Abstract
The choroid plexus (CP) plays a major role in controlling the entry of substances and immune cells into the brain as it forms the blood-cerebrospinal fluid barrier (BCSFB) in the brain ventricles. Dysregulated immune cell trafficking through the epithelial cell (EC) layer of CP is central for the pathogenesis of infectious diseases in the brain and many neurodegenerative disorders. In vitro studies elucidating the function of the CP have so far been limited to the monolayer culture of CP ECs. To mimic immune cell migration across the CP barrier, a three-dimensional model would be advantageous. Here, we present an in vitro platform for studies of the immune cell trafficking based on CP explants/organoids. The explants were generated from fragments of mouse CPs in Matrigel, where the cells formed luminal spaces and could be maintained in culture for at least 8 weeks. We demonstrate expression of the major CP markers in the explants, including transthyretin and aquaporin 1 as well as ZO1 and ICAM-1, indicating a capacity for secretion of cerebrospinal fluid (CSF) and presence of tight junctions. CP explants displayed CP-like cell polarization and formed an intact EC barrier. We also show that the expression of transthyretin, transferrin, occludin and other genes associated with various functions of CP was maintained in the explants at similar levels as in native CP. By using dendritic cells and neutrophils, we show that the migration activity of immune cells and their interactions with CP epithelium can be monitored by microscopy. Thereby, the three-dimensional CP explant model can be used to study the cellular and molecular mechanisms mediating immune cell migration through CP epithelium and other functions of choroid EC. We propose this platform can potentially be used in the search for therapeutic targets and intervention strategies to improve control of (drug) substances and (immune) cell entry into the central nervous system.
Highlights
The choroid plexus (CP), located in each of the brain ventricles, produces the cerebrospinal fluid (CSF), controls the access of substances and immune cells from the blood to the CSF and transfers substances from the CSF to the blood
CP explants were capable of self-organizing into polarized cell layers and forming their lumen with an intact epithelial barrier, the presence of which is supported by the retention of fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) inside of explants (Supplementary Figures S1A,B)
The CP explants/organoids were generated from dissociated fragments of adult mouse CP, could be passaged and could reorganize themselves and form a closed lumen
Summary
The choroid plexus (CP), located in each of the brain ventricles, produces the cerebrospinal fluid (CSF), controls the access of substances and immune cells from the blood to the CSF and transfers substances from the CSF to the blood. Several in vitro models have been invented and used for studies on CP structure and function—primary CP fragments (Inoue et al, 2015), monolayers (Baehr et al, 2006; Monnot and Zheng, 2013) and clusters (Sandrof et al, 2017) In these systems, the functions of CP cannot be monitored, as the functionality of the tissue depends on the ability to maintain 3D structure (Redzic, 2013; Takebe and Wells, 2019), i.e., appropriate cell polarization allowing formation of the lumen and thereby intact EC barrier. Cerebral organoids (Lancaster and Knoblich, 2015; Watanabe et al, 2017) are a recently developed advanced system, where induced pluripotent stem cells (iPSC) from human skin, are differentiated into a brain-like structure by ‘‘guidance’’ with specific growth factors These cultures are not applied for studies using transgenic mouse material and individual mouse tissue comparison, as well as close monitoring of CP epithelium
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