Comparative study of four immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, and optimization of culture conditions, for an in vitro blood-brain barrier model for drug permeability studies.
Comparative study of four immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, and optimization of culture conditions, for an in vitro blood-brain barrier model for drug permeability studies.
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DOI：
10.1186/2045-8118-10-33
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发表时间：
2013-11-22
影响因子：
7.3
通讯作者：
Oufir M
中科院分区：
文献类型：
--
作者：
Eigenmann DE;Xue G;Kim KS;Moses AV;Hamburger M;Oufir M
在早期药物发现和开发中迫切需要可靠的适合于高通量筛选的人体外血脑屏障（BBB）模型，以评估有前途的生物活性化合物克服BBB的能力。为了建立一个改进的人体外血脑屏障模型，我们比较了四个目前可用的和充分表征的永生化人脑毛细血管内皮细胞系，hCMEC/D3，hBMEC，TY 10和BB 19，在屏障紧密性和细胞旁渗透性。设计了使用永生化人星形胶质细胞（SVG-A细胞系）和永生化人周细胞（HBPCT细胞系）的共培养系统，目的是积极影响屏障紧密性。通过使用常规上皮伏安计（EVOM）和自动化CellZscope系统（其实时记录TEER和细胞层电容（CCL））的跨内皮电阻（TEER）测量来评估紧密连接（TJ）形成。使用具有低BBB渗透性的两种荧光标记化合物（荧光素钠（Na-F）和荧光黄（LY））评估细胞旁渗透性。通过筛选来自不同供应商的一系列24孔组织培养插入物来优化每种内皮细胞系的条件。对于hBMEC细胞，通过改变包被材料、包被程序、细胞接种密度和生长培养基组成进行进一步优化。对每个内皮细胞系进行细胞类型特异性跨膜粘附连接蛋白VE-钙粘蛋白和TJ蛋白ZO-1和claudin-5的生化表征。此外，在hBMEC细胞系中进行ZO-1的免疫染色。四种细胞系均表达内皮细胞类型特异性粘附连接蛋白VE-钙粘蛋白。TJ蛋白ZO-1在hCMEC/D3和hBMEC细胞中表达。免疫细胞化学染色证实ZO-1在hBMEC细胞中表达。在hCMEC/D3、TY 10中检测到Claudin-5的表达，并且在hBMEC细胞中以非常低的水平检测到Claudin-5的表达。当在Greiner Bio-one®的24孔组织培养插入物（透明PET膜，3.0 μm孔径）上培养时，用hBMEC细胞系的单培养物获得Na-F和LY的最高TEER值和最低细胞旁渗透性。在与SVG-A和HBPCT细胞的共培养模型中，未观察到TEER的增加，这表明所研究的内皮细胞系均未对来自永生化星形胶质细胞或周细胞的刺激作出阳性反应。在我们的实验中检查的条件下，hBMEC被证明是最合适的人细胞系，用于在24孔单培养系统中的体外BBB模型，该模型涉及用于更高通量的屏障紧密性。该BBB模型正在用几种化合物（已知穿过或不穿过BBB）进行验证，并可能被选择用于评估生物活性天然产物的BBB渗透。
Reliable human in vitro blood–brain barrier (BBB) models suitable for high-throughput screening are urgently needed in early drug discovery and development for assessing the ability of promising bioactive compounds to overcome the BBB. To establish an improved human in vitro BBB model, we compared four currently available and well characterized immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, with respect to barrier tightness and paracellular permeability. Co-culture systems using immortalized human astrocytes (SVG-A cell line) and immortalized human pericytes (HBPCT cell line) were designed with the aim of positively influencing barrier tightness. Tight junction (TJ) formation was assessed by transendothelial electrical resistance (TEER) measurements using a conventional epithelial voltohmmeter (EVOM) and an automated CellZscope system which records TEER and cell layer capacitance (CCL) in real-time. Paracellular permeability was assessed using two fluorescent marker compounds with low BBB penetration (sodium fluorescein (Na-F) and lucifer yellow (LY)). Conditions were optimized for each endothelial cell line by screening a series of 24-well tissue culture inserts from different providers. For hBMEC cells, further optimization was carried out by varying coating material, coating procedure, cell seeding density, and growth media composition. Biochemical characterization of cell type-specific transmembrane adherens junction protein VE-cadherin and of TJ proteins ZO-1 and claudin-5 were carried out for each endothelial cell line. In addition, immunostaining for ZO-1 in hBMEC cell line was performed. The four cell lines all expressed the endothelial cell type-specific adherens junction protein VE-cadherin. The TJ protein ZO-1 was expressed in hCMEC/D3 and in hBMEC cells. ZO-1 expression could be confirmed in hBMEC cells by immunocytochemical staining. Claudin-5 expression was detected in hCMEC/D3, TY10, and at a very low level in hBMEC cells. Highest TEER values and lowest paracellular permeability for Na-F and LY were obtained with mono-cultures of hBMEC cell line when cultivated on 24-well tissue culture inserts from Greiner Bio-one® (transparent PET membrane, 3.0 μm pore size). In co-culture models with SVG-A and HBPCT cells, no increase of TEER could be observed, suggesting that none of the investigated endothelial cell lines responded positively to stimuli from immortalized astrocytic or pericytic cells. Under the conditions examined in our experiments, hBMEC proved to be the most suitable human cell line for an in vitro BBB model concerning barrier tightness in a 24-well mono-culture system intended for higher throughput. This BBB model is being validated with several compounds (known to cross or not to cross the BBB), and will potentially be selected for the assessment of BBB permeation of bioactive natural products.