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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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's nerves2,3. Maintaining the integrity of the BBB can effectively prevent the development and progression of disorders affecting the central nervous ....

Protocol

NOTE: Mouse brain-derived Endothelial cells.3 (bEnd.3) were inoculated into the chambers of a 24-well plate to construct a simple in vitro model of BBB under specific medium conditions. The TEER of normal cells and hypoxic cells were measured by TEER meter (Figure 1 and Figure 2).

1. Solution preparation

  1. Prepare the DMEM cell culture medium containing FBS (10%, v/v), 100 U/mL penicillin, and 10 mg.......

Representative Results

This protocol allowed the recording of changes in the resistance values of cells according to the parameters set in the transendothelial resistor meter. The viability of bEnd.3 cells (number of live cells) treated with different concentrations of CoCl2 were screened by CCK-8 assay. Greater cell damage produced by CoCl2 was represented by lower cell viability. We found that 300 µM of CoCl2 was significantly cytotoxic in vitro, and this concentration was used for the next expe.......

Discussion

One of the most developed bodily organs, the brain controls a wide range of intricate physiological processes, including memory, cognition, hearing, smell, and movement27. The brain is one of the human body's most complicated and diseased organs at the same time. The occurrence of many central nervous system disorders shows a growing tendency year over year due to factors including air pollution, irregular eating patterns, and other factors27,

Acknowledgements

We appreciate the financial support from the National Natural Science Foundation of China (82274207 and 82104533), the Key Research and Development Program of Ningxia (2023BEG02012), and Xinglin Scholar Research Promotion Project of Chengdu University of TCM (XKTD2022013).

....

Materials

NameCompanyCatalog NumberComments
24-well transwell plateCorning (Corning 3470, 0.33 cm2, 0.4 µm)10522023
75 % ethanolChengDu Chron Chemicals Co,.Ltd2023052901
96-well plateGuangzhou Jet Bio-Filtration Co., Ltd220412-078-B
bEnd.3 cellsHunan Fenghui Biotechnology Co., LtdCL0049
Cell counting kit-8 (CCK-8)Boster Biological Technology Co., LtdBG0025
Cell culture dish (100mm)Zhejiang Sorfa Life Science Research Co., Ltd1192022
Cobalt Chloride (CoCl2)Sigma15862
DMSOBoster Biological Technology Co., LtdPYG0040
Dulbecco's modified eagle medium (1x)Gibco ThermoFisher Scientific8121587
Fetal bovine serumGibco ThermoFisher Scientific2166090RP
GraphPad Prism softwareGraphPad Software9.0.0(121)
Matrigel (Contains collagen IV)MedChemexpressHY-K6002
Microplate readerMolecular DevicesSpectraMax iD5
OriginPro 8 softwareOriginLab Corporationv8.0724(B724)
Penicillin-Streptomycin (100x)Boster Biological Technology Co., Ltd17C18B16
Phosphate buffered saline (PBS, 1x)Gibco ThermoFisher Scientific8120485
Sodium hypochloriteChengDu Chron Chemicals Co,.Ltd2022091501
Transmembrane resistance meterWorld Precision Instruments LLCVOM3 (verison 1.6)
Trypsin 0.25% (1x)HyCloneJ210045

References

  1. Kim, Y., et al. CLEC14A deficiency exacerbates neuronal loss by increasing blood-brain barrier permeability and inflammation. J Neuroinflammation. 17 (1), 48 (2020).
  2. Bagchi, S., et al.

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Blood brain BarrierBBB ModelBEnd 3 CellsTransendothelial Electrical Resistance TEERBarrier IntegrityCell PermeabilityIn Vitro ModelCentral Nervous SystemDrug ScreeningHypoxia

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