Name: Auteur in de schijnwerpers: Toepassingen van TEER-detectie om de integriteit van de celbarrière te beoordelen
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

We waarderen de financiële steun van de National Natural Science Foundation of China (82274207 en 82104533), het Key Research and Development Program van Ningxia (2023BEG02012) en het Xinglin Scholar Research Promotion Project van de Chengdu University of TCM (XKTD2022013).

OPMERKING: Van de hersenen van muizen afgeleide endotheelcellen.3 (bEnd.3) werden geïnoculeerd in de kamers van een plaat met 24 putjes om een eenvoudig in vitro model van BBB te construeren onder specifieke mediumomstandigheden. De TEER van normale cellen en hypoxische cellen werd gemeten met een TEER-meter (Figuur 1 en Figuur 2).
1. Voorbereiding van de oplossing
<ol><li>Bereid het DMEM-celkweekmedium da...

Transendotheliale weerstandsmeting om de integriteit van de celbarrière te beoordelen

<ol>
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De hersenen zijn een van de meest ontwikkelde lichaamsorganen en regelen een breed scala aan ingewikkelde fysiologische processen, waaronder geheugen, cognitie, gehoor, geuren beweging. De hersenen zijn tegelijkertijd een van de meest gecompliceerde en zieke organen van het menselijk lichaam. Het voorkomen van veel aandoeningen van het centrale zenuwstelsel vertoont jaar na jaar een groeiende tendens als gevolg van factoren zoals luchtvervuiling, onregelmatige eetpatronen en andere factoren 27,28,...

BBB is een unieke biologische interface tussen bloedcirculatie en zenuwweefsel, die is samengesteld uit vasculaire endotheelcellen, pericyten, astrocyten, neuronen en andere cellulairestructuren1. De stroom van ionen, chemicaliën en cellen tussen het bloed en de hersenen wordt strikt gereguleerd door deze barrière. Deze homeostase beschermt de zenuwweefsels tegen gifstoffen en ziekteverwekkers en maakt tegelijkertijd de juiste werking van de zenuwen van de hersenen mogelijk 2,3. Het handhaven van de integriteit van de BBB kan de ontwikkeling en progressie van aandoeningen die het centrale zenuwstelsel aantasten, zoals neuronale disfunctie, oedeem en neuro-inflammatie, effectief voorkomen. De unieke fysiologische eigenschappen van de BBB voorkomen echter dat meer dan 98% van de medicijnen met kleine moleculen en 100% van de macromoleculaire geneesmiddelen het centrale zenuwstelselbinnendringen. Daarom is het verhogen van de penetratie van medicijnen via de BBB tijdens de ontwikkeling van geneesmiddelen voor het centrale zenuwstelsel essentieel voor het bereiken van therapeutische werkzaamheid 6,7. Hoewel computersimulatiescreening van substraten de kans dat kandidaat-geneesmiddelen de BBB overschrijden aanzienlijk heeft verhoogd, zijn betrouwbare en betaalbare in vitro/in vivo BBB-modellen nog steeds nodig om aan de behoeften van wetenschappelijk onderzoek te voldoen8.
Een snelle en betaalbare techniek voor high-throughput drug screening is het in vitro model9. Om licht te werpen op de fundamentele processen van de effecten van geneesmiddelen op de BBB-functie en hun rol in de ontwikkeling en progressie van ziekten, is een reeks vereenvoudigde in vitro BBB-modellen gemaakt. Op dit moment zijn de gebruikelijke in vitro BBB-modellen de monolaag-, co-cultuur-, dynamische en microfluïdische modellen 10,11,12, geconstrueerd door vasculaire endotheelcellen en astrocyten, pericyten of microglia 13,14. Hoewel 3D-celculturen meer in overeenstemming zijn met de fysiologische structuur van BBB15, wordt hun toepassing als middel voor het screenen van geneesmiddelen op BBB nog steeds beperkt door hun ingewikkelde ontwerp en ondermaatse reproduceerbaarheid. Het monolayer in vitro model daarentegen wordt het meest gebruikt om de BBB te onderzoeken en is toepasbaar voor het bepalen van de expressie van membraantransporters en tight junction eiwitten in bepaalde cellen.
Transmembraan elektrische weerstandsmeting (TEER) is een techniek om de cellaag over de weerstand te evalueren en te bewaken en de celintegriteit en permeabiliteit van de barrière te evalueren. Door tegelijkertijd twee elektroden in het groeimedium of de bufferoplossing aan weerszijden van de monolaag te plaatsen, is het mogelijk om de wisselstroom of elektrische impedantie door de compacte laag van de cel te meten 16,17. Om te bepalen of het in vitro BBB-model op de juiste manier is gemaakt, zal de meting van TEER meestal worden gebruikt als de gouden standaard18. Aan de andere kant kan de trend van medicatie-actie op BBB-permeabiliteit nauwkeurig worden voorspeld door de verandering in elektrische weerstand van de cellaag na betrokkenheid van het geneesmiddel te meten19. Feng et al. ontdekten bijvoorbeeld dat catalpol (het primaire actieve monomeer van rehmanniae) de door lipopolysaccharide geïnduceerde neerwaartse regulatie van tight junction-eiwitten in de BBB effectief kon omkeren en de TEER-waarde van de endotheelcellaag20 van de muizenhersenen kon verhogen.
De neuro-inflammatoire reactie is meestal de belangrijkste oorzaak van een verstoring van de BBB-homeostase21. Hypoxische behandeling om neuro-inflammatoir letsel te induceren is de belangrijkste methode om de bloed-hersenbarrière te vernietigen, voornamelijk met inbegrip van fysische methoden en chemische reagensmethoden. De eerste maakt voornamelijk gebruik van een incubator met drie gassen om het zuurstofgehalte in de celgroeiomgeving te variëren om hypoxische omstandigheden te simuleren22, terwijl de laatste wordt bereikt door kunstmatig deoxyreagentia zoals CoCl2 in het celkweekmediumte introduceren 23. De cellen blijven zuurstofarm als Co2+ in het heem wordt vervangen door Fe2+. Als Fe2+ wordt gesubstitueerd door Co2+ in de katalytische groep, wordt de activiteit van prolinehydroxylase en aspartaathydroxylase geremd, wat resulteert in een accumulatie van hypoxie-induceerbare factor-1α (HIF-1α)24. Bij aanhoudende hypoxie veroorzaakt de defosforylering van HIF-1α in het cytoplasma celdood en activeert de vasculaire endotheliale groeifactor, die uiteindelijk de vasculaire permeabiliteit verhoogt. In eerdere studies 25,26 is goed aangetoond dat hypoxie de expressie van endotheliale tight junction-eiwitten aanzienlijk kan verminderen om de permeabiliteit van BBB te vergroten. In deze studie werd de tijdweerstandscurve van bEnd.3-cellen gezaaid in platen met 24 putjes gemeten om een eenvoudig BBB-model te creëren. Met behulp van dit model hebben we de veranderingen in cel-TEER na CoCl2-interventie gekarakteriseerd om een celmodel te construeren dat kan worden gebruikt om geneesmiddelen te screenen op BBB-bescherming.

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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>
		</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

De bloed-hersenbarrière (BBB) is een dynamische fysiologische structuur die bestaat uit microvasculaire endotheelcellen, astrocyten en pericyten. Door de interactie tussen beperkte doorvoer van schadelijke stoffen, opname van voedingsstoffen en metabolietklaring in de hersenen te coördineren, is de BBB essentieel voor het behoud van de homeostase van het centrale zenuwstelsel. Het bouwen van in vitro modellen van de BBB is een waardevol hulpmiddel voor het onderzoeken van de pathofysiologie van neurologische aandoeningen en het creëren van farmacologische behandelingen. Deze studie beschrijft een procedure voor het maken van een in vitro monolaag BBB-celmodel door bEnd.3-cellen in de bovenste kamer van een plaat met 24 putjes te zaaien. Om de integriteit van de celbarrièrefunctie te beoordelen, werd de conventionele epitheelcelvoltmeter gebruikt om de transmembraan elektrische weerstand van normale cellen en CoCl 2-geïnduceerde hypoxische cellen in realtime vast te leggen. We verwachten dat de bovenstaande experimenten effectieve ideeën zullen opleveren voor het maken van in vitro modellen van BBB en geneesmiddelen voor de behandeling van aandoeningen van ziekten van het centrale zenuwstelsel.

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.

Auteur in de schijnwerpers: Toepassingen van TEER-detectie om de integriteit van de celbarrière te beoordelen 

Opname van functionele integriteit van barrière op bEnd.3 vasculaire endotheelcellen via detectie van transendotheliale elektrische weerstand

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.

Dit protocol maakte het mogelijk om veranderingen in de weerstandswaarden van cellen te registreren volgens de parameters die waren ingesteld in de transendotheelweerstandsmeter. De levensvatbaarheid van bEnd.3-cellen (aantal levende cellen) die met verschillende concentraties CoCl2 werden behandeld, werd gescreend door middel van een CCK-8-test. Grotere celbeschadiging veroorzaakt door CoCl2 werd vertegenwoordigd door een lagere levensvatbaarheid van de cellen. We ontdekten dat 300 μM CoCl2</...

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

Dit protocol beschrijft een betrouwbaar en efficiënt in vitro model van de bloedbarrière van de hersenen. De methode maakt gebruik van cerebrale vasculaire endotheelcellen bEnd.3 van muizen en meet de transmembraan elektrische weerstand.

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

Huidige studies maken doorgaans gebruik van permeabiliteitstests en bijbehorende eiwitexpressie om de nauwkeurigheid van TEER-detectie te verifiëren. Het BBB-model van monolaagcellen is minder effectief voor het screenen van geneesmiddelen, en het gebruik van meer cellen maakt het moeilijker om het model te construeren. Het protocol van deze studie is relatief eenvoudig, omdat de niet-invasieve procedure het mogelijk maakt om de celmonsters na TEER-detectie in andere experimenten te gebruiken.De detectie van TEER om de permeabiliteit van celmodellen te beoordelen, kan de screening van geneesmiddelen voor behandeling en preventie van het centrale zenuwstelsel versnellen om het slagingspercentage van de ontwikkeling van nieuwe geneesmiddelen te vergroten. In de toekomst zullen we de expressie van tight junction-eiwit in de cellen van het bovenste compartiment van de borstwand detecteren om te verifiëren dat TEER-testresultaten de integriteit van de BBB-barrièrefunctie effectief kunnen weergeven.

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.

Kweek van bEnd.3-cellen en cellevensvatbaarheid na behandeling met CoCl2

In Vitro Blood-Brain Barrier Model Assembly for Measurement of TEER

To begin, rinse the upper chamber of 24 well plates with PBS. Mix the BEND3 cells with DMEM medium using a vortex mixer to make a suspension. Then, seed 200 microliters of BEND3 cell suspension on the PET membrane in the upper chamber of a 24 well plate.Add 1, 200 microliters of complete medium to the lower chamber of the plate to stabilize the osmotic pressure of the upper and lower chambers. Change the medium by slowly removing the old medium from one side with a negative-pressure pipette and adding a new medium along the wall during fluid exchange. Before measuring TEER, place the resistor, 5%sodium hypochlorite solution, 75%ethanol, and double distilled water on an ultra clean table.Turn on the UV irradiation for 30 minutes to eliminate residual bacteria and pathogens. Place the electrodes in 5%sodium hypochlorite solution with slow shaking for three to five seconds. Then immerse in 75%ethanol for 15 minutes.Finally, transfer to PBS or double distilled water until use. Next, turn on the switch at the back of the cell resistor meter. Click Select Plate and Select 24 well plate.According to the operation, select the appropriate detection sequence. Insert a kilo ohm resistor into the right plug to calibrate the instrument. If the calibration result is 1000 plus minus 5 ohms, consider the instrument accurate.If not, click on Mode Units on the main interface to select Ohms and then click Calibrate. Then, remove the kilo ohm resistor and replace it with the measuring electrode using a connecting wire. Place the electrode vertically into the 24 well plate containing only medium and click Blank Handling on the instrument.The background value of the plate resistance without seated cells should be about 134.4 ohm. Next, insert the two electrodes into the upper and lower chambers of the seated plate, such that the cell layer is between them. Record the resistance value by gently stepping on the pedal.Make sure that the electrode does not touch the cells in the upper chamber and the bottom of the lower chamber. Obtain TEER values by multiplying electrical resistance values with the bottom area of the upper chamber, as shown in the equation. Draw a line plot of TEER versus time in days.When the resistance value does not increase with time, the cells have formed a barrier. The resistance values plotted versus time for BEND3 cells showed that the cell TEER values began to stabilize on the fifth day.

In vitro bloed-hersenbarrièremodelassemblage voor het meten van 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.