Name: تسليط الضوء على المؤلف: تطبيقات اكتشاف TEER لتقييم سلامة حاجز الخلية
Uploaded: 2023-09-29T13:40:00
Description: Watch this Scientific Journal Video about Applications of TEER Detection to Assess Cell Barrier Integrity at JoVE.com

نحن نقدر الدعم المالي المقدم من المؤسسة الوطنية للعلوم الطبيعية في الصين (82274207 و 82104533) ، وبرنامج البحث والتطوير الرئيسي في نينغشيا (2023BEG02012) ، ومشروع تعزيز أبحاث Xinglin Scholar التابع لجامعة تشنغدو للطب الصيني التقليدي (XKTD2022013).

ملاحظة: تم تلقيح الخلايا البطانية المشتقة من دماغ الفأر.3 (bEnd.3) في غرف صفيحة 24 بئرا لبناء نموذج بسيط في المختبر من BBB في ظل ظروف متوسطة محددة. تم قياس TEER للخلايا الطبيعية وخلايا نقص الأكسجين بواسطة مقياس TEER (الشكل 1 والشكل 2).
1. إعداد الحل<...

قياس المقاومة عبر بطانة الرحم لتقييم سلامة حاجز الخلية

<ol>
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أحد أكثر أعضاء الجسم تطورا ، يتحكم الدماغ في مجموعة واسعة من العمليات الفسيولوجية المعقدة ، بما في ذلك الذاكرة والإدراك والسمع والشم والحركة27. الدماغ هو واحد من أكثر أعضاء جسم الإنسان تعقيدا ومرضا في نفس الوقت. يظهر حدوث العديد من اضطرابات الجهاز العصبي المركزي ميلا متزايدا ?...

BBB هي واجهة بيولوجية فريدة بين الدورة الدموية والأنسجة العصبية ، والتي تتكون من الخلايا البطانية الوعائية ، والخلايا المحيطة ، والخلايا النجمية ، والخلايا العصبية ، والهياكل الخلويةالأخرى 1. يتم تنظيم تدفق الأيونات والمواد الكيميائية والخلايا بين الدم والدماغ بشكل صارم بواسطة هذا الحاجز. يحمي هذا التوازن الأنسجة العصبية من السموم ومسببات الأمراض مع تمكين التشغيل المناسب لأعصاب الدماغ 2,3. يمكن أن يؤدي الحفاظ على سلامة BBB إلى منع تطور وتطور الاضطرابات التي تؤثر على الجهاز العصبي المركزي بشكل فعال ، مثل الخلل الوظيفي العصبي والوذمة والالتهاب العصبي4. ومع ذلك ، فإن الخصائص الفسيولوجية الفريدة ل BBB تمنع أكثر من 98٪ من الأدوية الجزيئية الصغيرة و 100٪ من الأدوية الجزيئية من دخول الجهاز العصبي المركزي5. لذلك ، فإن زيادة تغلغل الأدوية من خلال BBB أثناء تطوير الأدوية للجهاز العصبي المركزي أمر ضروري لتحقيق الفعالية العلاجية 6,7. على الرغم من أن فحص المحاكاة الحاسوبية للركائز قد زاد بشكل كبير من احتمال عبور الأدوية المرشحة ل BBB ، إلا أنه لا تزال هناك حاجة إلى نماذج BBB موثوقة وبأسعار معقولة في المختبر / في الجسم الحي لتلبية احتياجات البحث العلمي8.
تقنية سريعة وبأسعار معقولة لفحص الأدوية عالية الإنتاجية هي النموذج9 في المختبر. لإلقاء الضوء على العمليات الأساسية لتأثيرات الأدوية على وظيفة BBB ودورها في تطور المرض وتطوره ، تم إنشاء سلسلة من نماذج BBB المبسطة في المختبر. في الوقت الحاضر ، نماذج BBB الشائعة في المختبر هي النماذج أحادية الطبقة ، والثقافة المشتركة ، والديناميكية ، والموائع الدقيقة10،11،12 ، التي شيدتها الخلايا البطانية الوعائية والخلايا النجمية ، أو الخلايا المحيطة ، أو الخلايا الدبقية الصغيرة13،14. على الرغم من أن ثقافات الخلايا ثلاثية الأبعاد تتماشى بشكل أكبر مع التركيب الفسيولوجي ل BBB15 ، إلا أن تطبيقها كوسيلة لفحص الأدوية ل BBB لا يزال مقيدا بتصميمها المعقد واستنساخها دون المستوى. في المقابل ، فإن نموذج الطبقة الأحادية في المختبر هو النموذج الأكثر استخداما للبحث في BBB وهو قابل للتطبيق لتحديد تعبير ناقلات الغشاء وبروتينات الوصلة الضيقة في خلايا معينة.
قياس المقاومة الكهربائية عبر الغشاء (TEER) هو تقنية لتقييم ومراقبة طبقة الخلايا عبر المقاومة وتقييم سلامة الخلية ونفاذية الحاجز. من خلال إدخال قطبين كهربائيين في وقت واحد في وسط النمو أو محلول المخزن المؤقت على جانبي الطبقة الأحادية ، من الممكن قياس التيار المتردد أو المعاوقة الكهربائية من خلال الطبقة المدمجة للخلية16,17. من أجل تحديد ما إذا كان نموذج BBB في المختبر قد تم إنشاؤه بشكل صحيح ، سيتم استخدام قياس TEER عادة كمعيار ذهبي18. من ناحية أخرى ، يمكن التنبؤ بدقة باتجاه عمل الدواء على نفاذية BBB عن طريق قياس التغير في المقاومة الكهربائية لطبقة الخلية بعد إصابة الدواء19. على سبيل المثال ، اكتشف Feng et al. أن catalpol (المونومر النشط الأساسي ل rehmanniae) يمكن أن يعكس بشكل فعال التنظيم السفلي الناجم عن عديد السكاريد الشحمي لبروتينات الوصلات الضيقة في BBB ويرفع قيمة TEER لطبقة الخلايا البطانية لدماغ الفأر20.
عادة ما تكون الاستجابة الالتهابية العصبية هي السبب الرئيسي لاختلال توازن BBB21. علاج نقص الأكسجين للحث على إصابة التهاب الأعصاب هو الطريقة الرئيسية لتدمير الحاجز الدموي الدماغي ، بما في ذلك الطرق الفيزيائية وطرق الكاشف الكيميائي. يستخدم الأول في المقام الأول حاضنة ثلاثية الغازات لتغيير محتوى الأكسجين في بيئة نمو الخلايا لمحاكاة ظروف نقص الأكسجين22 ، بينما يتم تحقيق الأخير عن طريق إدخال كواشف deoxy بشكل مصطنع مثل CoCl2 إلى وسط زراعة الخلايا23. ستبقى الخلايا في حالة غير مؤكسجة إذا تم استبدال Fe2+ ب Co2+ في الهيم. إذا تم استبدال Fe2+ ب Co2+ في المجموعة التحفيزية ، تثبيط نشاط هيدروكسيلاز البرولين وهيدروكسيلاز الأسبارتات ، مما يؤدي إلى تراكم العامل المحرض لنقص الأكسجة -1α (HIF-1α)24. في ظل نقص الأكسجة المستمر ، يؤدي نزع الفسفرة من HIF-1α في السيتوبلازم إلى موت الخلايا وينشط عامل نمو بطانة الأوعية الدموية ، مما يؤدي في النهاية إلى زيادة نفاذية الأوعية الدموية. في الدراسات السابقة25،26 ، ثبت جيدا أن نقص الأكسجة يمكن أن يقلل بشكل كبير من التعبير عن بروتينات الوصلات الضيقة البطانية لزيادة نفاذية BBB. في هذه الدراسة ، تم قياس منحنى مقاومة الوقت لخلايا bEnd.3 المصنفة في 24 لوحة بئر من أجل إنشاء نموذج BBB مباشر. باستخدام هذا النموذج ، قمنا بتمييز التغييرات في TEER الخلوي بعد تدخل CoCl2 من أجل بناء نموذج خلية يمكن استخدامه لفحص الأدوية لحماية BBB.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24-well transwell plate</td>
			<td>Corning (Corning 3470, 0.33 cm2, 0.4 &#181;m)</td>
			<td>10522023</td>
			<td></td>
		</tr>
		<tr>
			<td>75 % ethanol</td>
			<td>ChengDu Chron Chemicals Co,.Ltd</td>
			<td>2023052901</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Guangzhou Jet Bio-Filtration Co., Ltd</td>
			<td>220412-078-B</td>
			<td></td>
		</tr>
		<tr>
			<td>bEnd.3 cells</td>
			<td>Hunan Fenghui Biotechnology Co., Ltd</td>
			<td>CL0049</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell counting kit-8 (CCK-8)</td>
			<td>Boster Biological Technology Co., Ltd</td>
			<td>BG0025</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture dish (100mm)</td>
			<td>Zhejiang Sorfa Life Science Research Co., Ltd</td>
			<td>1192022</td>
			<td></td>
		</tr>
		<tr>
			<td>Cobalt Chloride (CoCl2)</td>
			<td>Sigma</td>
			<td>15862</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Boster Biological Technology Co., Ltd</td>
			<td>PYG0040</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified eagle medium (1x)</td>
			<td>Gibco ThermoFisher Scientific</td>
			<td>8121587</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco ThermoFisher Scientific</td>
			<td>2166090RP</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism software</td>
			<td>GraphPad Software</td>
			<td>9.0.0(121)</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel (Contains collagen IV)</td>
			<td>MedChemexpress</td>
			<td>HY-K6002</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Molecular Devices</td>
			<td>SpectraMax iD5</td>
			<td></td>
		</tr>
		<tr>
			<td>OriginPro 8 software</td>
			<td>OriginLab Corporation</td>
			<td>v8.0724(B724)</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (100x)</td>
			<td>Boster Biological Technology Co., Ltd</td>
			<td>17C18B16</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS, 1x)</td>
			<td>Gibco ThermoFisher Scientific</td>
			<td>8120485</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hypochlorite</td>
			<td>ChengDu Chron Chemicals Co,.Ltd</td>
			<td>2022091501</td>
			<td></td>
		</tr>
		<tr>
			<td>Transmembrane resistance meter</td>
			<td>World Precision Instruments LLC</td>
			<td>VOM3 (verison 1.6)</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin 0.25% (1x)</td>
			<td>HyClone</td>
			<td>J210045</td>
			<td></td>
		</tr>
	</tbody>
</table>

barrier functional integrity recording on bend.3 vascular endothelial cells via transendothelial electrical resistance detection

الحاجز الدموي الدماغي (BBB) هو هيكل فسيولوجي ديناميكي يتكون من الخلايا البطانية الوعائية الدقيقة والخلايا النجمية والخلايا المحيطة. من خلال تنسيق التفاعل بين العبور المقيد للمواد الضارة ، وامتصاص المغذيات ، وإزالة المستقلب في الدماغ ، فإن BBB ضروري في الحفاظ على توازن الجهاز العصبي المركزي. يعد بناء نماذج في المختبر من BBB أداة قيمة لاستكشاف الفيزيولوجيا المرضية للاضطرابات العصبية وإنشاء علاجات دوائية. تصف هذه الدراسة إجراء لإنشاء نموذج خلية BBB أحادي الطبقة في المختبر عن طريق زرع خلايا bEnd.3 في الغرفة العلوية لصفيحة 24 بئرا. لتقييم سلامة وظيفة حاجز الخلية ، تم استخدام مقياس الفولتميتر الخلوي الظهاري التقليدي لتسجيل المقاومة الكهربائية عبر الغشاء للخلايا الطبيعية وخلايا نقص الأكسجين التي يسببها CoCl2 في الوقت الفعلي. نتوقع أن توفر التجارب المذكورة أعلاه أفكارا فعالة لإنشاء نماذج في المختبر من BBB والأدوية لعلاج اضطرابات أمراض الجهاز العصبي المركزي.

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.

تسليط الضوء على المؤلف: تطبيقات اكتشاف TEER لتقييم سلامة حاجز الخلية 

تسجيل السلامة الوظيفية للحاجز على bEnd.3 الخلايا البطانية الوعائية عبر اكتشاف المقاومة الكهربائية عبر بطانة الرحم

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.

سمح هذا البروتوكول بتسجيل التغييرات في قيم مقاومة الخلايا وفقا للمعايير المحددة في مقياس المقاوم عبر البطانة. تم فحص صلاحية خلايا bEnd.3 (عدد الخلايا الحية) المعالجة بتركيزات مختلفة من CoCl2 بواسطة مقايسة CCK-8. تم تمثيل تلف الخلايا الأكبر الناتج عن CoCl2 من خلال انخفاض صلاحية الخلية. وج...

Watch this Scientific Journal Video about Applications of TEER Detection to Assess Cell Barrier Integrity at JoVE.com

يصف هذا البروتوكول نموذجا يمكن الاعتماد عليه وفعالا في المختبر لحاجز الدم في الدماغ. تستخدم الطريقة الخلايا البطانية الوعائية الدماغية للفأر bEnd.3 وتقيس المقاومة الكهربائية عبر الغشاء.

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

تستخدم الدراسات الحالية عادة اختبارات النفاذية والتعبير البروتيني المرتبط بها للتحقق من دقة اكتشاف TEER. نموذج BBB للخلايا أحادية الطبقة في الفضيلة أقل فعالية لفحص المخدرات ، واستخدام المزيد من الخلايا يجعل من الصعب بناء النموذج. بروتوكول هذه الدراسة بسيط نسبيا ، حيث يسمح الإجراء غير الجراحي باستخدام عينات الخلايا في تجارب أخرى بعد اكتشاف TEER.يمكن أن يؤدي اكتشاف TEER لتقييم نفاذية نماذج الخلايا إلى تسريع فحص الأدوية للعلاج والوقاية من الجهاز العصبي المركزي لزيادة معدل نجاح تطوير دواء جديد. في المستقبل ، سنكتشف تعبير بروتين الوصلة الضيقة في خلايا الجزء العلوي من جدار الصدر للتحقق من أن نتائج اختبار TEER يمكن أن تمثل بشكل فعال سلامة وظيفة حاجز BBB.

barrier-functional-integrity-recording-on-bend3-vascular-endothelial

Research

JoVE Journal

Neuroscience

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.

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.

ثقافة خلايا bEnd.3 وصلاحية الخلية بعد علاج CoCl2

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.

في المختبر الجمعية النموذجية للحاجز الدموي الدماغي لقياس TEER

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.