A Novel Dynamic Neonatal Blood-Brain Barrier on a Chip.

Sudhir P Deosarkar,Barbara Krynska,Joel B Sheffield,Bin Wang,Balabhaskar Prabhakarpandian,Mohammad F Kiani,Mária A Deli

doi:10.1371/journal.pone.0142725

Abstract

Studies of neonatal neural pathologies and development of appropriate therapeutics are hampered by a lack of relevant in vitro models of neonatal blood-brain barrier (BBB). To establish such a model, we have developed a novel blood-brain barrier on a chip (B3C) that comprises a tissue compartment and vascular channels placed side-by-side mimicking the three-dimensional morphology, size and flow characteristics of microvessels in vivo. Rat brain endothelial cells (RBEC) isolated from neonatal rats were seeded in the vascular channels of B3C and maintained under shear flow conditions, while neonatal rat astrocytes were cultured under static conditions in the tissue compartment of the B3C. RBEC formed continuous endothelial lining with a central lumen along the length of the vascular channels of B3C and exhibited tight junction formation, as measured by the expression of zonula occludens-1 (ZO-1). ZO-1 expression significantly increased with shear flow in the vascular channels and with the presence of astrocyte conditioned medium (ACM) or astrocytes cultured in the tissue compartment. Consistent with in vivo BBB, B3C allowed endfeet-like astrocyte-endothelial cell interactions through a porous interface that separates the tissue compartment containing cultured astrocytes from the cultured RBEC in the vascular channels. The permeability of fluorescent 40 kDa dextran from vascular channel to the tissue compartment significantly decreased when RBEC were cultured in the presence of astrocytes or ACM (from 41.0±0.9 x 10−6 cm/s to 2.9±1.0 x 10−6 cm/s or 1.1±0.4 x 10−6 cm/s, respectively). Measurement of electrical resistance in B3C further supports that the addition of ACM significantly improves the barrier function in neonatal RBEC. Moreover, B3C exhibits significantly improved barrier characteristics compared to the transwell model and B3C permeability was not significantly different from the in vivo BBB permeability in neonatal rats. In summary, we developed a first dynamic in vitro neonatal BBB on a chip (B3C) that closely mimics the in vivo microenvironment, offers the flexibility of real time analysis, and is suitable for studies of BBB function as well as screening of novel therapeutics.

Highlights

Blood-brain barrier (BBB) is a physical and functional barrier formed by the brain vascular endothelial cells and perivascular cells [1, 2]
In vitro BBB models that closely mimic the in vivo BBB microenvironment are valuable tools for the study of neonatal BBB function as well as for screening of novel therapeutics
Transwell BBB models approximate several of the important aspects of the in vivo BBB and are routinely used for BBB studies and for screening neurotherapeutics [38, 39]

Summary

Introduction

Blood-brain barrier (BBB) is a physical and functional barrier formed by the brain vascular endothelial cells and perivascular cells [1, 2]. Transwell based BBB models have been improved to approximate several important aspects of the in vivo BBB including high electrical resistance and realistic cytoarchitecture [12,13,14,15,16] These in vitro BBB models often lack realistic morphological (e.g. realistic microvascular size and tube-like structure of vascular channels) and functional (e.g. physiological shear flow in the vascular compartment) features that allow for the development of a realistic in vivo-like microenvironment. Two-dimensional monocultures of primary adult endothelial cells used in simple BBB in vitro models over time lose many of the characteristics of the in vivo phenotype, e.g. tight junction formation These observations suggest that a proper microenvironment such as factors secreted by the perivascular cells and/or realistic shear forces from blood flow is required to maintain an optimally functioning neonatal in vitro BBB

Methods

Results

Conclusion