Name: In Vitro Blood-Brain Barrier Model Assembly for Measurement of TEER
Uploaded: 2023-09-29T13:40:00
Description: Watch this Scientific Journal Video about In Vitro Blood-Brain Barrier Model Assembly for Measurement of TEER at JoVE.com

in vitro blood-brain barrier model assembly for measurement of teer

Medición de la resistencia transendotelial para evaluar la integridad de la barrera celular

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.

Autor destacado: Aplicaciones de la detección de TEER para evaluar la integridad de la barrera celular 

Registro de la integridad funcional de la barrera en las células endoteliales vasculares bEnd.3 mediante detección de resistencia eléctrica transendotelial

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 In Vitro Blood-Brain Barrier Model Assembly for Measurement of TEER at JoVE.com

In Vitro Blood-Brain Barrier Model Assembly for Measurement of TEER  

Ensamblaje de un modelo de barrera hematoencefálica in vitro para la medición de TEER

Para comenzar, enjuague la cámara superior de las placas de 24 pocillos con PBS. Mezcle las celdas BEND3 con el medio DMEM usando un mezclador de vórtice para hacer una suspensión. A continuación, siembre 200 microlitros de suspensión celular BEND3 en la membrana de PET en la cámara superior de una placa de 24 pocillos.Agregue 1, 200 microlitros de medio completo a la cámara inferior de la placa para estabilizar la presión osmótica de las cámaras superior e inferior. Cambie el medio retirando lentamente el medio viejo de un lado con una pipeta de presión negativa y añadiendo un nuevo medio a lo largo de la pared durante el intercambio de fluidos. Antes de medir TEER, coloque la resistencia, la solución de hipoclorito de sodio al 5%, el etanol al 75% y el agua destilada doble sobre una mesa ultra limpia.Encienda la irradiación UV durante 30 minutos para eliminar las bacterias y patógenos residuales. Coloque los electrodos en una solución de hipoclorito de sodio al 5% agitando lentamente durante tres a cinco segundos. Luego sumérjalo en etanol al 75% durante 15 minutos.Finalmente, transfiera a PBS o agua destilada doble hasta su uso. A continuación, encienda el interruptor en la parte posterior del medidor de resistencia de celda. Haga clic en Seleccionar placa y Seleccionar placa de 24 pocillos.De acuerdo con la operación, seleccione la secuencia de detección adecuada. Inserte una resistencia de kilo ohmios en el enchufe derecho para calibrar el instrumento. Si el resultado de la calibración es 1000 más menos 5 ohmios, considere que el instrumento es preciso.De lo contrario, haga clic en Unidades de modo en la interfaz principal para seleccionar Ohmios y luego haga clic en Calibrar. Luego, retire la resistencia de kilo ohmios y reemplácela con el electrodo de medición con un cable de conexión. Coloque el electrodo verticalmente en la placa de 24 pocillos que contiene solo medio y haga clic en Manipulación de piezas en bruto en el instrumento.El valor de fondo de la resistencia de la placa sin celdas asentadas debe ser de aproximadamente 134,4 ohmios. A continuación, inserte los dos electrodos en las cámaras superior e inferior de la placa asentada, de modo que la capa de celda quede entre ellos. Registre el valor de resistencia pisando suavemente el pedal.Asegúrese de que el electrodo no toque las celdas de la cámara superior y la parte inferior de la cámara inferior. Obtenga los valores de TEER multiplicando los valores de resistencia eléctrica por el área inferior de la cámara superior, como se muestra en la ecuación. Dibuja un diagrama lineal de TEER frente al tiempo en días.Cuando el valor de resistencia no aumenta con el tiempo, las células han formado una barrera. Los valores de resistencia trazados en función del tiempo para las células BEND3 mostraron que los valores de TEER de las células comenzaron a estabilizarse al quinto día.

Research

JoVE Journal

Neuroscience

In Vitro Blood-Brain Barrier Model Assembly for Measurement of TEER

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 ...

Investigación

Neurociencias

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

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

Cultivo de células bEnd.3 y viabilidad celular después del tratamiento con CoCl2

Barrier Destruction and Statistical Analysis After TEER Measurement in an In Vitro Blood-Brain Barrier Model

Destrucción de la barrera y análisis estadístico después de la medición de TEER en un modelo de barrera hematoencefálica in vitro