An Advanced Co-Culture Model of the Human Blood-Cerebrospinal Fluid Barrier for Separate Analysis of Choroid Plexus Epithelium and Endothelium

Summary

A co-culture of human epithelial and endothelial cells of the choroid plexus was established to generate an advanced in vitro model of the blood-cerebrospinal fluid barrier. We describe the separate isolation of epithelium and endothelium for subsequent RNA preparation and transcriptional analysis under selected conditions.

Abstract

The highly perfused choroid plexus (CP), located in the ventricular system of the brain, consists of several structural components, including a cuboid epithelium and a fenestrated endothelium, and is the location of the blood-cerebrospinal fluid barrier (BCSFB). To investigate the CP in vitro, several different cell culture setups are conceivable. An inverted cell culture filter model with strong barrier function, based on human epithelial CP papilloma (HIBCPP) cells, was established by our laboratory and is used extensively. Recently, we were able to generate a stable immortalized human CP endothelial cell line (iHCPEnC). Combining both cell lines, we have assembled an advanced functional two-cell-type model that developed an increased barrier function compared to HIBCPP cells alone. Since this model represents both the CP epithelium and endothelium, it allows for the investigation of the interplay between the two cell types under selected conditions, such as infection, inflammation, or others. In this protocol, we describe methods to set up this two-cell-type model with subsequent separation of the epithelial and endothelial cells for transcriptional analyses. We describe PCR primers specific for epithelium and endothelium, which serve to detect possible cross-contamination between the two cell types. To investigate a possible impact of the CP endothelium on the epithelium in this model under inflammatory conditions, the expression levels of selected genes in the context of lipopolysaccharide (LPS) treatment were analyzed in the HIBCPP cell fraction. The advanced CP in vitro model and the possibility to separately analyze epithelial and endothelial responses are of significant interest for research on cellular processes involving biological and pathological functions of the CP.

Introduction

The choroid plexus (CP) is a highly vascularized conglomerate of different populations of cells located in all four ventricles of the brain. Among other functions, it serves as a physiological barrier, the so-called blood-cerebrospinal fluid barrier (BCSFB), between the central nervous system (CNS) and the blood, preventing the brain from, e.g., infectious substances circulating in the blood. The CP consists of different cell types, including the polarized epithelial cells, containing tight- and adherence junctions as well as desmosomes, sitting on a basement membrane. The epithelial cells' surface is enlarged by the presence of microvilli on the luminal surface. ....

Protocol

1. HIBCPP cells

NOTE: In the following, we describe the culture and maintenance of HIBCPP cells, as well as the preparation of inverted cell culture filter inserts with HIBCPP cells. This protocol is designed for the growth of HIBCPP cells on 12-well cell culture filter inserts. A detailed protocol with accompanying video for culture of HIBCPP cells on 24-well cell culture filter inserts has been published previously⁹.

1. Taking HIBCPP cells into culture
   1. Thaw a vial of HIBCPP cells at room temperature (RT).
   2. Resuspend the content of the vial with 10 mL of DMEM/HAMS-F12 ....

Results

In this protocol, we describe the step-by-step generation of a two-cell-type model of the human CP consisting of epithelium (HIBCPP cells) and endothelium (iHCPEnC) on cell culture filter inserts, as shown schematically in Figure 1. In this model system, HIBCPP cells are grown in an inverted fashion on the lower side of the filter, whereas iHCPEnC are seeded either on the upper side of the filter of the insert or on a second filter placed on top of the membrane of the insert.

Discussion

The human CP consists of several different cell types, including a barrier-forming epithelium and an endothelium of the extended vasculature³. Although the endothelial cells of the CP are fenestrated, recent research has demonstrated their contribution to barrier function at the BCSFB^4,7. To study the intricate interactions between the endothelium and epithelium in organs as the CP, advanced in vitro models allowing analysis of bo.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.25% Trypsin-EDTA	Life Technologies	25200056
12 well cell culture plate	StarLab	CC7682-7512
12 well filter inserts (3 µM pores)	Greiner Bio One	665631
24 well cell culture plate	StarLab	CC7672-7524
24 well filter insert  (0.4 µM pores)	Greiner Bio One	662641
24 well filter insert (3 µM pores)	Greiner Bio One	662631
6 well cell culture plate	StarLab	CC7682-7506
AffinityScript cDNA Synthesis Kit (cDNA synthesis )	Agilent	600559
Attachment Factor	Cell Systems	4ZO-500	Part of the Complete Classic Endothelial Medium kit.
Brilliant II SYBR Green qPCR Master Mix (real-time PCR )	Agilent	600828
Chopstick electrode	Millipore	MERSSTX01	This product is discontinued.
Complete Classic Endothelial Medium (iHCPEnC medium)	Cell Systems	4ZO-500	Kit, containing medium, supplements and Attachment Factor for coating.
DMEM/F-12 (1:1) w/ phenolred (10% HIBCPP medium)	Thermo Fisher	31330-038
DMEM/F-12 (1:1) w/o phenolred (1% HIBCPP medium)	Thermo Fisher	11039-021
Fetal Calf Serum (FCS) (1% and 10% HIBCPP medium)	Thermo Fisher	10270-106
Insulin (1% and 10% HIBCPP medium)	Sigma	I9278
Lipopolysaccharide (LPS)	Sigma	L3024
MilliCell ERS2	Millipore	MERS00002	This product is discontinued.
Nanodrop (Spectrophotometer)	Thermo Scientific	ND1000	This product is discontinued.
No. 11 perforating iron	Würth	880223211
PBS	Life Technologies	14190169
Primer designing tool	NCBI	https://www.ncbi.nlm.nih.gov/tools/primer-blast/
Primers	Sigma	VC00021
Qiagen RNeasy micro	Qiagen	74004
RLT+b-ME (part of Qiagen Rneasy micro kit)	Qiagen	74004
T25 cell culture flask	Sarstedt AG &Co KG	83,39,10,002
T75 cell culture flask	Sarstedt AG &Co KG	83,39,11,002
TRAKETCH PET 3.0 cc (separate membrane)	Sabeu	SK4580
UV light	Carl Roth	H469.1