Name: Applications of TEER Detection to Assess Cell Barrier Integrity (Video) | JoVE
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...

Transendothelial Resistance Measurement to Assess Cell Barrier Integrity

<ol>
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A., Shapiro, L. A., Huang, J. 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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.

<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-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.

Barrier Functional Integrity Recording on bEnd.3 Vascular Endothelial Cells via Transendothelial Electrical Resistance Detection

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

Applications of TEER Detection to Assess Cell Barrier Integrity (Video) | JoVE

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.

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

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.