Name: Culture of bEnd.3 Cells and Cell Viability After CoCl2 Treatment
Uploaded: 2023-09-29T13:40:00
Description: Watch this Scientific Journal Video about Culture of bEnd.3 Cells and Cell Viability After CoCl2 Treatment at JoVE.com

culture of bend.3 cells and cell viability after cocl2 treatment

Transendothelial Resistance Measurement to Assess Cell Barrier Integrity

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.

Author Spotlight: Applications of TEER Detection to Assess Cell Barrier Integrity 

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

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.

Watch this Scientific Journal Video about Culture of bEnd.3 Cells and Cell Viability After CoCl2 Treatment at JoVE.com

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.

Research

JoVE Journal

Neuroscience

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