Engineering 3D Cortical Spheroids for an In Vitro Ischemic Stroke Model

Abstract

Three-dimensional (3D) spheroids composed of brain cells have shown great potential to mimic the pathophysiology of the brain. However, a 3D spheroidal brain-disease model for cerebral ischemia has not been reported. This study investigated an ultralow attachment (ULA) surface-mediated formation of 3D cortical spheroids using primary rat cortical cells to recapitulate the cerebral ischemic responses in stroke by oxygen-glucose deprivation-reoxygenation (OGD-R) treatment. Comparison between two-dimensional (2D) and 3D cell culture models confirmed the better performance of the 3D cortical spheroids as normal brain models. The cortical cells cultured in 3D maintained their healthy physiological morphology of a less activated state and suppressed mRNA expressions of pathological stroke markers, S100B, IL-1β, and MBP, selected based on in vivo stroke model. Interestingly, the spheroids formed on the ULA surface exhibited striking aggregation dynamics involving active cell–substrate interactions, whereas those formed on the agarose surface aggregated passively by the convective flow of the media. Accordingly, ULA spheroids manifested a layered arrangement of neurons and astrocytes with higher expressions of integrin β1, integrin α5, N-cadherin, and fibronectin than the agarose spheroids. OGD-R-induced stroke model of the ULA spheroids successfully mimicked the ischemic response as evidenced by the upregulated mRNA expressions of the key markers for stroke, S100B, IL-1β, and MBP. Our study suggested that structurally and functionally distinct cortical spheroids could be generated by simply tuning the cell–substrate binding activities during dynamic spheroidal formation, which should be an essential factor to consider in establishing a brain-disease model.

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

“…The same could be concluded when thapsigargin was used as stimulus, after eliminating the possibility of reduced drug accessibility as a confounding factor [31]. In the context of the development of a 3D cortical spheroid model for cerebral ischaemia, Ko et al confirmed that 3D cell culture models represent better normal brain models, since the neural cells in 3D maintained their healthy physiological morphology of a less activated state and suppressed mRNA expressions of pathological stroke markers S100B, IL1-β and MBP [151]. Moreover, our studies demonstrated different behavioural responses of neural cells in 2D and 3D.…”

“…Iwasa et al subjected cerebral organoids to OGD and reoxygenation and identified peroxisome proliferator-activated receptor (PPAR) signalling and pyruvate kinase isoform M2 (PKM2) as key markers of neuronal cells in response to OGD and reoxygenation [150]. In addition, Ko et al described 3D cortical spheroids derived from primary rat cortical cells treated with OGD and reoxygenation as a model for cerebral ischaemia [151]. They demonstrated that their model successfully mimicked the ischaemic response as evidenced by the upregulated mRNA expressions of the key markers for stroke, S100B, IL-1β and MBP and additionally substantiate the role of transient cell-substrate interactions herein.…”

Spheroid Engineering in Microfluidic Devices

“…One conventional approach for creating spheroids is the use of ultralow attachment plates (ULA), which is based on employing hydrophobic substrates to hinder cell adhesion and cause the cells to interact with each other to form spheroids − (Figure E). The main limitation of this technique is the inhomogeneous size distribution of produced spheroids, which may lead to inconsistent results in experimental studies …”

Ischemic Stroke, Lessons from the Past towards Effective Preclinical Models

“…To date, few studies evaluate the damage caused by OGD in spheroid cultures. In a recent study, OGD was used to simulate stroke in spheroids produced from the mouse brain cortex [ 122 ]. Researchers evaluated the expression of some key proteins that are associated with stroke effects (S100B, interleukin-1β, and myelin binding protein).…”

“…Researchers evaluated the expression of some key proteins that are associated with stroke effects (S100B, interleukin-1β, and myelin binding protein). Thus, it proved that the OGD model of in vitro ischemia successfully works on cortical spheroids [ 122 ], opening gates for new research on stroke in vitro.…”