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

Fuhan Fan; Hong Jiang; Ya Hou; Yi Zhang; Qipeng Zhao; Yong Zeng; Xianli Meng; Xiaobo Wang

doi:10.3791/65938

Registrazione dell'integrità funzionale della barriera su cellule endoteliali vascolari bEnd.3

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Neuroscience

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JoVE Journal Neuroscience

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

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

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09:03 min

September 29, 2023

DOI: 10.3791/65938-v

Fuhan Fan¹, Hong Jiang¹, Ya Hou¹, Yi Zhang^1,2,3, Qipeng Zhao⁴, Yong Zeng^1,2, Xianli Meng^1,2,5, Xiaobo Wang²

¹School of Pharmacy/School of Modern Chinese Medicine Industry,Chengdu University of Traditional Chinese Medicine, ²Meishan Hospital of Chengdu University of Traditional Chinese Medicine, ³School of Ethnic Medicine,Chengdu University of Traditional Chinese Medicine, ⁴School of Pharmacy,Ningxia Medical University, ⁵Innovative Institute of Chinese Medicine and Pharmacy/Academy for Interdiscipline,Chengdu University of Traditional Chinese Medicine

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Overview

This study presents a reliable in vitro model of the blood-brain barrier (BBB) using mouse cerebral vascular endothelial cells (bEnd.3). The model assesses transmembrane electrical resistance (TER) to evaluate the integrity of the BBB, facilitating drug screening for central nervous system treatments.

Key Study Components

Area of Science

Cell Biology
Neuroscience
Pharmacology

Background

The blood-brain barrier is crucial for maintaining homeostasis in the central nervous system.
Traditional in vitro models are less effective for drug testing due to limitations in cellular complexity.
This study addresses those limitations by presenting a simpler, non-invasive method.

Purpose of Study

To improve the assessment of BBB integrity through TER measurement.
To facilitate the screening of drugs targeting central nervous system disorders.
To verify the relationship between TER values and tight junction protein expression.

Methods Used

The study utilized bEnd.3 cell culture, which was seeded and cultured in a 96-well plate format.
Key interventions included the addition of cobalt chloride to assess cellular viability and barrier integrity.
TEER values were recorded over time to determine the formation and stability of the BBB model.
Statistical analysis was employed to compare control and treated groups.

Main Results

Results indicated a significant reduction in TEER values upon treatment with cobalt chloride, indicating compromised BBB integrity.
The study showed the potential of using TER as a reliable method for assessing drug effects on the BBB.
Stability of the cell barrier was reached by day five, underscoring the method's effectiveness.

Conclusions

This study demonstrates a simplified and effective approach to assess BBB integrity in drug development.
Results could enhance understanding of therapeutic interventions for central nervous system diseases.
The methods outlined may be adapted for broader applications in pharmacological research.

Frequently Asked Questions

What are the advantages of using the bEnd.3 cell line?

The bEnd.3 cell line is an established model for studying the blood-brain barrier, providing relevant insights into endothelial function and drug permeability.

How is the cobalt chloride treatment implemented?

Cobalt chloride is added to the cell cultures to investigate its effects on cell viability and barrier function, specifically assessing changes in TER.

What measures are taken to ensure accurate TEER readings?

Calibration of the measurement instrument and proper handling of electrodes are critical for obtaining reliable TEER measurements before and during the experiments.

What types of data can be obtained from this method?

The method provides data on electrical resistance, supporting assessments of cell viability and barrier integrity essential for drug development.

How can this model be adapted for other studies?

This BBB model could be modified to test various compounds or treatments, enhancing its utility for pharmacological and toxicological research.

What are the limitations of this study?

While the model is reliable, it may not fully replicate in vivo conditions, signaling the need for complementary studies using more complex biological models.

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.

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.

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