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

跨内皮抗性测量以评估细胞屏障完整性

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.

作者聚焦：TEER 检测在评估细胞屏障完整性中的应用 

通过跨内皮电阻检测对bEnd.3血管内皮细胞进行屏障功能完整性记录

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  

bEnd.3 细胞的培养和 CoCl2 处理后的细胞活力

首先，制备含有 FBS、青霉素和链霉素的 DMEM 细胞培养基。此外，通过将 1.30 毫克氯化钴加入 100 微升 DMSO 中来制备 100 毫摩尔氯化钴储备溶液。接下来，在含有DMEM培养基的100毫升培养皿中接种一毫升BEND3细胞，并在37摄氏度的5%二氧化碳的湿润气氛中培养。每两到三天更换一次培养基，每周两次对细胞进行传代培养。BEND3细胞生长至80%汇合后，用0.25%胰蛋白酶消化细胞30秒。使用细胞计数器对细胞进行计数，并制成密度悬浮液 7 乘以每毫升 10 到第四个细胞。然后，将 100 微升该细胞悬液接种在 96 孔板中以进行细胞粘附。然后用 PBS 洗涤细胞并加入 100 微升含有氯化钴的培养基。培养细胞24小时。孵育后，取出培养基并用PBS洗涤。接下来，加入 100 微升 CCK-8 溶液。将板在 37 摄氏度下孵育一小时，然后使用酶标仪测量 450 纳米处的吸光度。根据该公式计算氯化钴诱导的细胞活力。选择与对照组相比细胞活力降低有显著差异的浓度。通过CoCl2分析了用不同浓度氯化钴处理的BEND3细胞的活力。结果表明，300微摩尔的氯化钴在体外表现出显著的细胞毒性。

culture-of-bend3-cells-and-cell-viability-after-cocl2-treatment

Research

JoVE Journal

Neuroscience

Culture of bEnd.3 Cells and Cell Viability After CoCl2 Treatment

122 次观看.  Chengdu University of Traditional Chinese Medicine. 首先，制备含有 FBS、青霉素和链霉素的 DMEM 细胞培养基。此外，通过将 1.30 毫克氯化钴加入 100 微升 DMSO 中来制备 100 毫摩尔氯化钴储备溶液。接下来，在含有DMEM培养基的100毫升培养皿中接种一毫升BEND3细胞，并在37摄氏度的5%二氧化碳的湿润气氛中培养。每两到三天更换一次培养基，每周两次对细胞进行传代培养。BEND3细胞生长至80%汇合后，用0.25%胰蛋白酶消化细胞30秒?...

视频: Culture of bEnd.3 Cells and Cell Viability After CoCl2 Treatment  

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.

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

科研

神经科学

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.

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.

用于测量TEER的体外血脑屏障模型组装

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.