn-Schranke (BHS) ist eine dynamische physiologische Struktur, die aus mikrovaskulären Endothelzellen, Astrozyten und Perizyten besteht. Durch die Koordination der Wechselwirkung zwischen dem eingeschränkten Transit von Schadstoffen, der Nährstoffaufnahme und der Metaboliten-Clearance im Gehirn ist die BHS für die Aufrechterhaltung der Homöostase des Zentralnervensystems unerlässlich. Die Erstellung von In-vitro-Modellen der BHS ist ein wertvolles Werkzeug zur Erforschung der Pathophysiologie neurologischer Erkrankungen und zur Entwicklung pharmakologischer Behandlungen. Diese Studie beschreibt ein Verfahren zur Erstellung eines in vitro Monolayer-BHS-Zellmodells durch Aussaat von bEnd.3-Zellen in die obere Kammer einer 24-Well-Platte. Um die Integrität der Zellbarrierefunktion zu beurteilen, wurde das konventionelle Epithelzellvoltmeter verwendet, um den transmembranellen elektrischen Widerstand von normalen Zellen und CoCl 2-induzierten hypoxischen Zellen in Echtzeit aufzuzeichnen. Wir gehen davon aus, dass die oben genannten Experimente effektive Ideen für die Erstellung von In-vitro-Modellen für BHS und Medikamente zur Behandlung von Erkrankungen des zentralen Nervensystems liefern werden.

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.

Autor im Rampenlicht: Anwendungen der TEER-Detektion zur Beurteilung der Integrität der Zellbarriere 

Aufzeichnung der funktionellen Barriereintegrität auf bEnd.3 vaskulären Endothelzellen mittels transendothelialer elektrischer Widerstandsdetektion

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.

Dieses Protokoll ermöglichte die Aufzeichnung von Änderungen der Widerstandswerte von Zellen gemäß den im transendothelialen Widerstandsmessgerät eingestellten Parametern. Die Viabilität von bEnd.3-Zellen (Anzahl der lebenden Zellen), die mit unterschiedlichen Konzentrationen von CoCl2 behandelt wurden, wurde mittels CCK-8-Assay untersucht. Eine größere Zellschädigung durch CoCl2 wurde durch eine geringere Zelllebensfähigkeit repräsentiert. Wir fanden heraus, dass 300 μM CoCl2 in vit...

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

Dieses Protokoll beschreibt ein zuverlässiges und effizientes In-vitro-Modell der Blutschranke des Gehirns. Die Methode verwendet zerebrale vaskuläre Endothelzellen der Maus bEnd.3 und misst den elektrischen Widerstand der Transmembran.

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

Aktuelle Studien verwenden in der Regel Permeabilitätstests und die damit verbundene Proteinexpression, um die Genauigkeit des TEER-Nachweises zu überprüfen. Das BHS-Modell von Monolayer-Zellen ist für das Wirkstoff-Screening weniger effektiv, und die Verwendung von mehr Zellen erschwert die Konstruktion des Modells. Das Protokoll dieser Studie ist relativ einfach, da das nicht-invasive Verfahren es ermöglicht, die Zellproben nach dem TEER-Nachweis in anderen Experimenten zu verwenden.Der Nachweis von TEER zur Beurteilung der Permeabilität von Zellmodellen kann das Screening von Medikamenten zur Behandlung und Prävention des zentralen Nervensystems beschleunigen, um die Erfolgsrate der Entwicklung neuer Medikamente zu erhöhen. In Zukunft werden wir die Expression des Tight-Junction-Proteins in Zellen des oberen Kompartiments der Brustwand nachweisen, um zu überprüfen, ob TEER-Testergebnisse die Integrität der BHS-Barrierefunktion effektiv darstellen können.

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.

Forschung

Neurowissenschaften

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.

Kultur von bEnd.3-Zellen und Zellviabilität nach CoCl2-Behandlung

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.

In-vitro-Blut-Hirn-Schranken-Modellaufbau zur Messung von TEER

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.