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Merck

Modeling ischemic stroke in a triculture neurovascular unit on-a-chip.

Fluids and barriers of the CNS (2021-12-16)
Nienke R Wevers, Arya Lekshmi Nair, Tania M Fowke, Maria Pontier, Dhanesh G Kasi, Xandor M Spijkers, Charlie Hallard, Gwenaëlle Rabussier, Remko van Vught, Paul Vulto, Helga E de Vries, Henriëtte L Lanz
ABSTRACT

In 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. We 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. Immunofluorescent 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). The 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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
hEGF, EGF, recombinant, expressed in E. coli, lyophilized powder, suitable for cell culture
Sigma-Aldrich
Staurosporine from Streptomyces sp., ≥98% (HPLC), film
Sigma-Aldrich
Zosuquidar hydrochloride, ≥98% (HPLC)
Sigma-Aldrich
N6,2′-O-Dibutyryladenosine 3′,5′-cyclic monophosphate sodium salt, ≥96% (HPLC), powder
Sigma-Aldrich
Antimycin A from Streptomyces sp.
Sigma-Aldrich
Fetal Bovine Serum, USA origin, Heat Inactivated, sterile-filtered, suitable for cell culture, suitable for insect cell culture, suitable for hybridoma