Name: Författare i fokus: Tillämpningar av TEER-detektion för att bedöma cellbarriärens integritet
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

Vi uppskattar det ekonomiska stödet från National Natural Science Foundation of China (82274207 och 82104533), Key Research and Development Program of Ningxia (2023BEG02012) och Xinglin Scholar Research Promotion Project vid Chengdu University of TCM (XKTD2022013).

OBS: Endotelceller från mushjärna.3 (bEnd.3) inokulerades i kamrarna på en 24-hålsplatta för att konstruera en enkel in vitro-modell av BBB under specifika mediumförhållanden. TEER för normala celler och hypoxiska celler mättes med TEER-mätare (figur 1 och figur 2).
1. Beredning av lösningen
<ol><li>Bered DMEM-cellodlingsmediet som innehåller FBS (10 %, v/v), 100 E/ml penicillin och 10 mg/ml str...

Mätning av transendotelresistens för att bedöma cellbarriärens integritet

<ol>
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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. 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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. 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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. 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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. 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Hjärnan är ett av de mest utvecklade kroppsorganen och styr ett brett spektrum av intrikata fysiologiska processer, inklusive minne, kognition, hörsel, luktoch rörelse. Hjärnan är ett av människokroppens mest komplicerade och sjuka organ på samma gång. Förekomsten av många sjukdomar i centrala nervsystemet visar en ökande tendens från år till år på grund av faktorer som luftföroreningar, oregelbundna matvanor och andra faktorer 27,28,29.<sup clas...

BBB är ett unikt biologiskt gränssnitt mellan blodcirkulation och nervvävnad, som består av vaskulära endotelceller, pericyter, astrocyter, neuroner och andra cellulära strukturer1. Flödet av joner, kemikalier och celler mellan blodet och hjärnan regleras strikt av denna barriär. Denna homeostas skyddar nervvävnaderna mot gifter och patogener samtidigt som den möjliggör lämplig drift av hjärnans nerver 2,3. Att upprätthålla BBB:s integritet kan effektivt förhindra utveckling och progression av sjukdomar som påverkar det centrala nervsystemet, såsom neuronal dysfunktion, ödem och neuroinflammation4. De unika fysiologiska egenskaperna hos BBB förhindrar dock att mer än 98 % av småmolekylära läkemedel och 100 % av makromolekylära läkemedel kommer in i det centrala nervsystemet5. Därför är det viktigt att öka penetrationen av läkemedel genom BBB under utvecklingen av läkemedel för det centrala nervsystemet för att uppnå terapeutisk effekt 6,7. Även om datorsimuleringsscreening av substrat avsevärt har ökat sannolikheten för att läkemedelskandidater passerar BBB, behövs fortfarande tillförlitliga och prisvärda in vitro/in vivo BBB-modeller för att möta behoven av vetenskaplig forskning8.
En snabb och prisvärd teknik för läkemedelsscreening med hög kapacitet är in vitro-modell 9. För att belysa de grundläggande processerna för läkemedels effekter på BBB-funktion och deras roll i utveckling och progression av sjukdom har en serie förenklade in vitro-modeller skapats. För närvarande är de vanliga in vitro BBB-modellerna monolager-, samkultur-, dynamiska och mikrofluidiska modeller 10,11,12, konstruerade av vaskulära endotelceller och astrocyter, pericyter eller mikroglia 13,14. Även om 3D-cellkulturer är mer i linje med den fysiologiska strukturen hos BBB15, är deras tillämpning som ett sätt att screena läkemedel för BBB fortfarande begränsad av deras intrikata design och undermåliga reproducerbarhet. Däremot är monolagermodellen in vitro den som oftast används för att undersöka BBB och är tillämplig för att bestämma uttrycket av membrantransportörer och tight junction-proteiner i vissa celler.
Mätning av transmembrant elektriskt motstånd (TEER) är en teknik för att utvärdera och övervaka cellskiktet över motståndet och utvärdera barriärens cellintegritet och permeabilitet. Genom att samtidigt föra in två elektroder i odlingsmediet eller buffertlösningen på vardera sidan av monolagret är det möjligt att mäta växelströmmen eller den elektriska impedansen genom cellens kompakta skikt16,17. För att avgöra om in vitro-BBB-modellen har skapats på ett korrekt sätt kommer mätningen av TEER vanligtvis att användas som guldstandard18. Å andra sidan kan trenden för läkemedelsverkan på BBB-permeabilitet exakt förutsägas genom att mäta förändringen i celskiktets elektriska motstånd efter läkemedelsinblandning19. Till exempel upptäckte Feng et al. att catalpol (den primära aktiva monomeren av rehmanniae) effektivt kunde vända den lipopolysackaridinducerade nedregleringen av tight junction-proteiner i BBB och höja TEER-värdet i mushjärnans endotelcellslager20.
Det neuroinflammatoriska svaret är vanligtvis den främsta orsaken till BBB-homeostasobalans21. Hypoxisk behandling för att inducera neuroinflammatorisk skada är den huvudsakliga metoden för att förstöra blod-hjärnbarriären, främst inklusive fysikaliska metoder och kemiska reagensmetoder. Den förstnämnda använder främst en tregasinkubator för att variera syrehalten i celltillväxtmiljön för att simulera hypoxiska förhållanden22, medan den senare uppnås genom att artificiellt introducera deoxireagenser såsom CoCl2 till cellodlingsmediet23. Cellerna kommer att förbli i ett syrefattigt tillstånd om Fe2+ ersätts med Co2+ i hemet. Om Fe2+ substitueras med Co2+ i den katalytiska gruppen hämmas prolinhydroxylas- och aspartathydroxylasaktiviteten, vilket resulterar i en ackumulering av hypoxi-inducerbar faktor-1α (HIF-1α)24. Under ihållande hypoxi utlöser defosforyleringen av HIF-1α i cytoplasman celldöd och aktiverar vaskulär endotelial tillväxtfaktor, vilket i slutändan ökar vaskulär permeabilitet. I tidigare studier 25,26 har det väl visats att hypoxi signifikant kan minska uttrycket av endoteliala tight junction-proteiner för att öka permeabiliteten av BBB. I denna studie mättes tidsresistanskurvan för bEnd.3-celler sådda i 24-hålsplattor för att skapa en enkel BBB-modell. Med hjälp av denna modell karakteriserade vi förändringarna i cell-TEER efter CoCl2-intervention för att konstruera en cellmodell som kan användas för att screena läkemedel för BBB-skydd.

<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

Blod-hjärnbarriären (BBB) är en dynamisk fysiologisk struktur som består av mikrovaskulära endotelceller, astrocyter och pericyter. Genom att samordna interaktionen mellan begränsad passage av skadliga ämnen, näringsupptag och metaboliteliminering i hjärnan är BBB avgörande för att bevara centrala nervsystemets homeostas. Att bygga in vitro-modeller av BBB är ett värdefullt verktyg för att utforska patofysiologin för neurologiska sjukdomar och skapa farmakologiska behandlingar. Denna studie beskriver en procedur för att skapa en in vitro monolager BBB-cellmodell genom att plantera bEnd.3-celler i den övre kammaren på en 24-håls platta. För att bedöma integriteten hos cellbarriärfunktionen användes den konventionella epitelcellvoltmetern för att registrera det transmembrana elektriska motståndet hos normala celler och CoCl2-inducerade hypoxiska celler i realtid. Vi förväntar oss att ovanstående experiment kommer att ge effektiva idéer för skapandet av in vitro-modeller av BBB och läkemedel för att behandla sjukdomar i centrala nervsystemet.

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.

Författare i fokus: Tillämpningar av TEER-detektion för att bedöma cellbarriärens integritet 

Registrering av barriärfunktionell integritet på bEnd.3 vaskulära endotelceller via transendotelial elektrisk motståndsdetektering

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.

Detta protokoll gjorde det möjligt att registrera förändringar i cellernas resistansvärden enligt de parametrar som ställts in i transendotelresistormätaren. Viaabiliteten hos bEnd.3-celler (antal levande celler) som behandlades med olika koncentrationer av CoCl2 screenades med CCK-8-analys. Större cellskador orsakade av CoCl2 representerades av lägre cellviabilitet. Vi fann att 300 μM CoCl2 var signifikant cytotoxiskt in vitro, och denna koncentration användes för de ...

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

Detta protokoll beskriver en pålitlig och effektiv in vitro-modell av hjärnans blodbarriär. Metoden använder cerebrala vaskulära endotelceller bEnd.3 från möss och mäter transmembrant elektriskt motstånd.

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

Aktuella studier använder vanligtvis permeabilitetstester och tillhörande proteinuttryck för att verifiera noggrannheten i TEER-detektionen. BBB-modellen med monolagerceller är i kraft mindre effektiv för läkemedelsscreening, och användningen av fler celler gör det svårare att konstruera modellen. Protokollet för denna studie är relativt enkelt, eftersom det icke-invasiva förfarandet gör att cellproverna kan användas i andra experiment efter TEER-detektion.Detektion av TEER för att bedöma permeabiliteten hos cellmodeller kan påskynda screening av läkemedel för behandling och förebyggande av det centrala nervsystemet för att öka framgångsgraden för utveckling av nya läkemedel. I framtiden kommer vi att detektera uttrycket av tight junction-protein i övre kompartmentceller i bröstväggen för att verifiera att TEER-testresultat effektivt kan representera integriteten hos BBB-barriärfunktionen.

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.

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.

Odling av bEnd.3-celler och cellviabilitet efter CoCl2-behandling

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 blod-hjärnbarriärmodell för mätning av 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.