Name: in vitro permeation of fitc-loaded ferritins across a rat blood-brain barrier: a model to study the delivery of nanoformulated molecules
Uploaded: 2016-08-22T15:00:00.000+00:00
Duration: 11 min 13 s
Description: Watch this Scientific Journal Video about In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules at JoVE.com

The authors acknowledge Assessorato alla Sanit&#224;, Regione Lombardia and Sacco Hospital (NanoMeDia Project) for research funding.

1. BBB 모델 구축 참고 : 우리가 시판 차 RBMECs와 RCA 단자를 사용하는 것이 좋습니다 BBB를 모델을 구축하십시오. 모든 단계를 층류 후드에서 멸균 처리 시약 및 일회용으로 수행되어야한다. <ol><li> 세포 배양 <ol><li> 폴리 -L- 라이신 100 μg의 / ㎖ (RT에서 1 시간) 또는 피브로넥틴 50 ㎍ / ㎖ (37 ° C에서 1 시간)와 코트 세포 배양 플라스크는 각각 RCA 단자 또는 RBMECs의 부착을 촉진. 이어서, 5 % 소 태아 혈청, 1 %의 내피 세포 성장 보충와 1 % 페니실린 / 스트렙토 마이신 (SECM)으로 보충 된 1 × 106 RCA 단자와 내피 세포 배지에서 5 × 105 RBMECs, 해동. 10 ml의 SECM의 T75 플라스크에서 20 ㎖의 SECM과 RBMECs의 T175 플라스크에 종자 RCA 단자. 주 : 해동 및 RCA 단자와 RBMECs 시드의 통로는 배양 세포 밀도와 시간의 조건으로 조정할 필요는 BBB 모델에서 시험 될 실험 조건의 수에 따라. 처음으로1 × 106 RCA 단자 1 바이알에 5 × 105 RBMECs 1 바이알로 보내고, 그것을 20 BBB 베어링 삽입까지 얻을 수있다. </li><li> RCA 단자에 대해 80 %가 합류 할 때까지 37 ° C, 5 %, 약 6 일 동안 가습 분위기 중의 CO 2에서 세포를 유지하고 RBMECs 90 % 이상 합류. 5 분 (T75 플라스크 1 ㎖의 트립신과 T175 플라스크 2 ㎖)에 대해 : 트립신 EDTA 용액 (250 트립신 1)를 사용하여 세포를 분리합니다. SECM와 트립신 활동 정지 (2 : 1)로 5 분 동안 750 × g에서 원심 분리하고 세포 현탁액 60 ml를 SECM에 펠릿을 재현 탁. </li><li> 삽입에 파종 전에 다른 3 일간 SECM 3 T175 플라스크와 문화에 RBMECs을 분할합니다. BURKER 챔버 내에 광학 현미경 하에서 트립 판 블루로 1 : 1 희석하여 세포 현탁액을 관찰함으로써, RCA 단자 생활의 총 수를 계산. </li></ol></li><li> 삽입 위에 셀 시드 <ol><li> 6 멀티 웰 transpar의 폴리에틸렌 테레 프탈레이트의 일측 치료 (PET) 멤브레인100 μg의 / ㎖ 폴리 -L- 리신 피브로넥틴를 50㎍ / ㎖로 상대방과 ENT 인서트 RCA 단자와 BMECs의 부착을 허용한다. 애완 막과의 접촉을 피하기 위하여 핀셋을 삽입 핸들. <ol><li> 상부 챔버로 피브로넥틴 용액 (최소 500 μl를) 6 웰 플레이트에 삽입을 놓고 추가 할 수 있습니다. 37 ° C에서 배양 1 시간 후, 피브로넥틴 용액을 제거 멀티 웰 플레이트 떨어져 삽입을하고 대한 멸균 지원 여기에 사용되는 150cm 2 페트리 접시의 바닥에 거꾸로 넣어 이미 삽입 코팅. </li><li> 부드럽게 (도 1a에 도시 된 바와 같이, RCA 단자 시드 라 함)를 인서트의 바닥면에 폴리 -L- 라이신 800 μL를 추가하고, 실온에서 1 시간 동안 인서트의 해결책을 유지한다. </li><li> 솔루션을 대기음하고 삽입은 15 ~ 30 분 동안 실온에서 건조 할 수 있습니다. 인서트는 이제 셀 시딩에 대한 준비뿐만 아니라, 여러 가지의 멀티 웰 플레이트에 저장 될 수있다BBB를 건설을 진행하기 전에 4 ° C에서 일. 참고 : FD40 유량 측정에 의해 BBB 설립의 유효성을 검사하는 컨트롤로 사용되는 세포에서 무료로 최소 3 코팅 삽입을 유지해야합니다. </li></ol></li><li> 시드 RCA 단자 삽입 (도 1a)의 아래에서 위쪽에 세포 현탁액 800 ㎕를 적하하여 폴리 -L- 리신 코팅 된 인서트의 바닥면 상 (35,000 / cm 2). 막에 세포의 효과적인 부착을 허용하기 위해, RT에서 4 시간 동안 인서트의 RCA 현탁액을 떠난다. </li><li> 잔류 용액을 흡인 SECM 2 ㎖을 함유하는 웰에 삽입 배치하고 매 2 일 SECM을 변경 가습 분위기에서 37 ℃, 5 % CO 2의 멀티 웰 플레이트를 유지한다. </li><li> 삼일 후에 때 RCA 단자는 인서트의 하부면을 코팅 한 다음이 단계 인서트의 상부면에 시드 RBMECs : <ol><li> T175 플라스크에서 RBMECs를 분리하고 계산총 살아있는 세포 단계 1.1.2과 1.1.3에보고. </li><li> 아스트로 사이트만을 가진 3 삽입 이탈 SECM (1000 μL) (도 1B)에 삽입 체의 상면에 씨앗 RBMECs (60,000 / cm 2), 티이 배경으로 사용될 수있다. 표준 배양 조건에서 멀티 웰 플레이트를 놓습니다. 2 일마다 내측 하부 챔버 내의 SECM 변경 적어도 3 일간 배양하는 시스템을 유지한다. </li></ol></li></ol></li></ol> 2. BBB 검증 <ol><li> 봉사자 측정 <ol><li> 공동 배양 3 일째에, 적절한 챔버로 BBB 베어링 삽입물을 삽입하여 티이을 확인 (즉, 캡을 가지고 있으며, 챔버 뚜껑 모두 전압 검출 및 전류 쌍의 전극을 포함 할 경우) (4) 충전을 SECM ml의. 상피 조직의 볼트 / 저항계로 연결합니다. </li><li> 함께 셀 BBB-시스템 티이 측정에 의해, RCA 단자 번째 베어링 3 인서트 봉사자 값을 기록에서 BBBs 얻은 값에서 차감됩니다. Ω cm의 X의 2와 같은 결과를 표현하기 위하여 인서트 (4.2 cm 2)의 표면에 의해 얻어진 티이 값을 곱한다. </li><li> 공동 문화의 3 번째 날부터 매일 봉사자를 측정한다. 참고 : 봉사자가 증가 초기 기간 후 기록 된 값 (보통 5 일 및 공동 문화의 일곱 번째 일 사이에) 적어도 두 개의 연속 일 동안 안정적으로 유지해야한다. 그 시점에서 BBB 검증 (2.2 절) 및 / 또는 트랜스 BBB의 실험의 두 번째 단계에 대한 준비가되어 있습니다. 참고 : 2.1 절에서 설명하는 절차는 다음 투과성 실험 (3 절)에 전념 같은 BBB-시스템에서 수행 할 수 있습니다. 무균 조건에서 작동합니다. </li></ol></li><li> 의 트랜스 BBB 플럭스 FITC-덱스 트란 40 (FD40) <ol><li> 3 빈 인서트 걸쳐 그 비교 BBB 모델의 하부 챔버 상부에서 FD40 광속 측정다음 단계에 따라 (섹션 1.2.1의 주 참조) <ol><li> 1 ㎎을 추가 / ㎖ FD40는 BBBs의 상부 구획으로 (SECM 희석) 및 λ를, 그들을 1, 2, 3 시간 후, 하부 챔버 200 μL의 SECM 인출 및 λ의 예를 (spectrofluorimeter으로 488 nm의 형광 강도를 측정 515 nm의) 5 슬릿. </li><li> 동일한 분석 매개 변수를 사용하여 처녀 매체의 500 μl를 측정 fluorescenceby SECM 배경에 대한 값을 가져옵니다. 모든 FD40 형광 값의 SECM 배경 형광을 뺍니다. </li><li> 공지 농도 (즉. 0.312, 0.625, 1.25, 2.5 μg의 / ㎖) SECM 500 μL에 용해 형광 추적자로 제조 한 검량선과 관찰 된 형광 값의 비교에 의해 투과 FD40의 양을 결정한다. </li><li> 에 따라 평균 플럭스 값의 외관상 투과 계수 (P 응용 프로그램)을 계산한다 : P 앱 = J / AC <brJ 분자 (몰 / 초)의 유량이고 /&gt;, A는 투과 면적 (cm 2)이고, C는 상기 상부 구획실 (몰 / cm 3)의 분자의 농도이다. 참고 : 적절한 봉사자 값에 도달했습니다 3 개 이상 BBB-시스템은 독점적으로이 검증 절차에 전념해야하며, 다음과 같은 투과성 실험 (섹션 3)에 이용되어서는 안된다. 불임은 필수가 아닙니다. </li></ol></li></ol></li></ol> 3. 횡단 BBB FITC로드 Ferritins의 침투 (FNN) 참고 : 다른 형광 분자의 캡슐화 nanocages (FNN)에서 대장균에서 생산 및 조립 인간 페리틴의 재조합 변형 (FN)이,의 교수 스페리의 NanoBioLab (대학 밀라노 - 비 코카, 이탈리아)에서 구할 수 있습니다. FNN은 FITC로드 이전에 설명 프로토콜 (13) 모두 Fn을로드 분자의 농도에 따라 정확하게 determin되어 있습니다에디션. <ol><li> FITC의 트랜스 BBB 플럭스 및 FITC-FNN <ol><li> 항에있어서, 배양 7 및 24 시간 후에 자유 염료에 비하여, - (CO 배양 7 일째 통상 5 일에서) 유효성 BBB 모델의 하부 챔버 상부에서 FITC-FNN 광속 측정 다음 단계 : <ol><li> FITC-FNN은 (50 μg의가 / ㎖ FNN 1.1 μM FITC) 순으로 각 제제에 대해 적어도 6 BBB 시스템의 상부 챔버로 프리 FITC의 동일한 양의 하부 챔버로부터 SECM 용액 2 ㎖을 인출 추가 7 시간 후, 24 시간 후 다른 3 인서트의 하부 챔버에서 최소 3 삽입. </li><li> spectrofluorimeter에 의해 수집 된 시료 500 μL의 형광 강도를 측정 (λ의 전 488 nm의, λ 안에 515 나노 미터, 5 슬릿). </li><li> 동일한 분석 매개 변수를 사용하여 매체의 500 μl를 측정하여 SECM 배경 형광을 얻습니다. SECM 배경 형광을 빼기모든 FITC 또는 FITC-FNN 형광 값에서. </li><li> 500 μL SECM에 용해 유리 또는 nanoformulated 염료의 공지 된 농도 (즉. 6.87, 13.75, 27.5, 55 ㎚)로 제조 된 두 개의 서로 다른 캘리브레이션 곡선 얻어진 형광 값을 비교하여 투과 FITC 또는 FITC-FNN의 농도를 결정한다. </li></ol></li></ol></li><li> RBMECs에 FITC-FNN 현지화 <ol><li> 상기 BBB 시스템의 상부 챔버로부터 SECM 제거 PBS로 삽입 씻어 10 상부 구획에 500 ㎕의 파라 포름 알데히드 (인산염 완충 식염수 PBS 4 %)을 첨가하여, 각 실험군에 대해 적어도 두 개의 인서트에 RBMECs 해결 RT에서 분. </li><li> 잔류 파라 포름 알데히드를 제거하는 PBS로 삽입을 세 번 씻으십시오. 참고 :이 시점에서, 불임이 필요하지 않습니다. </li><li> 애완 막의 적합한 부분을 절단하고, 다음의 단계를 따라 세포를 진행 immunodecoration : <ol><li> Permeabi10 분 동안 PBS에서 0.1 % 트리톤 X-100 리제 RBMECs. PBS 2 % 소 혈청 알부민 (BSA), 2 % 염소 혈청을 함유하는 용액으로 실온에서 1 시간 동안 차단하는 단계를 수행한다. RT에서 2 시간 동안 1:20 희석 항 폰 빌레 브란트 인자 (VWF)로 샘플을 인큐베이션. </li><li> PBS로 3 회 세척 한 후, 2 % BSA로 RT에서 2 시간 동안 세포를 노출하는 하나에 적합한 차 항체를 함유하는 2 % 염소 혈청 용액 : 위해 300 희석 (A)에서 안티 VWR 및 DAPI를 공개 1 : 핵 탐지를위한 1,500 희석. </li></ol></li><li> antifade 장착 솔루션 (삽입 조각 당 하나의 드롭)를 사용하여 현미경 슬라이드 상에 삽입 조각을 탑재, 다음, 커버 슬립과 함께 샘플을 닫습니다. 40X 배율, 1.5 줌과 1,024 X 1,024 픽셀 해상도에서 기름 침지 렌즈를 사용하여 공 초점 현미경으로 분석합니다. </li></ol></li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+models+of+the+blood-brain+barrier+for+the+study+of+drug+delivery+to+the+brain.">In vitro models of the blood-brain barrier for the study of drug delivery to the brain.</a> Mol Pharm. 11 (7), 1949-1963 (2014).</li><li>Bellini, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+nanocages+for+self-triggered+nuclear+delivery+of+DNA-targeted+chemotherapeutics+in+Cancer+Cells.">Protein nanocages for self-triggered nuclear delivery of DNA-targeted chemotherapeutics in Cancer Cells.</a> J. Control. Release. 196, 184-196 (2014).</li><li>Fiandra, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoformulation+of+antiretroviral+drugs+enhances+their+penetration+across+the+blood+brain+barrier+in+mice.">Nanoformulation of antiretroviral drugs enhances their penetration across the blood brain barrier in mice.</a> Nanomedicine. 11 (6), 1387-1397 (2015).</li><li>Molino, Y., Jab&#232;s, F., Lacassagne, E., Gaudin, N., Khrestchatisky, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.</a> J. Vis. Exp. , e51278 (2014).</li><li>Perri&#232;re, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+in+vitro+model+of+rat+blood-brain+barrier+for+molecular+analysis+of+efflux+transporters.">A functional in vitro model of rat blood-brain barrier for molecular analysis of efflux transporters.</a> Brain Res. 1150, 1-13 (2007).</li></ol>

The authors declare that they have no competing financial interests.

여기에 설명 된 체외 방법은 나노 입자와 nanoformulation에 따라 형광 분자의 트랜스 BBB 전달을 연구하는 데 유용한 검증 된 접근 방식을 나타냅니다. 여기에서 우리는 BBB에서화물 분자의 전위를 연구 할 수있는 좋은 후보를 나타냅니다 FNN를 사용합니다. 그것은 구체적으로 BMECs의 내강 막에 표현 수용체 매개 내재화 경로를 사용하여 나노 입자의 흡수를 매개하는 TfR1 수용체에 의해 인식되기 ?...

약물 요법에 대한 중추 신경계의 저항 (CNS) 질환 (예. 암, 간질, 우울증, 정신 분열증 및 HIV 관련 신경 장애)로 인해 뇌 혈관 장벽을 통과 곤란한 약물 투과 포함하는 다양한 다른 메커니즘이다 (BBB) . BBB를 혈액 순환 물질로부터 뇌 조직을 분리 경계이다. 이 배리어 내 혈관 주위 세포와 성상 세포의 endfeet 지원 뇌 미세 혈관 내피 세포 (BMECs)의 층은, 분자량이 400 이상인 다 1 hydrosoluble 이들 약물에 BBB의 높은 선택성을 담당한다. 또 다른 약물 관련 저항기구는 CNS에 약물 침투를 감소시키고 뇌 (2)로부터 자신의 압출을 용이하게하기 위해 협력 약물 유출 컨베이어 (P 당 단백질 및 약제 내성 단백질)의 BMECs에 존재 연결된다. 지난 10 년간, 나노 기술 방법 중 다수는 BBB 3-6 걸쳐 약물 전달의 임상 적 및 생물학적 과제를 해결하기 위해 개발되었다. 이러한 상황에서, 나노 페리틴 (FNN)는 완전히 혁신적이고 유망한 솔루션을 나타낸다. FNN 8 nm의 내경 중공 구형 구조로 배열되어 자기 조립 (24) 페리틴 (FN) 모노머, 12 nm의 구형이다. 페리틴 소단위는 산성 pH에서 분해 및 다양한 유기 분자를 캡슐화 할 수 있도록 중립성 pH를 가져와 형상 기억 방식으로 조립 될 수있다. 따라서, FNN 다기능 약물 전달 시스템 7,8의 개발이 흥미있는 모델을 나타낸다. 또한, FNN이 셀 (9)의 내강 막 상에 발현되는 트랜스페린 수용체 (TFR) (1)의 구체적인 포상 BMECs 감사와 상호 작용할 수있다. 지금까지, BBB의 시험 관내 모델이 다른 순으로 하였다 개발되어왔다R은 다양한 약물의 독성 BBB 향해 또는 유출 수송차와 분자의 상호 작용 트랜스 BBB 투과성 해명. 사실,이 모델은 생체 내 연구를 진행하기 전에 활성 분자의 신속한 검사를 위해 접근 체외에서 유효한 것으로 간주됩니다. 이 모델 동물 (쥐, 마우스, 돼지와 소) 또는 인간의 세포 라인 10,11,12에서 얻은 BMECs 또는 공동 배양 BMECs 및 성상 세포 (드물게 혈관 주위 세포)의 단일 내피 층으로 구성되어 있습니다. TransEndothelial 전기 저항 (봉사자)과 정의 분자량 추적자의 명백한 침투성 (P 응용 프로그램)은 체외 모델의 품질을 결정하는 데 사용되는 두 가지 중요한 매개 변수를 나타냅니다. 여기에 우리가 쥐 BMECs (RBMECs)의 공동 문화를 기반으로 BBB 체외 모델의 고용을 설명하고 쥐 대뇌 피질 성상 세포 (RCA 단자)는 형광 isothi을 캡슐화 페리틴 nanocages의 트랜스 BBB 투과를 공부ocyanate (FITC).

<table><tbody><tr><td>Rat Brain Microvascular Endothelial Cells&amp;nbsp;</td><td>Innoprot</td><td>P10308</td><td>isolated from Sprague Dawley rat brain tissue, cryopreserved at passage one and delivered frozen</td></tr><tr><td>Cortical Astrocytes&amp;nbsp;</td><td>Innoprot</td><td>P10202</td><td>isolated from 2 days rat brain tissue, cryopreserved at passage one and delivered frozen.</td></tr><tr><td>Endothelial Cell Medium kit</td><td>Innoprot</td><td>P60104</td><td>ECM (500 ml) and fetal bovin serum (25 ml), endothelial cell growth supplement (5 ml) and penicillin/streptomycin (5 ml). Warm in 37 &amp;deg;C water bath before use and protect from light</td></tr><tr><td>Trypsin-EDTA without Phenol Red</td><td>EuroClone</td><td>ECM0920D</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Fluorescein isothiocyanate-dextran 40,000</td><td>Sigma</td><td>FD40S</td><td>protect from light</td></tr><tr><td>paraformaldehyde&amp;nbsp;</td><td>Sigma</td><td>158127</td><td>diluition in chemical hood</td></tr><tr><td>Dulbecco&#039;s phosphate buffer saline w/o Ca and Mg</td><td>EuroClone</td><td>ECB4004L</td><td></td></tr><tr><td>Triton&amp;nbsp;X-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>bovine serum albumin&amp;nbsp;</td><td>Sigma</td><td>A7906</td><td></td></tr><tr><td>goat serum&amp;nbsp;</td><td>EuroClone</td><td>ECS0200D</td><td></td></tr><tr><td>mouse monoclonal anti-Von Willebrand Factor</td><td>Dako</td><td>M0616</td><td></td></tr><tr><td>AlexaFluor 546-conjugated antibody against mouse IgGs</td><td>ThermoFischer Scientific</td><td>A-11003</td><td>protect from light</td></tr><tr><td>DAPI (4&amp;rsquo; ,6-diamidino-2-phenylindole)&amp;nbsp;</td><td>ThermoFischer Scientific</td><td>D1306</td><td>protect from light</td></tr><tr><td>ProLong Gold Antifade Mountant</td><td>ThermoFischer Scientific</td><td>P36934</td><td></td></tr><tr><td>Poly-L-lysine Hydrobromide</td><td>Sigma</td><td>P1274</td><td>the same solution can be used several times</td></tr><tr><td>fibronectin from bovine plasma&amp;nbsp;</td><td>Sigma</td><td>F1141</td><td>the same solution can be used several times</td></tr><tr><td>Polyethylene terephthalate (PET) inserts</td><td>Falcon</td><td>F3090</td><td>Transparent Polyethylene terephthalate (PET) membranes; surface area: 4.2 cm&lt;sup&gt;2&lt;/sup&gt;; pore size 0.4 &amp;micro;m/surface area</td></tr><tr><td>T75 Primo TC flask</td><td>EuroClone</td><td>ET7076</td><td></td></tr><tr><td>T175 Primo TC flask</td><td>EuroClone</td><td>ET7181</td><td></td></tr><tr><td>EVOM2 Epithelial Tissue Volt/Ohmmeter&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>World Precision Instruments Germany</td><td>EVOM2</td><td></td></tr><tr><td>Endohm- 24SNAP cup</td><td>World Precision Instruments Germany</td><td>ENDOHM-24SNAP</td><td></td></tr><tr><td>Light/fluorescence microscope with camera</td><td>Leica Microsystems</td><td>DM IL LED Fluo/ ICC50 W Camera Module</td><td>&amp;nbsp;inverted microscope for live cells with camera&amp;nbsp;</td></tr><tr><td>Confocal Microscope</td><td>Leica Microsystems</td><td>TCS SPE</td><td></td></tr><tr><td>Spectrofluorimeter</td><td>Jasco</td><td>FP-8000</td><td></td></tr></tbody></table>

in vitro permeation of fitc-loaded ferritins across a rat blood-brain barrier: a model to study the delivery of nanoformulated molecules

Brain microvascular endothelial cells, supported by pericytes and astrocytes endfeet, are responsible for the low permeation of large hydrosoluble drugs through the blood-brain barrier (BBB), causing difficulties for effective pharmacological therapies. In recent years, different strategies for promoting brain targeting have aimed to improve drug delivery and activity at this site, including innovative nanosystems for drug delivery across the BBB. In this context, an in vitro approach based on a simplified cellular model of the BBB provides a useful tool to investigate the effect of nanoformulations on the trans-BBB permeation of molecules. This study describes the development of a double-layer BBB, consisting of co-cultured commercially available primary rat brain microvascular endothelial cells and astrocytes. A multiparametric approach for the validation of the model, based on the measurement of the transendothelial electrical resistance and the apparent permeability of a high molecular weight dextran, is also described. As proof of concept for the employment of this BBB model to study the effect of different nanoformulations on the translocation of fluorescent molecules across the barrier, we describe the use of fluorescein isothiocyanate (FITC), loaded into ferritin nanoparticles. The ability of ferritins to improve the trans-BBB permeation of FITC was demonstrated by flux measurements and confocal microscopy analyses. The results suggest this is a useful system for validating nanosystems for delivery of drugs across the BBB.

인서트의 BBB 모델, 세포 부착 및 성장의 확립 중에 PET 막의 특성상 투명 광학 현미경 감사를 사용하여 모니터링 할 수있다. 35,000 세포 / cm 2, RT (도 2A)에서 배양 4 시간 후, 인서트의 바닥면에 효과적으로 부착의 밀도로 시딩 RCA 단자와, 스핀들 형상의 형태를 가지고, 일에 막 표면을 덮도록 성장 (그림 2B). 60,000 세포 / ㎠의 밀도로 접...

Watch this Scientific Journal Video about In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules at JoVE.com

In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules

A method to establish an in vitro model of blood-brain barrier based on a co-culture of rat brain microvascular endothelial cells and astrocytes is described and validated. This system proved to be a valid tool to study the effect of nanoformulation on the trans-barrier permeation of fluorescent molecules.

쥐 뇌 혈관 벽의 맞은 편에 FITC로드 Ferritins의 생체 외 투과 년 : 모델 Nanoformulated 분자의 배달을 공부하기

Brain microvascular endothelial cells, supported by pericytes and astrocytes endfeet, are responsible for the low permeation of large hydrosoluble drugs ...

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Bioengineering

In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules

7.8K 조회수.  Università di Milano. A method to establish an in vitro model of blood-brain barrier based on a co-culture of rat brain microvascular endothelial cells and astrocytes is described and validated. This system proved to be a valid tool to study the effect of nanoformulation on the trans-barrier permeation of fluorescent molecules.

비디오: In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

핵산, 단백질과 작은 분자의 Reagentless 감지를위한 전기 -의 DNA Biosensors의 제작

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

연구

생체공학

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

의 순환 압력 생물 반응기 설계 예 VIVO</em대동맥 심장 밸브> 학습

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Predicting In Vivo Payloads Delivery using a Blood-brain Tumor-barrier in a Dish

Highly selective by nature, the blood-brain barrier (BBB) is essential for the brain homeostasis in physiological conditions. However, in the context of brain tumors, the molecular selectivity of BBB also shields the neoplastic cells by blocking the delivery of peripherally administered chemotherapies. The development of novel drugs (including nanoparticles) targeting malignant brain tumors ideally requires the use of preclinical animal models to study the drug&#8217;s transcytosis and antitumor efficacy. In order to comply with the 3R principle (refine, reduce, and replace) to reduce the number of laboratory animals in experimental setup and perform the high-throughput screening of a large library of antitumor agents, we developed a reproducible in vitro human and murine mimic of the blood-brain tumor-barrier (BBTB) using three-layered cultures of endothelial cells, astrocytes, and patient-derived glioblastoma spheres. For higher scalability and reproducibility, commercial cell lines or immortalized cells have been used in tailored conditions to allow the formation of a barrier resembling the actual BBB. Here we describe a protocol to obtain a BBTB mimic by culturing endothelial cells in contact with astrocytes at specific cell densities on inserts. This BBTB mimic can be used, for instance, for the quantification and confocal imaging of the nanoparticle passage through the endothelial and astrocytic barriers, in addition to the evaluation of the tumor cell targeting within the same assay. Moreover, we show that the obtained data can be used to predict the behavior of nanoparticles in preclinical animal models. In a broader perspective, this in vitro model could be adapted to other neurodegenerative diseases for the determination of the passage of new therapeutic molecules through the BBB and/or be supplemented with brain organoids to directly evaluate the efficacy of drugs.

Drug targeting to central nervous system tumors is a major challenge. Here we describe a protocol to produce an in vitro mimic of the blood-brain tumor-barrier using murine and/or human cells and discuss their relevance for the predictability of central nervous system tumor targeting in vivo.

예측의 Vivo 페이로드 배달 접시에 혈액 종양-배리어를 사용 하 여

Highly selective by nature, the blood-brain barrier (BBB) is essential for the brain homeostasis in physiological conditions. However, in the context of ...

암 연구학

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

방법 혈액 - 뇌 장벽의 인간 세포 기반의 연락 모델의 준비를 위해 개선

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

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

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm&#183;cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 &#177;&#160;0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

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

The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.

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 ...

의학

Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay between perturbed BBB homeostasis and the pathogenesis of brain disorders needs further investigation, the development and validation of a reliable procedure to accurately detect BBB alterations may be crucial and represent a useful tool for potentially predicting disease progression and developing targeted therapeutic strategies.
Here, we present an easy and efficient procedure for evaluating BBB leakage in a neurodegenerative condition like that occurring in a preclinical mouse model of Huntington disease, in which defects in the permeability of BBB are clearly detectable precociously in the disease. Specifically, the high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin, which is able to cross the BBB only when the latter is impaired, is acutely infused into a mouse jugular vein and its distribution in the vascular or parenchymal districts is then determined by fluorescence microscopy.
Accumulation of green fluorescent-albumin in the brain parenchyma functions as an index of aberrant BBB permeability and, when quantitated by using Image J processing software, is reported as Green Fluorescence Intensity.

In this study, we present an easy and efficient procedure for evaluating the disruption of the blood-brain barrier under neurodegenerative conditions. To achieve our objective, we infused high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin into mouse jugular vein and evaluated its leakage into the brain parenchyma by fluorescence microscopy.

FITC 표시 된 알 부 민 신경 질환의 마우스 모델에서의 정 맥 주입에 의해 혈액-뇌 장벽 침투성의 평가

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay ...

신경과학

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains brain homeostasis. The principal sites of the barrier are endothelial cells of the brain capillaries whose barrier function results from tight intercellular junctions and efflux transporters expressed on the plasma membrane. This function is regulated by pericytes and astrocytes that together form the neurovascular unit (NVU). Several neurological diseases such as stroke, Alzheimer&#39;s disease (AD), brain tumors are associated with an impaired BBB function. Assessment of the BBB permeability is therefore crucial in evaluating the severity of the neurological disease and the success of the treatment strategies employed.
We present here a simple yet robust permeability assay that have been successfully applied to several mouse models both, genetic and experimental. The method is highly quantitative and objective in comparison to the tracer fluorescence analysis by microscopy that is commonly applied. In this method, mice are injected intraperitoneally with a mix of aqueous inert fluorescent tracers followed by anesthetizing the mice. Cardiac perfusion of the animals is performed prior to harvesting brain, kidneys or other organs. Organs are homogenized and centrifuged followed by fluorescence measurement from the supernatant. Blood drawn from the cardiac puncture just before perfusion serves for normalization purpose to the vascular compartment. The tissue fluorescence is normalized to the wet weight and serum fluorescence to obtain a quantitative tracer permeability index. For additional confirmation, the contralateral hemi-brain preserved for immunohistochemistry can be utilized for tracer fluorescence visualization purposes.

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Here we present a mouse brain vascular permeability assay using intraperitoneal injection of fluorescent tracers followed by perfusion that is applicable to animal models of blood-brain barrier dysfunction. One hemi-brain is used for assessing permeability quantitatively and the other for tracer visualization/immunostaining. The procedure takes 5 - 6 h for 10 mice.

Vivo에서 혈액-뇌 장벽 침투성 분석 결과 사용 하 여 마우스에 붙일 추적기 표시

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains ...

Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability

Dysfunction of the blood-retinal barrier (BRB) contributes to the pathophysiology of several vascular eye diseases, often resulting in retinal edema and subsequent vision loss. The inner blood-retinal barrier (iBRB) is mainly composed of retinal vascular endothelium with low permeability under physiological conditions. This feature of low permeability is tightly regulated and maintained by low rates of paracellular transport between adjacent retinal microvascular endothelial cells, as well as transcellular transport (transcytosis) through them. The assessment of retinal transcellular barrier permeability may provide fundamental insights into iBRB integrity in health and disease. In this study, we describe an endothelial cell (EC) transcytosis assay, as an in vitro model for evaluating iBRB permeability, using human retinal microvascular endothelial cells (HRMECs). This assay assesses the ability of HRMECs to transport transferrin and horseradish peroxidase (HRP) in receptor- and caveolae-mediated transcellular transport processes, respectively. Fully confluent HRMECs cultured on porous membrane were incubated with fluorescent-tagged transferrin (clathrin-dependent transcytosis) or HRP (caveolae-mediated transcytosis) to measure the levels of transferrin or HRP transferred to the bottom chamber, indicative of transcytosis levels across the EC monolayer. Wnt signaling, a known pathway regulating iBRB, was modulated to demonstrate the caveolae-mediated HRP-based transcytosis assay method. The EC transcytosis assay described here may provide a useful tool for investigating the molecular regulators of EC permeability and iBRB integrity in vascular pathologies and for screening drug delivery systems.

Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability

This protocol illustrates an in vitro endothelial cell transcytosis assay as a model to evaluate inner blood-retinal barrier permeability by measuring the ability of human retinal microvascular endothelial cells to transport horseradish peroxidase across cells in caveolae-mediated transcellular transport processes.

내피 세포 트랜스사이토시스 분석법(Endothelial Cell Transcytosis assay)은 내부 혈액-망막 장벽 투과성을 평가하기 위한 시험관 내 모델로서 분석합니다.

Dysfunction of the blood-retinal barrier (BRB) contributes to the pathophysiology of several vascular eye diseases, often resulting in retinal edema and ...

Blood-Brain Barrier Mimic Permeability Assay: An In Vitro Assay to Assess the Permeability of BBB Mimic to Tracer Molecules

Source: Le Joncour, V. et al. <a href="https://www.jove.com/v/59384/predicting-vivo-payloads-delivery-using-blood-brain-tumor-barrier">Predicting In Vivo Payloads Delivery using a Blood-Brain Tumor-Barrier in a Dish.</a> J. Vis. Exp. (2019)
This video describes a permeability assay to evaluate the permeability of in vitro blood-brain barrier using sodium fluorescein molecules.

Blood-Brain Barrier 모방 투과성 분석: 추적자 분자에 대한 BBB 모방의 투과성을 평가하기 위한 체외 분석

Source: Le Joncour, V. et al. Predicting In Vivo Payloads Delivery using a Blood-Brain Tumor-Barrier in a Dish. J. Vis. Exp. (2019)
This video describes a ...

JoVE Encyclopedia of Experiments

Cancers of the Nervous System

In vitro studies

Studying the Permeation of FITC and FITC-Ferritin through an In Vitro Blood-Brain Barrier Model

Source: Fiandra, L., et al. <a href="https://app.jove.com/v/54279">In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules.</a> J. Vis. Exp. (2016).
This video demonstrates a method to study permeation through an in vitro blood-brain barrier (BBB) model using astrocytes and endothelial cells on a membrane insert, with free FITC and FITC-loaded ferritin. FITC-ferritin bound to receptors on endothelial cells and crossed the membrane barrier to the lower chamber while free FITC showed restricted permeation and remained in the upper chamber.

쥐 뇌 혈관 벽의 맞은 편에 FITC로드 Ferritins의 생체 외 투과 년 : 모델 Nanoformulated 분자의 배달을 공부하기

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