The epithelial cells of the choroid plexus (CP) form the blood-cerebrospinal fluid barrier (BCSFB). An in vitro model of the BCSFB employs human choroid plexus papilloma (HIBCPP) cells. This article describes culturing and basolateral infection of HIBCPP cells using a cell culture filter insert system.
The epithelial cells of the choroid plexus (CP), located in the ventricular system of the brain, form the blood-cerebrospinal fluid barrier (BCSFB). The BCSFB functions in separating the cerebrospinal fluid (CSF) from the blood and restricting the molecular exchange to a minimum extent. An in vitro model of the BCSFB is based on cells derived from a human choroid plexus papilloma (HIBCPP). HIBCPP cells display typical barrier functions including formation of tight junctions (TJs), development of a transepithelial electrical resistance (TEER), as well as minor permeabilities for macromolecules. There are several pathogens that can enter the central nervous system (CNS) via the BCSFB and subsequently cause severe disease like meningitis. One of these pathogens is Neisseria meningitidis (N. meningitidis), a human-specific bacterium. Employing the HIBCPP cells in an inverted cell culture filter insert system enables to study interactions of pathogens with cells of the BCSFB from the basolateral cell side, which is relevant in vivo. In this article, we describe seeding and culturing of HIBCPP cells on cell culture inserts. Further, infection of the cells with N. meningitidis along with analysis of invaded and adhered bacteria via double immunofluorescence is demonstrated. As the cells of the CP are also involved in other diseases, including neurodegenerative disorders like Alzheimer`s disease and Multiple Sclerosis, as well as during the brain metastasis of tumor cells, the model system can also be applied in other fields of research. It provides the potential to decipher molecular mechanisms and to identify novel therapeutic targets.
血-脑脊髓液屏障(BCSFB)是血液和脑1之间三个屏障站点之一。其形态相关成分是脉络丛(CP)2,3-上皮细胞,内皮上皮回旋,这强烈血管和位于脑的脑室。将CP用来产生脑脊液(CSF),以及后者从血液中分离出来。为了实现屏障功能,在CP的上皮细胞显示出低的吞饮活性,表达特定转运,并且通过紧密连接(紧密连接)2,3-的连续网络密集连接。
人类脉络丛乳头状瘤(HIBCPP)细胞,从日本女子4的恶性脉络丛乳头状瘤衍生的,用于构建在 BCSFB的体外模型的功能。 HIBCPP细胞显示一对夫妇的功能BCSFB的特点,为TJ形成股线,可以判断为跨上皮电阻(TEER)高跨上皮膜电位的发展,以及对大分子次要渗透率。此外,HIBCPP细胞表达的特点转运,这可能会起到调节离子微环境,并显示心尖/基底极性5,6,7。
该BCSFB已显示作为一个条目的网站的病原体(细菌,病毒和真菌)进入中枢神经系统(CNS)8。病原体,包括脑膜炎奈瑟氏球菌 ( 脑膜炎奈瑟氏球菌 ),一种革兰氏阴性细菌,入侵能引起严重的疾病如脑膜炎。 证据表明,它克服了CP的保护上皮屏障是由组织病理学观察患者支持与脑膜炎球菌性疾病表现出增加的血管和CP上皮细胞9,10-脑膜炎球菌的量。要进入宿主细胞巴cteria经常挟持内吞机制,其介导或由位于宿主细胞特异性表面受体触发。由于具有这些受体的病原体的相互作用可以是物种特异性11,动物模型只能协商,以一个受限制的程度。该HIBCPP细胞系提供了机会,研究入侵过程中,以及在人体模型系统的分子机制。采用细胞培养插入使我们能够分析病原体相互作用与宿主细胞从两个不同的细胞两侧。许多细菌,包括N。脑膜炎 ,强烈受重力的期间感染测定法的影响。对于与检测期间HIBCPP细胞病原体最佳交互,该细菌最初添加到细胞培养滤芯系统的上隔室。到从心尖或基底细胞侧,分别在体外系统的两种变化已经埃斯塔使感染blished:在标准系统HIBCPP细胞接种到过滤器插入件的上部隔室,模仿情况时微生物位于CSF的侧,并获得与所述细胞( 图1A,C)的顶面接触。与此相反,使用HIBCPP细胞在倒置的细胞培养滤芯系统反映当细菌进入血流的条件。微生物传播在从基底外侧侧( 图1B,D)的血液和遭遇的CP上皮细胞。值得注意的是,在该模型系统,已经表明,细菌侵入HIBCPP细胞极性的方式专门从基底细胞侧5,7。
随后向CP的感染,所述入侵病原体可通过结扎先天免疫系统对模式识别受体(的PRRs)识别。蓝耳的良好描述成员属于Toll样受体(TLR)家族。 Toll样受体能斌d,来感染性微生物的特性的结构,其被称为病原体相关分子模式(PAMP)。受体的结扎导致触发的细胞因子和趋化因子12,其反过来横跨BCSFB 13,14刺激免疫细胞的轮回的表达宿主细胞的活化信号级联。它已经显示HIBCPP细胞表达在mRNA水平数的TLR和感染N.脑膜炎导致多种细胞因子和趋化因子,包括CXCL1-3,IL6,IL8和TNFα15,16的分泌。
在这里,我们描述了一个模仿BCSFB倒置细胞培养插管系统种植和人细胞系HIBCPP的感染。此模型系统使得来研究与体内的相关基底细胞侧以及随后的细胞反应的病原体的相互作用。
CP的上皮细胞形成分隔从血液2,3脑脊液的BCSFB。我们最近成立了HIBCPP细胞系作为BCSFB的功能人体模型。细胞显示在体外 BCSFB的重要屏障功能,包括一个高的膜电位的发展,一个低渗透性的大分子,以及紧密连接5的连续股线的存在。的TJ蛋白向细胞的顶/底外侧极性。极性是表面受体的定向定位特定的转运蛋白高的重要性,以及。我们已经证明了受体ECAD和Met以及ATP结合盒的部件…
The authors have nothing to disclose.
The authors would like to thank Prof. Hartwig Wolburg for performing the electron microscopy.
0.25% Trypsin-EDTA | Gibco | 25200-056 | |
4´,6 diamidino-2-phenylindole (DAPI) | Life Technologies | D1306 | |
12-well plates | Starlab | CC7682-7512 | |
24-well plates | Starlab | CC7682-7524 | |
Anti Neisseria meningitidis α-OMP | This antibody was a gift from Drs. H. Claus and U. Vogel (University of Würzburg, Germany) | ||
Alexa Fluor 488 (chicken anti rabbit) | Invitrogen | A21441 | |
Alexa Fluor 594 (chicken anti rabbit) | Invitrogen | A21442 | |
Alexa Fluor 660 Phalloidin | Invitrogen | A22285 | |
Bovine serum albumine (BSA) | Calbiochem | 12659 | |
Chocolate agar plates | Biomerieux | 43109 | |
Cytochalasin D | Sigma | C8273 | |
DMEM/F12 + L-Glut + 15 mM HEPES | Gibco | 31330-095 | |
DMEM/F12 + L-Glut + 15 mM HEPES w/o Phenolred | Gibco | 11039-047 | |
Dimethyl sulfoxide | Sigma | D2650 | |
Fetal calf serum (FCS) | Life Technologies | 10270106 | |
FITC-Inulin | Sigma | F3272 | |
Insulin | Sigma | 19278 | |
MgCl2 | Sigma | 2393 | |
NaHCO3 | Sigma | 55761 | |
PBS + Mg +Ca | Gibco | 14040-174 | |
Penicillin/Streptomycin | MP Biomedicals | 1670049 | |
Polyvitex | Biomerieux | 55651 | |
Proteose peptone | BD | 211684 | |
Serum-free medium | Gibco | 10902-096 | |
Thincert cell culture inserts for 24-well plates, pore size 3 µm | Greiner | 662630 | |
Tissue culture flask 75 cm² red cap sterile | Greiner | 658175 | |
Triton X-100 | Sigma | T8787 | |
Volt-Ohm Meter Millicell-ERS2 with MERSSTX01 electrode | Millipore | MERSSTX00 |