A novel dynamic multicellular co-culture system for studying individual blood-brain barrier cell types in brain diseases and cytotoxicity testing

Patricia Miranda-Azpiazu,Sikha Saha,Gin Jose,Stavros Panagiotou

doi:10.1038/s41598-018-26480-8

Patricia Miranda-Azpiazu, Sikha Saha + Show 2 more

Open Access

https://doi.org/10.1038/s41598-018-26480-8

Copy DOI

Journal: Scientific Reports	Publication Date: Jun 8, 2018
Citations: 34	License type: open-access

Affiliation: University of Leeds

Abstract

Blood brain barrier (BBB) cells play key roles in the physiology and pathology of the central nervous system (CNS). BBB dysfunction is implicated in many neurodegenerative diseases, including Alzheimer’s disease (AD). The BBB consists of capillary endothelial cells, pericytes encircling the endothelium and surrounding astrocytes extending their processes towards it. Although there have been many attempts to develop in vitro BBB models, the complex interaction between these cell types makes it extremely difficult to determine their individual contribution to neurotoxicity in vivo. Thus, we developed and optimised an in vitro multicellular co-culture model within the Kirkstall Quasi Vivo System. The main aim was to determine the optimal environment to culture human brain primary endothelial cells, pericytes and astrocytes whilst maintaining cellular communication without formation of a barrier in order to assess the contribution of each cell type to the overall response. As a proof of concept for the present system, the effects of amyloid-beta 25-35 peptide (Aβ25-35), a hallmark of AD, were explored. This multicellular system will be a valuable tool for future studies on the specific roles of individual BBB cell type (while making connection with each other through medium) in CNS disorders as well as in cytotoxicity tests.

Highlights

The blood brain barrier (BBB) is a specialised structure separating the central nervous system (CNS) from the peripheral blood circulation
Brain endothelial cells are responsible for formation of tight junctions, both pericytes and astrocytes have been shown to participate in their formation[3,4,5,6,7], and are critical for maintaining normal Blood brain barrier (BBB) physiology and function as a barrier
The ability of endothelial cells to form tight junctions under static conditions was confirmed by antibody labelling of the tight junction marker, zonula occludens (ZO1), thereby demonstrating they were able to express this marker even in the absence of flow (Fig. 1D)

Summary

Introduction

The blood brain barrier (BBB) is a specialised structure separating the central nervous system (CNS) from the peripheral blood circulation. It is crucial for maintaining the homeostasis of the brain microenvironment and prevention of entry of toxic substances into the CNS1,2. Brain endothelial cells are responsible for formation of tight junctions, both pericytes and astrocytes have been shown to participate in their formation[3,4,5,6,7], and are critical for maintaining normal BBB physiology and function as a barrier. Despite the fact that several BBB barrier models have been created, most lack the ability to study individual BBB cell types separately, whilst maintaining communication between them. Images were taken from an optical microscope (Nikon Eclipse TS 100) at 20× augmentation

Objectives

Methods

Results

Conclusion