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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • 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 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' 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'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-1α (HIF-1α)24. Under persistent hypoxia, the dephosphorylation of HIF-1α 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.

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/mL streptomycin (see Table of Materials).
  2. Prepare 100 mM CoCl2 stock solution by adding 1.30 mg CoCl2 to 100 µL DMSO solution.
    NOTE: All the above solutions were stored at 4 °C condition, and the stock solution was diluted according to the desired concentration before use.
  3. Prepare 5% sodium hypochlorite solution (v/v) by adding 2 mL of sodium hypochlorite solution to 38 mL of double distilled water.

2. Cell culture and cell viability

  1. Seed 1 mL of bEnd.3 cells in culture dish (100 mm) containing DMEM medium at a density of 1 x 106 cells/mL and culture at 37 °C in a humidified atmosphere of 5% CO2. Change the medium every 2 to 3 days and subculture the cells 2x a week.
  2. After bEnd.3 cells grow to 80 % confluence, digest the cells with 0.25% trypsin for 30 s.
  3. Make a suspension of bEnd.3 cells at density of 7 x 104 cells/mL using DMEM medium through a cell counter. Then, seed 100 µL of bEnd.3 cell suspension in a 96-well plate.
  4. After cell adhesion, clean the cells with PBS and culture the cells with 100 µL of culture medium or drug-containing medium (100 µM, 200 µM, 300 µM, 400 µM, 500 µM CoCl2) for 24 h under the same conditions. After removing the medium inside the well plate and cleaning it with PBS, add 100 µL of CCK-8 solution.
    NOTE: Do not generate bubbles in the well, which will affect the values of optical density (OD).
  5. Put 96-well plates at 37 °C environment and incubate for 1 h. Measure the absorbance of the 96-well plates at 450 nm using a microplate reader.
    NOTE: To avoid intra-group differences, the well plates were cooled to room temperature before detection.
  6. Calculate the cell viability induced by different concentrations of CoCl2 according to the formula [ODDrug - ODBlank] / [ODControl-ODBlank] x 100%. Select the CoCl2 concentration with a significant difference in cell viability reduction compared with the control group for the next experiment.

3. Model assembly

  1. Rinse the upper chamber of the 24-well plates with PBS.
    NOTE: Cells grew and fused at the bottom of the upper chamber of the 24-well plate, and tight junctions between cells gradually formed to play a barrier role. If the endothelial cells used in some studies are not able to form a complete barrier on PET membranes independently, coating the membranes with collagen IV solution is required prior to cell inoculation.
  2. Mix the bEnd.3 cells with DMEM medium to make a suspension at a density of 5 x 105 cells/mL using a vortex mixer. Then, seed 200 µL of bEnd.3 cell suspension on the PET membrane in the upper chamber of a 24-well plate (0.33 cm2, 0.4 µm membrane pore size).
    NOTE: The pilot study of this protocol found no difference in results when using 5 to 10 passages of bEnd.3 cells for experiments. To ensure the probability of successful modeling, please refer to the cell number interval of this protocol.
  3. Add 1200 µL of complete medium to the lower chamber of the plate to ensure that the osmotic pressure of the upper and lower chambers tends to stabilize. There are differences in the volume of medium added depending on the type; ensure that the liquid level of the upper and lower chambers is flush.
  4. Change the medium of the upper chamber and the lower chamber at a fixed time every day and monitor the resistance value at the same time.
    1. When changing the medium, the integrity of the cell layer will be damaged by artificial touch or excessive movement. To avoid the above situation, slowly remove the old medium from one side with a negative pressure pipette and slowly add a new medium along the wall during fluid exchange.

4. Measurement of TEER

  1. Before work begins, place the resistor, 5% sodium hypochlorite solution, 75% ethanol, and double distilled water solution in an ultra-clean table. Turn on the UV irradiation for 30 min to eliminate residual bacteria and pathogens.
  2. Place the electrodes in 5% sodium hypochlorite solution with slow shaking for 3 s to 5 s, and then immerse in 75% ethanol for 15 min. Finally, transfer to PBS or double distilled water solution until use.
  3. Turn ON the switch on the back of the cell resistor meter, click Select Plate according to the parameters in Table 1, and select 24-Well Plate.
  4. According to the operation needs, select the appropriate detection sequence. We selected A1 procedure in this study.
  5. Insert a kΩ resistor into the right plug to calibrate the instrument. If the calibration result is 1000 ± 5 Ω, consider the accuracy of the instrument to be normal.
    1. If the calibration result is not 1000 Ω, click on Mode Units on the main interface to select OHMS, and then click Calibrate on the main screen to re-calibrate the instrument.
  6. Pull out the kΩ resistor on the right side and replace the measuring electrode with a connecting wire.
  7. Place the electrode into the 24-well plate without seeding cells vertically and click Blank Handling on the main screen of the instrument. The background value of the plate resistance without seeded cells is about 134.4 Ω.
  8. Insert the two electrodes into the upper and lower chambers of the seeded plate with cells so that the cell layer is between them, and then record the resistance value by gently stepping on the Pedal.
    1. Make sure that the electrode does not touch the cells in the upper chamber and the bottom of the lower chamber. The soaking time of the electrode in the solution has no effect on the resistance value, and it is only necessary to make sure that the electrode is in the correct position.
  9. Obtain TEER values (Ωcm2) by multiplying electrical resistance values (ohms) with the bottom area (cm2) of the upper chamber, as in the equation:
    TEER (Ωcm2) = Resistance (Ω) x Sinsert (cm2)
  10. Draw a line plot of TEER-Time (in days). When the resistance value does not increase further with time (measured over days), consider the cell formed a barrier.
    NOTW: The monolayer BBB model of bEnd.3 cells will be successfully constructed by culturing bEND.3 cells with the parameters in this study for about 6 days. The TEER of bEnd.3 cell monolayer model ranged from 16.49 ± 2.12 Ωcm2 to 27.59 ± 1.50 Ωcm2 within 6 days (n=8, mean ± SD).

5. Barrier destruction and statistical analysis

  1. According to the TEER - time curve, select the wells with barrier function and divide them into the control group and CoCl2 group (n = 4).
  2. Add the culture medium and culture medium containing 300 µM CoCl2 (200 µL) into the control group and CoCl2 group upper chamber, respectively.
  3. Detect the resistance values of the control and CoCl2 groups at 12 h and 24 h of incubation at 37 °C using the procedure described in step 4.
  4. Use commercial software to map the trend of TEER values of the barrier of cells cultured with or without 300 µM CoCl2.
  5. Use statistical analysis software to analyze the difference in resistance values between CoCl2-treated cells and normal cells (n=4, *p<0.05, **p<0.01, ***p<0.001).

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,

Disclosures

The authors have nothing to disclose.

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

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Barrier Functional IntegrityTransendothelial Electrical ResistanceTEER DetectionBlood brain BarrierBBB ModelBEnd 3 CellsDrug ScreeningPermeability TestsNeurological DisordersTight Junction ProteinsIn Vitro ModelsCentral Nervous SystemCell Barrier FunctionPharmacological Treatments

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