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

레온 타이 (Leon Tai)는 일리노이 대학 시카고 스타트 업 펀드가 자금을 지원합니다.

모든 실험은 시카고 기관 동물 관리 및 사용위원회의 일리노이 대학 (University of Illinois)을 따른다. 1. 일반 준비 참고 : 뇌 미세 혈관 내피 세포주 (hCMEC / D3)는 광범위하게 특성화 된 불멸화 인간 BEC 라인 14 , 15 , 16 , 18 , 19 입니다. 기저...

<ol>
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기술 된 방법은 뇌 혈관 커버리지를 둘러싼 몇 가지 근본적인 생물학적 문제를 해결하기 위해 활용 될 수있다. 구체적으로, 그들은 수용체 및 신호 전달 경로가 혈관 신생, 암 조직에서의 혈관 확장 및 뇌와 관련된 말초 내피 세포에서 역할을하는지 확인할 수있다. 예를 들면, 혈관 신생 성장 인자 수용체, 산화 질소, 마이 토겐 활성화 단백질 키나아제 신호 전달 및 칼슘 신호 전달 25,26,27이 있습니...

<html> 대뇌 모세 혈관의 혈액 뇌 장벽 (BBB)은 혈액 대 뇌 접촉의 가장 큰 경계면이며 중추 신경계 (CNS) 1 , 2 의 항상성에 중심 역할을합니다. BBB의 동적 인 과정은 혈액에서 원하지 않는 분자의 섭취를 막고, CNS에서 폐기물을 제거하고, 필수 영양소와 신호 분자를 CNS에 공급하고, 신경 염증 1 , 2 , 3 , 4 , 5를 조절 합니다. BBB 손상은 노화 및 알쯔하이머 병 (AD), 다발성 경화증 및 뇌졸중 1 , 2 , 3 , 4 , 5 ,ass = &quot;xref&quot;&gt; 6. 따라서 BBB 기능 장애는 치료 목표를 포함 해 신경 퇴행성 장애에 핵심적인 역할을 할 수 있습니다. 혈관 커버리지를 유지하는 것은 BBB의 항상성 기능에 중요합니다. 그러나, 생체 내 및 시험 관내 자료가 신경 퇴행성 장애에 관련된 과정이 AD에서 특히 BBB 적용 범위를 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13으로 증가시키는 지에 대해 충돌합니다. 따라서 BBB 범위의 역학을 평가하고보다 포괄적으로 이해하기 위해 관련 세포 유형을 사용 하는 시험 관내 모델의 개발에 대한 강력한 근거가 있습니다. 대뇌 모세 혈관은 성상 세포, 혈관 주위 세포 및 뇌 내피 세포 (BEC) 3 입니다. 일반적으로 BBB 기능을 평가하기위한 세포 배양 기술은 스트레스 원 14 , 15 , 16 모두를 첨가 한 후 필터 삽입물에서 키운 세포에서 수행 된 투과성 분석 또는 주요 BEC 단백질의 수준 평가입니다. 중요한 것은이 검사는 뇌 혈관 검사에 초점을 두지 않는다는 것입니다. 여기에서 우리의 이전 방법 17 은 불멸화 된 인간 BEC의 단일 배양, 일차 마우스 BEC의 단일 배양 및 인간화 된 삼중 배양을 사용하여 BEC 적용 범위 및 망사 형 구조를 평가하기 위해 상세화된다모델 (BECs, astrocytes 및 pericytes) BBB. 목표는 BEC 적용 범위에서 AD 진행에 중요한 공헌자로 간주되는 oAβ의 해로운 영향을 입증하는 것이 었습니다. 표피 성장 인자 (EGF)의 보호 효과는 치료 선별 도구로서의 기술의 잠재력을 강조합니다. 이 기술은 1) 혈관 신생 및 혈관 범위에 대한 특정 경로의 역할을 묘사하는 것, 2) 혈관 신생 및 혈관 형성에 질병 및 노화 관련 요인의 영향을 평가하는 것, 3) 약리학 적 특성을 확인하는 것 목표.

<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

Blood-brain barrier (BBB) ​​범위는 중추 신경계 (CNS)의 항상성에 중요한 역할을합니다. BBB는 성상 세포, 혈관 주위 세포 및 뇌 내피 세포 (BECs)에 의해 동적으로 유지됩니다. 여기, 우리는 불멸의 인간 BEC의 단일 문화, 기본 마우스 BEC의 단일 문화, BBB의 인간화 트리플 문화 모델 (BECs, astrocytes 및 pericytes)를 사용하여 BBB 범위를 평가하는 방법을 자세히 설명합니다. 질병 상태에 대한 분석법의 적용 가능성을 강조하기 위해 우리는 BBB 적용 범위에서 알츠하이머 병 (AD) 진행에 중요한 기여를하는 oligomeric amyloid-β (oAβ)의 효과에 대해 설명합니다. 또한 우리는이 기술의 약물 스크리닝 가능성을 조명하기 위해 표피 성장 인자 (EGF)를 이용합니다. 우리의 결과는 단일 및 삼중 배양 BECs은 기초 조건 하에서 meshwork와 같은 구조를 형성하고, oAβ는이 세포 망 조직 형성을 방해하고 예비 형성된 메쉬 구조를 퇴화시키고,EGF는 이러한 혼란을 차단합니다. 따라서 기술 된 기술은 BBB 적용 범위를 조절하는 근본적이고 질병 관련 프로세스를 해부하는 데 중요합니다.

단일 문화에서는 hCMEC / D3 세포 ( 그림 3A )와 기본 마우스 BEC ( 그림 3B ) 모두 우물 전체에 망사 모양의 구조를 형성합니다. 구조는 상호 연결된 노드들의 망목을 특징으로합니다 ( 그림 3 ). 설명 된 모든 패러다임 ( 그림 1 )에서, meshwork와 같은 구조는 24 시간 후 대조군에서 유사하여 ?...

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

혈액 뇌 장벽의 범위를 유지하는 것은 중추 신경계의 항상성을 유지하기위한 열쇠입니다. 이 프로토콜은 혈액 - 뇌 장벽 범위를 조정하는 근본적인 병적 과정을 묘사하는 체외 기술을 설명합니다.

혈액 뇌 장벽 메쉬와 유사한 혈관 형성 및 분열을 평가하기위한 In vitro 시험 법

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

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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

쥐 모델에서 Transcranial 집중 초음파를 사용하여 혈액 - 뇌 장벽의 MRI를 유도 장애

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

연구

의학

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

혈액 - 뇌 장벽 장애의 쥐 모델 Neurovascular 치료학 타겟이 분명한 허용하려면

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

<em&gt; 생체 내</em혈관 투과율을 테스트하려면&gt; 분석

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

로 뇌 미세 혈관 내피 세포 (CEnd를)에 기지개<em&gt; 체외</em혈액 뇌 장벽의&gt; 외상성 뇌 손상 모델

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

설정 - 업<em&gt; 체외</em쥐 뇌 혈관 벽의&gt; 모델 (BBB)​​ : BBB 불 침투성 및 수용체 매개 운송에 초점

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

소설<em&gt; 생체</em&gt; 유전자 전달 기술과<em&gt; 시험관</em근골격계 질환의 뼈 손실의 연구를위한&gt; 셀 기반 분석 실험

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

같은 산소 포도당 박탈과 재산 소화<em&gt; 체외</em&gt; 허혈 - 재관류 손상 모델 공부 뇌 혈관 장벽 부전

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

인간 태반 생리학의 통합 연구에 대한 4 혈관 샘플링 접근법<em&gt; In Vivo</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

생체 외에서 혈액-뇌 장벽 돼지 뇌 내 피 세포를 기반으로 하는 모델의 설립에 대 한 방법 개선

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

사기 질 기관 이용과 환경에 노출 된 쥐의 악의 니에서의 마이크로 해 부

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