Name: 著者スポットライト:細胞バリアの完全性を評価するためのTEER検出の応用
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

中国国家自然科学基金会(82274207および82104533)、寧夏回族自治区重点研究開発プログラム(2023BEG02012)、成都中華大学興林奨学生研究促進プロジェクト(XKTD2022013)からの財政的支援に感謝します。

注:マウス脳由来内皮細胞.3(bEnd.3)を24ウェルプレートのチャンバーに接種し、特定の培地条件下でBBBの単純な in vitro モデルを構築しました。正常細胞と低酸素細胞のTEERをTEERメーターで測定しました(図1 、 図2)。
1. 溶液調製
<ol><li>FBS(10%、v/v)、100 U/mLペニシリン、および10 mg/mLストレプトマイシンを含むDMEM?...

細胞バリアの完全性を評価するための経内皮抵抗測定

<ol>
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最も発達した身体器官の1つである脳は、記憶、認知、聴覚、嗅覚、運動など、さまざまな複雑な生理学的プロセスを制御しています27。脳は、人体で最も複雑で病気の器官の1つであると同時に、人体で最も複雑で病気の器官の1つです。多くの中枢神経系疾患の発生は、大気汚染、不規則な食事パターン、およびその他の要因を含む要因により、年々増加傾向を示していま?...

BBBは、血管内皮細胞、周皮細胞、星状膠細胞、ニューロン、その他の細胞構造で構成される血液循環と神経組織の間のユニークな生物学的インターフェースです1。血液と脳の間のイオン、化学物質、細胞の流れは、このバリアによって厳密に制御されています。このホメオスタシスは、毒素や病原体から神経組織を保護すると同時に、脳の神経の適切な操作を可能にします2,3。BBBの完全性を維持することで、神経機能障害、浮腫、神経炎症など、中枢神経系に影響を与える疾患の発症と進行を効果的に防ぐことができます4。しかし、BBBのユニークな生理学的特性により、低分子医薬品の98%以上、高分子医薬品の100%が中枢神経系に入るのを防ぎます5。したがって、中枢神経系の薬剤の開発中にBBBを介した薬剤の浸透性を高めることは、治療効果を達成するために不可欠です6,7。基質のコンピューターシミュレーションスクリーニングにより、医薬品候補がBBBを通過する確率が大幅に向上しましたが、科学研究のニーズを満たすためには、信頼性が高く手頃な価格のin vitro/in vivo BBBモデルが依然として必要です8。
ハイスループット薬物スクリーニングのための迅速で手頃な技術は、in vitroモデル9です。医薬品がBBB機能に及ぼす影響の基本的なプロセスと、疾患の発症と進行における医薬品の役割を明らかにするために、一連の簡略化されたin vitro BBBモデルが作成されました。現在、一般的なin vitro BBBモデルは、血管内皮細胞とアストロサイト、ペリサイト、またはミクログリアによって構築される単層、共培養、動的、およびマイクロ流体モデル10,11,12である13,14。3D細胞培養はBBB15の生理学的構造とほぼ一致していますが、BBBの薬物スクリーニングの手段としての応用は、その複雑なデザインと標準以下の再現性によって依然として制約されています。対照的に、単分子膜in vitroモデルは、BBBの研究に最も頻繁に使用されるモデルであり、特定の細胞における膜トランスポーターおよびタイトジャンクションタンパク質の発現を決定するために適用できます。
膜貫通電気抵抗(TEER)測定は、抵抗を横切る細胞層を評価および監視し、バリアの細胞の完全性と透過性を評価する技術です。単分子膜の両側にある増殖培地または緩衝液に2つの電極を同時に挿入することにより、セルのコンパクト層16,17を通る交流または電気インピーダンスを測定することができる。in vitro BBBモデルが適切に作成されているかどうかを判断するために、TEERの測定は通常、ゴールドスタンダード18として採用されます。一方、BBB透過性に対する薬物作用の傾向は、薬物関与後の細胞層の電気抵抗の変化を測定することによって正確に予測することができる19。例えば、Fengらは、カタルポール(レフマニアエの主要な活性モノマー)が、BBBのタイトジャンクションタンパク質のリポ多糖誘導性ダウンレギュレーションを効果的に逆転させ、マウス脳内皮細胞層のTEER値を上昇させることができることを発見しました20。
神経炎症反応は、通常、BBBホメオスタシスの不均衡の主な原因である21。神経炎症性損傷を誘発する低酸素治療は、血液脳関門を破壊する主な方法であり、主に物理的方法と化学試薬法が含まれます。前者は、主に3ガスインキュベーターを利用して、細胞増殖環境中の酸素含有量を変化させて低酸素状態をシミュレートし22、後者は、CoCl2などのデオキシ試薬を細胞培養培地に人工的に導入することによって達成される23。ヘム中のCo2+がFe2+に置換されている場合、細胞は脱酸素状態のままになります。触媒基でFe2+がCo2+に置換されると、プロリンヒドロキシラーゼとアスパラギン酸ヒドロキシラーゼ活性が阻害され、低酸素誘導因子-1α(HIF-1α)が蓄積する24。持続的な低酸素状態では、細胞質におけるHIF-1αの脱リン酸化が細胞死を引き起こし、血管内皮増殖因子を活性化し、最終的に血管透過性を高めます。以前の研究25,26では、低酸素症が内皮タイトジャンクションタンパク質の発現を大幅に低下させてBBBの透過性を高めることが十分に実証されています。本研究では、24ウェルプレートに播種したbEnd.3細胞の時間抵抗曲線を測定し、簡単なBBBモデルを作成しました。このモデルを用いて、CoCl2介入後の細胞TEERの変化を特徴づけ、BBB保護のための薬物のスクリーニングに使用できる細胞モデルを構築しました。

<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

血液脳関門(BBB)は、微小血管内皮細胞、星状細胞、および周皮細胞で構成される動的な生理学的構造です。BBBは、有害物質の輸送制限、栄養素の吸収、および脳内の代謝産物クリアランスの間の相互作用を調整することにより、中枢神経系の恒常性を維持するために不可欠です。BBBの in vitro モデルの構築は、神経疾患の病態生理学を探求し、薬理学的治療法を作成するための貴重なツールです。この研究では、bEnd.3細胞を24ウェルプレートの上部チャンバーに播種することにより、 in vitro 単層BBB細胞モデルを作成する手順について説明します。細胞バリア機能の完全性を評価するために、従来の上皮細胞電圧計を使用して、正常細胞とCoCl2誘導低酸素細胞の膜貫通電気抵抗をリアルタイムで記録しました。以上の実験は、BBBの in vitro モデルや中枢神経系疾患の治療薬の創製に有効なアイデアを提供することが期待されます。

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.

著者スポットライト:細胞バリアの完全性を評価するためのTEER検出の応用 

経内皮電気抵抗検出 による bEnd.3血管内皮細胞のバリア機能完全性記録

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.

このプロトコルにより、経内皮抵抗計に設定されたパラメータに従って、細胞の抵抗値の変化を記録することができました。異なる濃度のCoCl2で処理したbEnd.3細胞(生細胞数)の生存率をCCK-8アッセイでスクリーニングしました。CoCl2 によって引き起こされる細胞損傷が大きいほど、細胞生存率が低くなることが示されました。300 μMのCoCl2がin vitroで有意に細胞傷?...

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

このプロトコルは脳の血液関門の信頼できる、有効な 生体外 モデルを記述する。この方法では、マウスの脳血管内皮細胞bEnd.3を使用し、膜貫通電気抵抗を測定します。

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

現在の研究では、通常、透過性試験と関連するタンパク質発現を使用して、TEER検出の精度を検証しています。単層細胞のBBBモデルは、薬物スクリーニングにはあまり効果がなく、より多くの細胞を使用すると、モデルの構築が難しくなります。この研究のプロトコルは、非侵襲的な手順により、TEER検出後に細胞サンプルを他の実験で使用できるため、比較的簡単です。細胞モデルの透過性を評価するためのTEERの検出は、中枢神経系の治療と予防のための薬物のスクリーニングを加速し、新薬開発の成功率を高めることができます。将来的には、胸壁の上部コンパートメント細胞におけるタイトジャンクションタンパク質の発現を検出し、TEERテスト結果がBBBバリア機能の完全性を効果的に表すことができることを確認します。

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.

神経科学

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.

bEnd.3細胞の培養とCoCl2 処理後の細胞生存率

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.

TEER測定のためのin vitro血液脳関門モデルアセンブリ

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.