Name: Forfatter Spotlight: Anvendelser af TEER-detektion til vurdering af cellebarriereintegritet
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 sætter pris på den økonomiske støtte fra National Natural Science Foundation of China (82274207 og 82104533), Key Research and Development Program of Ningxia (2023BEG02012) og Xinglin Scholar Research Promotion Project fra Chengdu University of TCM (XKTD2022013).

BEMÆRK: Musehjerneafledte endotelceller.3 (bEnd.3) blev podet ind i kamrene på en 24-brønds plade for at konstruere en simpel in vitro-model af BBB under specifikke mediumbetingelser. TEER for normale celler og hypoxiske celler blev målt med TEER-meter (figur 1 og figur 2).
1. Forberedelse af opløsning
<ol><li>Forbered DMEM-cellekulturmediet indeholdende FBS (10%, v / v), 100 E / ml penicillin og 10 mg...

Transendotelial modstandsmåling for at vurdere cellebarriereintegritet

<ol>
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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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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>

Et af de mest udviklede kropsorganer, hjernen styrer en bred vifte af indviklede fysiologiske processer, herunder hukommelse, kognition, hørelse, lugt og bevægelse27. Hjernen er et af menneskekroppens mest komplicerede og syge organer på samme tid. Forekomsten af mange lidelser i centralnervesystemet viser en stigende tendens år efter år på grund af faktorer, herunder luftforurening, uregelmæssige spisemønstre og andre faktorer 27,28,29....

BBB er en unik biologisk grænseflade mellem blodcirkulation og nervevæv, som består af vaskulære endotelceller, pericytter, astrocytter, neuroner og andre cellulære strukturer1. Strømmen af ioner, kemikalier og celler mellem blodet og hjernen er strengt reguleret af denne barriere. Denne homeostase beskytter nervevævet mod toksiner og patogener, samtidig med at det muliggør en passende funktion af hjernens nerver 2,3. Opretholdelse af BBB's integritet kan effektivt forhindre udvikling og progression af lidelser, der påvirker centralnervesystemet, såsom neuronal dysfunktion, ødem og neuroinflammation4. BBB's unikke fysiologiske egenskaber forhindrer imidlertid mere end 98% af lægemidler med små molekyler og 100% af makromolekylære lægemidler i at komme ind i centralnervesystemet5. Derfor er det vigtigt at øge penetrationen af medicin gennem BBB under udviklingen af lægemidler til centralnervesystemet for at opnå terapeutisk effekt 6,7. Selv om computersimuleringsscreening af substrater har øget sandsynligheden for, at lægemiddelkandidater krydser BBB, er der stadig behov for pålidelige og overkommelige in vitro/in vivo BBB-modeller for at imødekomme behovene inden for videnskabelig forskning8.
En hurtig og overkommelig teknik til high-throughput lægemiddelscreening er in vitro model9. For at belyse de grundlæggende processer for lægemidlers virkninger på BBB-funktionen og deres rolle i udviklingen og udviklingen af sygdom er der skabt en række forenklede in vitro BBB-modeller. På nuværende tidspunkt er de almindelige in vitro BBB-modeller monolags-, co-kultur-, dynamiske og mikrofluidiske modeller 10,11,12, konstrueret af vaskulære endotelceller og astrocytter, pericytter eller mikroglia13,14. Selvom 3D-cellekulturer er mere i overensstemmelse med den fysiologiske struktur af BBB15, er deres anvendelse som et middel til lægemiddelscreening for BBB stadig begrænset af deres indviklede design og subpar reproducerbarhed. I modsætning hertil er monolags in vitro-modellen den, der hyppigst anvendes til forskning i BBB og er anvendelig til bestemmelse af ekspressionen af membrantransportører og tætte forbindelsesproteiner i bestemte celler.
Måling af transmembran elektrisk modstand (TEER) er en teknik til evaluering og overvågning af cellelaget over modstanden og evaluering af barrierens celleintegritet og permeabilitet. Ved samtidig at indsætte to elektroder i vækstmediet eller bufferopløsningen på hver side af monolaget er det muligt at måle vekselstrømmen eller den elektriske impedans gennem cellens kompakte lag16,17. For at afgøre, om in vitro BBB-modellen er oprettet korrekt, vil målingen af TEER normalt blive anvendt som guldstandard18. På den anden side kan tendensen til medicinvirkning på BBB-permeabilitet forudsiges nøjagtigt ved at måle ændringen i cellelagets elektriske modstand efter lægemiddelinvolvering19. For eksempel opdagede Feng et al., at catalpol (den primære aktive monomer af rehmanniae) effektivt kunne vende den lipopolysaccharid-inducerede nedregulering af tight junction-proteiner i BBB og hæve TEER-værdien af musehjernens endotelcellelag20.
Det neuroinflammatoriske respons er normalt hovedårsagen til BBB homeostase ubalance21. Hypoxisk behandling for at fremkalde neuroinflammatorisk skade er den vigtigste metode til at ødelægge blod-hjerne-barrieren, hovedsageligt inklusive fysiske metoder og kemiske reagensmetoder. Førstnævnte anvender primært en tre-gas-inkubator til at variere iltindholdet i cellevækstmiljøet for at simulere hypoxiske forhold22, mens sidstnævnte opnås ved kunstigt at indføre deoxyreagenser såsom CoCl2til cellekulturmediet23. Cellerne forbliver i en deoxygeneret tilstand, hvis Fe2 + erstattes af Co2 + i hæmmen. Hvis Fe2+ erstattes af Co2+ i den katalytiske gruppe, hæmmes prolinhydroxylase og aspartathydroxylaseaktivitet, hvilket resulterer i en akkumulering af hypoxi-inducerbar faktor-1α (HIF-1α)24. Under vedvarende hypoxi udløser dephosphoryleringen af HIF-1α i cytoplasma celledød og aktiverer vaskulær endotelvækstfaktor, hvilket i sidste ende øger vaskulær permeabilitet. I tidligere undersøgelser 25,26 er det blevet godt demonstreret, at hypoxi signifikant kan reducere ekspressionen af endoteltætte forbindelsesproteiner for at øge permeabiliteten af BBB. I denne undersøgelse blev tidsmodstandskurven for bEnd.3-celler podet i plader med 24 brønde målt for at skabe en ligetil BBB-model. Ved hjælp af denne model karakteriserede vi ændringerne i celle TEER efter CoCl2-intervention for at konstruere en cellemodel, der kan bruges til at screene lægemidler til BBB-beskyttelse.

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	<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>
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		<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-hjerne-barrieren (BBB) er en dynamisk fysiologisk struktur sammensat af mikrovaskulære endotelceller, astrocytter og pericytter. Ved at koordinere samspillet mellem begrænset transit af skadelige stoffer, næringsstofabsorption og metabolitclearance i hjernen er BBB afgørende for at bevare centralnervesystemets homeostase. Opbygning af in vitro-modeller af BBB er et værdifuldt værktøj til at udforske patofysiologien af neurologiske lidelser og skabe farmakologiske behandlinger. Denne undersøgelse beskriver en procedure til oprettelse af en in vitro monolags BBB-cellemodel ved såning af bEnd.3-celler i det øverste kammer på en 24-brøndplade. For at vurdere integriteten af cellebarrierefunktionen blev det konventionelle epitelcellevoltmeter brugt til at registrere den transmembrane elektriske modstand af normale celler og CoCl 2-inducerede hypoxiske celler i realtid. Vi forventer, at ovenstående forsøg vil give effektive ideer til oprettelse af in vitro-modeller af BBB og lægemidler til behandling af lidelser i centralnervesystemet.

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.

Forfatter Spotlight: Anvendelser af TEER-detektion til vurdering af cellebarriereintegritet 

Registrering af barrierefunktionel integritet på bEnd.3 Vaskulære endotelceller via transendotelial elektrisk modstandsdetektion

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.

Denne protokol tillod registrering af ændringer i cellernes modstandsværdier i henhold til parametrene indstillet i transendotelmodstandsmåleren. Levedygtigheden af bEnd.3-celler (antal levende celler) behandlet med forskellige koncentrationer afCoCl2 blev screenet ved CCK-8-assay. Større celleskader produceret afCoCl2 var repræsenteret ved lavere cellelevedygtighed. Vi fandt, at 300 μMCoCl2 var signifikant cytotoksisk in vitro, og denne koncentration blev anvendt til de n?...

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

Denne protokol beskriver en pålidelig og effektiv in vitro-model af hjernens blodbarriere. Metoden anvender musecerebrale vaskulære endotelceller bEnd.3 og måler transmembran elektrisk modstand.

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

Aktuelle undersøgelser bruger typisk permeabilitetstest og tilhørende proteinekspression for at verificere nøjagtigheden af TEER-detektion. BBB-modellen af enkeltlagsceller i dyd er mindre effektiv til lægemiddelscreening, og brugen af flere celler gør det vanskeligere at konstruere modellen. Protokollen for denne undersøgelse er relativt enkel, da den ikke-invasive procedure gør det muligt at bruge celleprøverne i andre eksperimenter efter TEER-detektion.Påvisning af TEER til vurdering af cellemodellers permeabilitet kan fremskynde screeningen af lægemidler til behandling og forebyggelse af centralnervesystemet for at øge succesraten for udvikling af nye lægemidler. I fremtiden vil vi detektere ekspressionen af tight junction-protein i øvre rumceller i brystvæggen for at verificere, at TEER-testresultater effektivt kan repræsentere integriteten af BBB-barrierefunktionen.

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.

Dyrkning af bEnd.3 Celler og cellelevedygtighed 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-hjerne barriere model samling til måling af 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.