Blood–brain-barrier organoids for investigating the permeability of CNS therapeutics

Abstract

In vitro models of the blood brain barrier (BBB) are crucial tools for the study of BBB transport and development of drugs that can reach the CNS. Brain endothelial cells grown in culture are often used to model the BBB however it is challenging to maintain reproducible BBB properties and function. “BBB organoids” are obtained following co-culture of endothelial cells, pericytes and astrocytes under low adhesion conditions. These organoids reproduce many features of the BBB, including the expression of tight junctions, molecular transporters and drug efflux pumps and hence can be used to model drug transport across the BBB. This protocol provides a comprehensive description of the techniques required to culture and maintaina BBB organoids. We also describe two separate detection approaches that can be used to analyze drug penetration into the organoids: confocal fluorescence microscopy and mass spectrometry imaging. Using our protocol, BBB organoids can be established within 2–3 days. An additional day is required to analyse drug permeability. The BBB organoid platform represents an accurate, versatile and cost-effective in vitro tool. It can easily be scaled to a high-throughput format, offering a tool for BBB modeling that could accelerate therapeutic discovery for the treatment of various neuro-pathologies.

Emerging links between cerebrovascular and neurodegenerative diseases—a special role for pericytes

“…The pericyte represents a cell type of particular therapeutic interest, given its critical role in controlling BBB integrity via regulation of endothelial barrier function. Although not perfect mimics of the in vivo situation, organoid-based in vitro BBB systems for endothelium-pericyte interactions have recently been developed [171,172]. Pericytes exhibit plasticity after experimental injury [169], supporting the notion that their function may be improved or restored in an injury situation.…”

“…[170]). Although not perfect mimics of the in vivo situation, organoid-based in vitro BBB systems for endothelium-pericyte interactions have recently been developed [171,172]. Such three-dimensional tissue-mimicking systems may be better approximations of the in vivo situation than their two-dimensional counterparts, and it will be interesting to learn whether these systems will accelerate the discovery of therapeutic agents and provide new insights into mechanisms affecting BBB integrity.…”

3D brain angiogenesis model to reconstitute functional human blood–brain barrier in vitro

“…Although the ultimate goal of regulating these proteins is to increase the efficacy of human CNS drug delivery, most tests have been performed using murine models (Bihorel, Camenisch, Lemaire, & Scherrmann, ; A. H. Schinkel et al, ), and human cell‐based platforms have been restricted to 2D culture assays (Gaillard et al, ; Holló et al, ; D. G. Kim & Bynoe, ) with low physiological relevance. Recently, a few 3D in vitro BBB spheroids with a functional efflux transport system have been introduced (Bergmann et al, ; C. F. Cho et al, ), but they lack a 3D perfusable vascular network, limiting their potential for drug application through the intraluminal region. Park et al () also described 2D microfluidic BBB model with a functional efflux pump.…”

“…G. Kim & Bynoe, 2016) with low physiological relevance. Recently, a few 3D in vitro BBB spheroids with a functional efflux transport system have been introduced (Bergmann et al, 2018;C. F. Cho et al, 2017), but they lack a 3D perfusable vascular network, limiting their potential for drug application through the intraluminal region.…”

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“…• Random positioning of the developing brain structures Cerebral organoids develop in the absence of extraembryonic tissues that provide essential cues to set up the antero-posterior axis of the embryonic brain. As a result, cerebral organoids lack normal [8] embryonic axis formation, an important mechanism that instructs the ordered formation of specific brain regions in the correct orientation.…”