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

Hücre Bariyeri Bütünlüğünü Değerlendirmek için Transendotelyal Direnç Ölçümü

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.

Yazar Spotlight: Hücre Bariyeri Bütünlüğünü Değerlendirmek için TEER Algılama Uygulamaları 

bEnd.3 Transendotelyal Elektriksel Direnç Tespiti Yoluyla Vasküler Endotel Hücrelerinde Bariyer Fonksiyonel Bütünlük Kaydı

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  

TEER Ölçümü için In Vitro Kan-Beyin Bariyeri Model Düzeneği

Başlamak için, 24 oyuklu plakanın üst odasını PBS ile durulayın. Bir süspansiyon yapmak için bir vorteks karıştırıcı kullanarak BEND3 hücrelerini DMEM ortamı ile karıştırın. Daha sonra, 24 oyuklu bir plakanın üst odasındaki PET membran üzerine 200 mikrolitre BEND3 hücre süspansiyonu tohumlayın.Üst ve alt odaların ozmotik basıncını stabilize etmek için plakanın alt odasına 1.200 mikrolitre tam ortam ekleyin. Sıvı değişimi sırasında eski ortamı bir taraftan negatif basınçlı bir pipetle yavaşça çıkararak ve duvar boyunca yeni bir ortam ekleyerek ortamı değiştirin. TEER'i ölçmeden önce direnci, %5 sodyum hipoklorit çözeltisini, %75 etanolü ve çift damıtılmış suyu ultra temiz bir masaya yerleştirin.Artık bakteri ve patojenleri ortadan kaldırmak için UV ışınlamasını 30 dakika boyunca açın. Elektrotları% 5 sodyum hipoklorit çözeltisine üç ila beş saniye boyunca yavaşça çalkalayarak yerleştirin. Daha sonra 15 dakika boyunca% 75 etanole daldırın.Son olarak, kullanana kadar PBS'ye veya çift damıtılmış suya aktarın. Ardından, hücre direnç ölçerin arkasındaki anahtarı açın. Plaka Seç'e tıklayın ve 24 kuyulu plakayı seçin.İşleme göre uygun algılama sırasını seçin. Cihazı kalibre etmek için sağ fişe bir kilo ohm direnç takın. Kalibrasyon sonucu 1000 artı eksi 5 ohm ise, cihazın doğru olduğunu düşünün.Değilse, Ohm'u seçmek için ana arayüzde Mod Birimleri'ne tıklayın ve ardından Kalibre Et'e tıklayın. Ardından, kilo ohm direncini çıkarın ve bir bağlantı kablosu kullanarak ölçüm elektroduyla değiştirin. Elektrotu yalnızca orta içeren 24 oyuklu plakaya dikey olarak yerleştirin ve cihaz üzerinde Boş Kullanım'a tıklayın.Oturmuş hücreler olmadan plaka direncinin arka plan değeri yaklaşık 134.4 ohm olmalıdır. Daha sonra, iki elektrotu, hücre tabakası aralarında olacak şekilde, oturmuş plakanın üst ve alt odalarına yerleştirin. Pedala hafifçe basarak direnç değerini kaydedin.Elektrotun üst bölmedeki ve alt bölmenin altındaki hücrelere temas etmediğinden emin olun. Denklemde gösterildiği gibi elektrik direnç değerlerini üst haznenin alt alanı ile çarparak TEER değerlerini elde edin. TEER'in gün cinsinden zamana karşı bir çizgi grafiği çizin.Direnç değeri zamanla artmadığında, hücreler bir bariyer oluşturmuştur. BEND3 hücreleri için zamana karşı çizilen direnç değerleri, hücre TEER değerlerinin beşinci günde stabilize olmaya başladığını gösterdi.

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 ...