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

Abstract

Background 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. Methods 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. Results 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). Conclusions 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.

Promising Strategies for the Development of Advanced In Vitro Models with High Predictive Power in Ischaemic Stroke Research

“…The chemical method relies on inhibition of the mitochondrial electron transport chain and has been regularly applied to cell cultures to study ischaemic stroke. For instance, sodium azide and antimycin are commonly used chemical-hypoxia inducers in these studies [21][22][23][24]. Less common is the enzymatic induction of hypoxia, which relies on manipulating the glucose oxidase and catalase (GOX/CAT) system [25][26][27].…”

“…Due to implementation of novel technologies in in vitro stroke model development, recently, researchers were able to recapitulate another factor besides oxygen and glucose depletion, namely the interrupted blood flow, by employing microfluidic systems [23,29]. This appears to be another factor affecting the downstream ischaemic cascade by reducing the integrity of the BBB and thereby allowing it to mimic in vivo stroke even more closely.…”

“…A first study using human iPSC-derived neurons was performed in 2020 by Juntunen et al, where the effect of OGD and potential protection by adipose stem cells was investigated [79]. Furthermore, human iPSC-derived cells are also increasingly being employed in the context of BBB/NVU models [23,[80][81][82]. It should be noted that, though iPSC-derived in vitro platforms have boosted research in many fields and hold great promise for the future, there are still challenges associated with the use of iPSCs.…”

“…Moreover, microfluidic platforms are associated with limited scalability [105]. Currently, novel platforms are being developed, allowing the culture of multiple chips in parallel [23]. Finally, the use of microfluidics typically requires smaller amounts of media and cells compared to traditional cell culture systems.…”

Advances in brain barriers and brain fluids research in 2021: great progress in a time of adversity

“…Progress continues to be made on modeling the NVU using brain microfluidic chips seeded with various vascular cells and subjected to conditions reflective of different disease states. For example, Lyu et al [ 95 ] and Wevers et al [ 96 ] have developed NVU-on-a chip models for examining therapeutic strategies for stroke. Pediaditakis et al [ 97 ] have devised brain-chips with fluid flow through vascular channels that model Parkinson’s disease and related synucleinopathies.…”