Name: Autore in primo piano: Applicazioni del rilevamento TEER per valutare l'integrità della barriera cellulare
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

Apprezziamo il sostegno finanziario della National Natural Science Foundation of China (82274207 e 82104533), del Key Research and Development Program di Ningxia (2023BEG02012) e dello Xinglin Scholar Research Promotion Project dell'Università di Chengdu di MTC (XKTD2022013).

NOTA: Le cellule endoteliali derivate dal cervello di topo.3 (bEnd.3) sono state inoculate nelle camere di una piastra a 24 pozzetti per costruire un semplice modello in vitro di BBB in condizioni specifiche del terreno. Il TEER delle cellule normali e delle cellule ipossiche è stato misurato con il misuratore TEER (Figura 1 e Figura 2).
1. Preparazione della soluzione
<ol><li>Preparare il terreno di coltu...

Misurazione della resistenza transendoteliale per valutare l'integrità della barriera cellulare

<ol>
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A., Shapiro, L. A., Huang, J. 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Uno degli organi corporei più sviluppati, il cervello controlla un'ampia gamma di intricati processi fisiologici, tra cui la memoria, la cognizione, l'udito, l'olfattoe il movimento. Il cervello è uno degli organi più complicati e malati del corpo umano allo stesso tempo. L'insorgenza di molti disturbi del sistema nervoso centrale mostra una tendenza crescente anno dopo anno a causa di fattori quali l'inquinamento atmosferico, i modelli alimentari irregolari e altri fattori 27,28,29.<sup class=...

La BBB è un'interfaccia biologica unica tra la circolazione sanguigna e il tessuto nervoso, composto da cellule endoteliali vascolari, periciti, astrociti, neuroni e altre strutture cellulari1. Il flusso di ioni, sostanze chimiche e cellule tra il sangue e il cervello è strettamente regolato da questa barriera. Questa omeostasi protegge i tessuti nervosi dalle tossine e dagli agenti patogeni, consentendo anche il corretto funzionamento dei nervi cerebrali 2,3. Mantenere l'integrità della BBB può prevenire efficacemente lo sviluppo e la progressione di disturbi che colpiscono il sistema nervoso centrale, come la disfunzione neuronale, l'edema e la neuroinfiammazione4. Tuttavia, le proprietà fisiologiche uniche della BBB impediscono a oltre il 98% dei farmaci a piccole molecole e al 100% dei farmaci macromolecolari di entrare nel sistema nervoso centrale5. Pertanto, aumentare la penetrazione dei farmaci attraverso la BBB durante lo sviluppo di farmaci per il sistema nervoso centrale è essenziale per raggiungere l'efficacia terapeutica 6,7. Anche se lo screening con simulazione al computer dei substrati ha aumentato significativamente la probabilità che i candidati farmaci attraversino la BEE, sono ancora necessari modelli di BBB in vitro/in vivo affidabili e convenienti per soddisfare le esigenze della ricerca scientifica8.
Una tecnica rapida ed economica per lo screening dei farmaci ad alto rendimento è il modello in vitro 9. Per far luce sui processi fondamentali degli effetti dei farmaci sulla funzione della BBB e sul loro ruolo nello sviluppo e nella progressione della malattia, è stata creata una serie di modelli semplificati di BBB in vitro. Attualmente, i modelli comuni di BBB in vitro sono i modelli monostrato, di co-coltura, dinamici e microfluidici 10,11,12, costruiti da cellule endoteliali vascolari e astrociti, periciti o microglia 13,14. Sebbene le colture cellulari 3D siano più in linea con la struttura fisiologica della BBB15, la loro applicazione come mezzo di screening farmacologico per la BBB è ancora limitata dal loro design intricato e dalla riproducibilità scadente. Al contrario, il modello monostrato in vitro è quello più frequentemente utilizzato per la ricerca sulla BBB ed è applicabile per determinare l'espressione dei trasportatori di membrana e delle proteine a giunzione stretta in particolari cellule.
La misurazione della resistenza elettrica transmembrana (TEER) è una tecnica per valutare e monitorare lo strato di cellule attraverso la resistenza e valutare l'integrità cellulare e la permeabilità della barriera. Inserendo contemporaneamente due elettrodi nel mezzo di crescita o nella soluzione tampone su entrambi i lati del monostrato, è possibile misurare la corrente alternata o l'impedenza elettrica attraverso lo strato compatto della cella16,17. Al fine di determinare se il modello BBB in vitro è stato creato correttamente, la misurazione del TEER sarà solitamente impiegata come gold standard18. D'altra parte, l'andamento dell'azione dei farmaci sulla permeabilità della BBB può essere previsto con precisione misurando la variazione della resistenza elettrica dello strato cellulare dopo il coinvolgimento del farmaco19. Ad esempio, Feng et al. hanno scoperto che il catalpol (il monomero attivo primario delle rehmanniae) potrebbe invertire efficacemente la down-regulation indotta dai lipopolisaccaridi delle proteine a giunzione stretta nella BBB e aumentare il valore TEER dello strato20 delle cellule endoteliali del cervello di topo.
La risposta neuroinfiammatoria è di solito la causa principale dello squilibrio dell'omeostasi della BBB21. Il trattamento ipossico per indurre lesioni neuroinfiammatorie è il metodo principale per distruggere la barriera emato-encefalica, includendo principalmente metodi fisici e metodi di reagenti chimici. Il primo utilizza principalmente un incubatore a tre gas per variare il contenuto di ossigeno nell'ambiente di crescita cellulare per simulare le condizioni ipossiche22, mentre il secondo si ottiene introducendo artificialmente reagenti deossi come CoCl2 nel terreno di coltura cellulare23. Le cellule rimarranno in una condizione di deossigenazione se Fe2+ viene sostituito con Co2+ nell'eme. Se Fe2+ viene sostituito con Co2+ nel gruppo catalitico, l'attività della prolina idrossilasi e dell'aspartato idrossilasi sarà inibita, con conseguente accumulo di fattore 1α inducibile dall'ipossia (HIF-1α)24. In condizioni di ipossia persistente, la defosforilazione di HIF-1α nel citoplasma innesca la morte cellulare e attiva il fattore di crescita dell'endotelio vascolare, che alla fine aumenta la permeabilità vascolare. In precedenti studi 25,26, è stato ben dimostrato che l'ipossia può ridurre significativamente l'espressione delle proteine endoteliali della giunzione stretta per aumentare la permeabilità della BBB. In questo studio, è stata misurata la curva tempo-resistenza delle cellule bEnd.3 seminate in piastre a 24 pozzetti al fine di creare un modello BBB semplice. Utilizzando questo modello, abbiamo caratterizzato i cambiamenti nel TEER cellulare dopo l'intervento di CoCl2 al fine di costruire un modello cellulare che può essere utilizzato per lo screening dei farmaci per la protezione della BBB.

<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

La barriera emato-encefalica (BBB) è una struttura fisiologica dinamica composta da cellule endoteliali microvascolari, astrociti e periciti. Coordinando l'interazione tra il transito limitato di sostanze nocive, l'assorbimento dei nutrienti e la clearance dei metaboliti nel cervello, la BBB è essenziale per preservare l'omeostasi del sistema nervoso centrale. La costruzione di modelli in vitro della BBB è uno strumento prezioso per esplorare la fisiopatologia dei disturbi neurologici e creare trattamenti farmacologici. Questo studio descrive una procedura per la creazione di un modello di cellula BBB monostrato in vitro seminando cellule bEnd.3 nella camera superiore di una piastra a 24 pozzetti. Per valutare l'integrità della funzione di barriera cellulare, il voltmetro convenzionale a cellule epiteliali è stato utilizzato per registrare in tempo reale la resistenza elettrica transmembrana delle cellule normali e delle cellule ipossiche indotte da CoCl2. Prevediamo che gli esperimenti di cui sopra forniranno spunti efficaci per la creazione di modelli in vitro di BBB e farmaci per il trattamento dei disturbi delle malattie del sistema nervoso centrale.

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.

Autore in primo piano: Applicazioni del rilevamento TEER per valutare l'integrità della barriera cellulare 

Registrazione dell'integrità funzionale della barriera su cellule endoteliali vascolari bEnd.3 tramite rilevamento della resistenza elettrica transendoteliale

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.

Questo protocollo ha permesso di registrare le variazioni dei valori di resistenza delle cellule in base ai parametri impostati nel resistore transendoteliale. La vitalità delle cellule bEnd.3 (numero di cellule vive) trattate con diverse concentrazioni di CoCl2 è stata esaminata mediante test CCK-8. Il maggior danno cellulare prodotto da CoCl2 è stato rappresentato da una minore vitalità cellulare. Abbiamo scoperto che 300 μM di CoCl2 erano significativamente citotossici in vitr...

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

Questo protocollo descrive un modello in vitro affidabile ed efficiente della barriera emato-encefalica. Il metodo utilizza cellule endoteliali vascolari cerebrali di topo bEnd.3 e misura la resistenza elettrica transmembrana.

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

Gli studi attuali utilizzano in genere test di permeabilità e l'espressione proteica associata per verificare l'accuratezza del rilevamento di TEER. Il modello BBB di cellule monostrato in virtù è meno efficace per lo screening dei farmaci e l'uso di più cellule rende più difficile la costruzione del modello. Il protocollo di questo studio è relativamente semplice, in quanto la procedura non invasiva consente di utilizzare i campioni di cellule in altri esperimenti dopo il rilevamento di TEER.Il rilevamento di TEER per valutare la permeabilità dei modelli cellulari può accelerare lo screening dei farmaci per il trattamento e la prevenzione del sistema nervoso centrale per aumentare il tasso di successo dello sviluppo di nuovi farmaci. In futuro, rileveremo l'espressione della proteina della giunzione stretta nelle cellule del compartimento superiore della parete toracica per verificare che i risultati del test TEER possano rappresentare efficacemente l'integrità della funzione di barriera della BBB.

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.

Ricerca

Neuroscienze

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.

Coltura di cellule bEnd.3 e vitalità cellulare dopo il trattamento con 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.

Assemblaggio di modelli di barriera emato-encefalica in vitro per la misurazione del 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.