Abstract
BackgroundIn ischemic stroke, the function of the cerebral vasculature is impaired. This vascular structure is formed by the so-called neurovascular unit (NVU). A better understanding of the mechanisms involved in NVU dysfunction and recovery may lead to new insights for the development of highly sought therapeutic approaches. To date, there remains an unmet need for complex human in vitro models of the NVU to study ischemic events seen in the human brain.MethodsWe here describe the development of a human NVU on-a-chip model using a platform that allows culture of 40 chips in parallel. The model comprises a perfused vessel of primary human brain endothelial cells in co-culture with induced pluripotent stem cell derived astrocytes and neurons. Ischemic stroke was mimicked using a threefold approach that combines chemical hypoxia, hypoglycemia, and halted perfusion.ResultsImmunofluorescent staining confirmed expression of endothelial adherens and tight junction proteins, as well as astrocytic and neuronal markers. In addition, the model expresses relevant brain endothelial transporters and shows spontaneous neuronal firing. The NVU on-a-chip model demonstrates tight barrier function, evidenced by retention of small molecule sodium fluorescein in its lumen. Exposure to the toxic compound staurosporine disrupted the endothelial barrier, causing reduced transepithelial electrical resistance and increased permeability to sodium fluorescein. Under stroke mimicking conditions, brain endothelial cells showed strongly reduced barrier function (35-fold higher apparent permeability) and 7.3-fold decreased mitochondrial potential. Furthermore, levels of adenosine triphosphate were significantly reduced on both the blood- and the brain side of the model (4.8-fold and 11.7-fold reduction, respectively).ConclusionsThe NVU on-a-chip model presented here can be used for fundamental studies of NVU function in stroke and other neurological diseases and for investigation of potential restorative therapies to fight neurological disorders. Due to the platform’s relatively high throughput and compatibility with automation, the model holds potential for drug compound screening.
Highlights
In ischemic stroke, the function of the cerebral vasculature is impaired
The entire structure contributing to blood–brain barrier (BBB) function is referred to as the neurovascular unit (NVU) and includes brain endothelial cells, astrocytes, pericytes, neurons, oligodendrocytes, microglia, and the basement membrane [4]
Human brain microvascular endothelial cell (HBMEC) were used between passage 4 and 10. induced pluripotent stem cell (iPSC)-derived human astrocytes (01434, FujiFilm-CDI) were expanded in T75 flasks (734–2705, Corning) coated with GelTrex (A15696-01, Gibco) in medium comprised of DMEM (31966-021, Gibco) supplemented with 10% fetal bovine serum (FBS, F4135, Sigma), 1 × N2 supplement (17502048, ThermoFisher), and 1% penicillin/streptomycin (P4333, Sigma)
Summary
The function of the cerebral vasculature is impaired This vascular structure is formed by the so-called neurovascular unit (NVU). The vasculature of the brain is Wevers et al Fluids and Barriers of the CNS (2021) 18:59 surrounded by perivascular cells that support and maintain healthy BBB functioning. Among these supporting cells are astrocytes and pericytes, which strengthen the inter-endothelial adherens junctions and tight junctions and maintain proper BBB transport function [2, 3]. The entire structure contributing to BBB function is referred to as the neurovascular unit (NVU) and includes brain endothelial cells, astrocytes, pericytes, neurons, oligodendrocytes, microglia, and the basement membrane [4]. While the NVU’s barrier is essential for healthy brain functioning, it poses a major challenge for drug delivery into the brain, as many drugs can’t freely enter the brain or are removed by efflux transporters [7]
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