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

Summary

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.

Abstract

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.

Introduction

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.

Protocol

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 .......

Discussion

The epithelial cells of the CP form the BCSFB that separates the CSF from the blood^2,3. 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.......

Materials

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