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Abstract

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Materials

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Medicine

A Choroid Plexus Epithelial Cell-based Model of the Human Blood-Cerebrospinal Fluid Barrier to Study Bacterial Infection from the Basolateral Side

Published: May 6th, 2016

DOI:

10.3791/54061

1Department of Pediatrics, Medical Faculty Mannheim, Heidelberg University, 2Department of NDU Life Sciences, Nippon Dental University

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.

The blood-cerebrospinal fluid barrier (BCSFB) is one of the three barrier sites between the blood and the brain1. Its morphological correlate are the epithelial cells of the choroid plexus (CP)2,3, an endothelial-epithelial convolute, which is strongly vascularized and located in the ventricles of the brain. The CP serves to produce the cerebrospinal fluid (CSF) as well as to separate the latter from the blood. In order to achieve barrier function, the CP epithelial cells show a low pinocytotic activity, express specific transporters, and are densely connected by a continuous network of tight junctions (TJs)2,3.

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1. Prepare Cell Culture Filter Inserts for Seeding HIBCPP Cells in an Inverted Model System

  1. Pre-warm DMEM/ F12 (Ham) supplemented with 5 µg/ml insulin, 100 U/ml penicillin, 100 µg/ml streptomycin and 10% fetal calf serum (FCS).
  2. Use sterile forceps to place 0.33 cm² growth area cell culture filter inserts with a pore size of 3 µm upside down into a 12-well plate (Figure 1E).
  3. Fill medium into the lower compartment of the cell culture filter insert (about .......

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Here we describe culturing and infection of HIBCPP cells in an inverted cell culture insert system. This model allows us to study invasion mechanisms and the underlying molecular signaling pathways from the basolateral cell side, reproducing a physiological situation of bacteria disseminating and entering epithelial cells via the blood stream (Figure 1).

The HIBCPP cells display certain barrier functions, which .......

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The epithelial cells of the CP form the BCSFB that separates the CSF from the blood2,3. We recently established the HIBCPP cell line as a functional human model of the BCSFB. The cells display important barrier functions of the BCSFB in vitro, including the development of a high membrane potential, a low permeability for macromolecules, as well as the presence of continuous strands of TJs5. The TJ proteins contribute to an apical/basolateral polarity of the cells. The polarity is of high im.......

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The authors would like to thank Prof. Hartwig Wolburg for performing the electron microscopy.

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Name Company Catalog Number Comments
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

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