Name: in vitro assays to assess blood-brain barrier mesh-like vessel formation and disruption
Uploaded: 2017-06-20T14:00:00
Description: Watch this Scientific Journal Video about In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption at JoVE.com

レオン・タイは、イリノイ大学シカゴの創業資金によって資金提供を受けています。

すべての実験は、イリノイ大学シカゴ研究所の動物用ケアおよび使用委員会のプロトコールに従う。 1.一般的な準備注：脳微小血管内皮細胞系（hCMEC / D3）は、広範に特徴付けられた不死化ヒトBEC系統14,15,16,18,19である。基底内皮成長基質培地中のコラーゲンタイプI（子牛皮膚、Ca 2+およびMg 2+を含有するハンクス平衡塩類溶液（HBSS）?...

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記載された方法は、脳血管カバレッジを取り巻くいくつかの基本的な生物学的問題に対処するために利用することができる24 。具体的には、どの受容体およびシグナル伝達経路が、血管新生、癌組織における血管カバレッジ、および脳に関連する末梢内皮細胞において役割を果たすかを同定することができる。例としては、血管新生増殖因子受容体、酸化窒素、マイトジェ?...

<html>脳毛細血管の血液脳関門（BBB）は、血液と脳の接触の最大の界面であり、中枢神経系（CNS） 1,2の恒常性において中心的な役割を果たす。 BBBでのダイナミックなプロセスは、血液からの不要な分子の取り込みを防ぎ、中枢神経系からの老廃物を除去し、必須の栄養素とシグナル伝達分子をCNSに供給し、神経炎症を調節する1,2,3,4,5。 BBB損傷は、加齢およびアルツハイマー病（AD）、多発性硬化症および脳卒中1,2,3,4,5,6,7,8,9,10,11,12,13,14 、ass = &quot;xref&quot;&gt; 6。したがって、BBB機能不全は、治療標的を含む神経変性疾患において重要な役割を果たす可能性がある。 船舶のカバレッジを維持することは、BBBの恒常性機能にとって重要である。しかし、 インビボおよびインビトロでのデータ衝突は、神経変性疾患に関与するプロセスが、特にADにおいて、BBB適用範囲を6,7,8,9,10,11,12,13より高くまたは低くするかどうかについて競合する。したがって、BBBカバレッジのダイナミクスを評価し、より包括的に理解するために、関連する細胞タイプを使用したインビトロモデルの開発には強い根拠があります。脳毛細血管は、星状細胞、周皮細胞および脳内皮細胞（BEC）から構成され、 3 。一般に、BBB機能を評価するための細胞培養技術は、ストレッサー14,15,16の添加後の両方において、フィルターインサート上で増殖させた細胞、または重要なBECタンパク質のレベルを評価する浸透性アッセイである。重要ではあるが、これらのアッセイは脳血管カバレッジに焦点を当てていない。 ここで、我々の以前の方法17は、不死化ヒトBECの単一培養、初代マウスBECの単一培養、およびヒト化三重培養を使用して、BEC適用範囲および網状様構造を評価するために詳述されているBBBのモデル（BEC、星状細胞および周皮細胞）を同定した。目標は、AECがAD進展の重要な要因であると考えられている有害作用がBECの対象範囲に及ぼす影響を実証することでした。表皮成長因子（EGF）の保護効果は、治療スクリーニングツールとしてのこの技術の可能性を強調している。この技術は、1）血管新生および血管カバレッジにおける特定の経路の役割の描写、2）血管新生および血管カバレージに対する疾患および加齢関連因子の影響の評価、および3）薬理学的目標。

<table border="0" cellpadding="0" cellspacing="0" width="847">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">hCMEC/D3 cells</td>
			<td style="width:183px;">Milipore</td>
			<td style="width:119px;">SCC066</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">EBM-2 basal media&#160;</td>
			<td style="width:183px;">Lonza</td>
			<td>CC-3156</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Collagen Type 1</td>
			<td style="width:183px;">ThermoFisher</td>
			<td style="width:119px;">A1064401&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">HBSS, calcium, magnesium, no phenol red</td>
			<td style="width:183px;">ThermoFisher</td>
			<td style="width:119px;">14025092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">HBSS, no calcium, no magnesium, no phenol red</td>
			<td style="width:183px;">ThermoFisher</td>
			<td style="width:119px;">14175095</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Trypsin-EDTA (0.25%)</td>
			<td style="width:183px;">ThermoFisher</td>
			<td style="width:119px;">25200056</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:379px;">Final concentrations of the SingleQuot growth factor supplements for EBM2 media</td>
			<td style="width:183px;">Lonza</td>
			<td>CC-4147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5% FBS</td>
			<td style="width:183px;">Lonza</td>
			<td>CC-4147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10% Ascorbic acid</td>
			<td style="width:183px;">Lonza</td>
			<td>CC-4147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10% Gentamycin sulphate</td>
			<td style="width:183px;">Lonza</td>
			<td>CC-4147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">25% Hydrocortisone</td>
			<td style="width:183px;">Lonza</td>
			<td>CC-4147</td>
			<td></td>
		</tr>
		<tr height="78">
			<td height="78" style="height:78px;width:379px;">1/4 volume of the supplied growth factors: fibroblast growth factor, epidermal growth factor, insulin-like growth factor, vascular endothelial growth factor</td>
			<td style="width:183px;">Lonza</td>
			<td>CC-4147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Puromycin hydrochloride</td>
			<td style="width:183px;">VWR</td>
			<td style="width:119px;">80503-312</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">MEM-HEPES&#160;</td>
			<td style="width:183px;">Thermo Scientific&#160;</td>
			<td style="width:119px;">12360-038</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Papain cell dissociation system (papain and DNase1)</td>
			<td style="width:183px;">Worthington Biochemical</td>
			<td style="width:119px;">LK003150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Human pericytes</td>
			<td style="width:183px;">Sciencell</td>
			<td>1200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Pericyte basal media</td>
			<td style="width:183px;">Sciencell</td>
			<td>1201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Pericyte growth supplement</td>
			<td style="width:183px;">Sciencell</td>
			<td>1252</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Human Astrocytes</td>
			<td style="width:183px;">Sciencell</td>
			<td>1800</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Astrocyte media</td>
			<td style="width:183px;">Sciencell</td>
			<td>1801</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Astrocyte growth supplement</td>
			<td style="width:183px;">Sciencell</td>
			<td>1852</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Basement membrane (Matrigel Growth Factor Reduced)&#160;</td>
			<td style="width:183px;">Corning</td>
			<td style="width:119px;">356231</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Angiogenesis m-plates (96-well)</td>
			<td style="width:183px;">ibidi</td>
			<td style="width:119px;">89646</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Human Epidermal growth factor&#160;</td>
			<td style="width:183px;">Shenendoah Biotechnology</td>
			<td style="width:119px;">100-26</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellTracker green&#160;</td>
			<td style="width:183px;">ThermoFisher</td>
			<td>C7025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellTracker orange</td>
			<td style="width:183px;">ThermoFisher</td>
			<td>C34551</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellTracker blue&#160;</td>
			<td style="width:183px;">ThermoFisher</td>
			<td>C2110</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Poly-l-lysine</td>
			<td style="width:183px;">Sciencell</td>
			<td>0403</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">10% Neutral Buffered Formalin</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td>HT5012-60ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">C57BL mice</td>
			<td style="width:183px;">Jackson Laboratory</td>
			<td style="width:119px;">na</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">PCR tube strips</td>
			<td style="width:183px;">GeneMate</td>
			<td style="width:119px;">T-3014-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Zeiss stereo discover v.8 dissecting microscope</td>
			<td style="width:183px;">Zeiss</td>
			<td style="width:119px;">na</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro assays to assess blood-brain barrier mesh-like vessel formation and disruption

血液脳関門（BBB）の適用範囲は、中枢神経系（CNS）の恒常性において中心的な役割を果たす。 BBBは、星状細胞、周皮細胞および脳内皮細胞（BEC）によって動的に維持される。ここでは、不死化ヒトBECの単一培養、初代マウスBECの単一培養、およびBBBのヒト化三重培養モデル（BEC、星状細胞および周皮細胞）を使用して、BBBカバレッジを評価する方法を詳述する。疾患状態に対するアッセイの適用性を強調するために、我々は、BBB適用範囲におけるアルツハイマー病（AD）進行の重要な要因であるオリゴマーアミロイドβ（oAβ）の効果を記載する。さらに、本発明者らは、この技術の薬物スクリーニング能力を明らかにするために、上皮増殖因子（EGF）を利用する。本発明者らの結果は、単一および三重培養BECが基底条件下で網目様構造を形成し、oAβがこの細胞網形成を破壊し、予備形成された網構造を変性させ、EGFはこの中断を阻止します。したがって、記載された技術は、BBBカバレッジを調節する基本的かつ疾患関連プロセスを解剖するために重要である。

単一培養では、hCMEC / D3細胞（ 図3A ）および初代マウスBEC（ 図3B ）の両方が、ウェル全体にわたって網目状の構造を形成する。構造は、相互リンクされたノードの網目構造によって特徴付けられます （ 図3 ）。記載されたすべてのパラダイム（ 図1 ）にお...

Watch this Scientific Journal Video about In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption at JoVE.com

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

血液脳関門のカバレッジを維持することは、中枢神経系の恒常性にとって重要である。このプロトコルは、血液脳関門のカバレッジを調節する基本的および病理学的プロセスを描写するためのインビトロ技術を記載する 。

血液脳関門メッシュ様血管形成および崩壊を評価するためのインビトロアッセイ

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by ...

The goals of this assay are to dissect the role of disease-relevant processes in blood brain-barrier coverage disruption, identify the underlying mechanisms, and test potential treatments in vitro.This method can answer key basic and applied research questions in the blood-brain barrier and neuro-degeneration fields.For example, by addressing whether disease-relevant stresses disrupt preformed network structures and identifying the underlying pathways.Through further screening, treatments can be identified that prevent stress-induced blood-brain barrier disruption for future in vivo testing.The implications of this technique extend towards mechanistic understanding of and treatment for Alzheimer's disease, because of the importance of the homeostatic functions of the blood-brain barrier on cognition.Though this method can provide insight into the blood-brain barrier dysfunction in Alzheimer's disease, it can also be applied to other neurodegenerative disorders with blood-brain barrier dysfunction, such as stroke.Begin by passaging hCMEC/D3 immortal endothelial cells at a ratio of one to five by incubating the cell sheet with calcium and magnesium-free HBSS for three minutes.Then detach the cells using five milliliters of 0.25%trypsin EDTA for five minutes.Neutralize the trypsin by adding five milliliters of EBM-2 complete medium.Collect the cells, and centrifuge at 290 times g for five minutes at four degrees Celsius.Following centrifugation, discard the supernatant, and resuspend the pellet in EBM-2 complete medium.Then, replate the cells at a one to five ratio.To prepare endothelial cells from the mouse brain, remove the brain stems and cerebella from seven euthanized mice with forceps.Then, detach the meninges by carefully rolling the brains on gauss.Then, place the brain tissue into a petri dish containing minimal essential medium with HEPES, and use a sterile razor blade to mince the remaining brain tissue.Then transfer the minced brain tissue to a 15 milliliter conical tube.And centrifuge at 290 times g for five minutes at four degrees Celsius.After removing the supernatant, add papain and DNase solution, and incubate in a 37 degree Celsius water bath for one hour to digest the tissue.Following the incubation, triturate the homogenate using a 19-gauge needle, and then a 21-gauge needle.Once triturated, add one volume of 22%bovine serum albumin solution.Then centrifuge at 1, 360 times g for 10 minutes at four degrees Celsius.Resuspend the resulting pellet in one milliliter of EBM-2 complete, and then centrifuge at 290 times g for five minutes at four degrees Celsius.After the centrifugation, again use one milliliter of EBM-2 complete medium to resuspend the cells, and plate the cells at one milliliter per well in collagen-coated, six-well plates.24 hours later, replace the medium with EBM-2 complete medium containing four micrograms per milliliter of puromycin hydrochloride, and continue to culture the cells until they reach full confluence at one times 10 to the fifth cells per square centimeter.Lastly, 24 hours prior to the assay, incubate the fully confluent brain endothelial cells in EBM-2 basal medium.On the day of the assay, pipette 10 microliters of basement membrane matrix into each well of a 96-well plate, and allow to set for one hour at 37 degrees Celsius.Suspend the lyophilized green cell tracking dye in 10 microliters of DMSO to generate a 10-millimolar stock solution.Dilute this solution to 10-micromolar in EBM-2 basal medium.Remove the medium from the six-well plate containing brain endothelial cells, and replace with EBM-2 basal containing the green cell tracking dye.Incubate the cells for 20 to 30 minutes at 37 degrees Celsius.Following the incubation, remove the medium with cell tracking dye, wash the cell sheet, and attach the cells with trypsin.Neutralize the trypsin with EBM-2 containing 10%FBS, and transfer the cells to centrifuge tubes.Centrifuge at 240 time g for five minutes at four degrees Celsius.Following centrifugation, resuspend the cell pellets in one milliliter of EBM-2 basal medium, plate the labeled cells in multiple 96-well plates at 10, 000 cells per well, and return the cells to the incubator until oligomeric oa

Research

JoVE Journal

Medicine

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

ラットモデルでの経頭蓋集束超音波を用いた血液脳関門のMRI誘導破壊

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a ...

医学

Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics

血液脳関門の破壊のラットモデルは、対象となる神経血管治療を許可する

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain ...

An in vivo Assay to Test Blood Vessel Permeability

An in vivo Assay to Test Blood Vessel Permeability

アン<em生体内で&gt;</em血管透過性をテストする&gt;アッセイ

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under ...

Stretch in Brain Microvascular Endothelial Cells (cEND) as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

Stretch in Brain Microvascular Endothelial Cells cEND as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

として脳微小血管内皮細胞（CEND）でストレッチ<em&gt;インビトロ</em血液脳関門の&gt;外傷性脳損傷モデル

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying ...

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

Setting-up an In Vitro Model of Rat Blood-brain Barrier BBB: A Focus on BBB Impermeability and Receptor-mediated Transport

設定アップ<em&gt;体外</emラット血液脳関門の&gt;モデル（BBB）：BBB不透過性および受容体媒介輸送に注力

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and ...

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

小説<em&gt; in vivoで</em&gt;遺伝子導入技術と<em&gt; in vitroで</em筋骨格障害における骨損失の検討のための&gt;細胞ベースのアッセイ

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

などの酸素グルコース欠乏および再酸素化<em&gt;インビトロ</em&gt;虚血再灌流傷害モデル血液脳関門の機能障害を研究するための

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

ヒト胎盤生理学の統合研究への4容器サンプリングアプローチ<em&gt;インビボ</em

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

体外血液脳関門ブタ脳血管内皮細胞に基づくモデルの確立のための改良法

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Micro-dissection of Enamel Organ from Mandibular Incisor of Rats Exposed to Environmental Toxicants

環境有害物質に暴露したラットの下顎からエナメル器官の微細解剖

Enamel defects resulting from environmental conditions and ways of life are public health concerns because of their high prevalence. These defects result ...