Introduction of 3D Cortex Spheroids

Stroke accounts for the greatest number of disability and the second greatest number of deaths worldwide. Due to the intrinsic complexity and poor accessibility of the brain, coupled with the complex pathologic events following stroke, there are currently no effective treatments to restore function after brain injury, and therefore new therapeutic strategies need to be developed. The development of a suitable model that accurately summarizes the pathophysiological events of cerebral ischemia and facilitates intuitive and rapid assessment is essential.

Three-dimensional (3D) spheroids composed of brain cells show great potential in modeling brain pathophysiology. Compared to traditional two-dimensional (2D) cell cultures, tightly bound 3D cell aggregates represent denser neurons and tight intercellular interactions between various constituent cells in the brain. In addition, by mixing cells of different lineages or assembling spheres of different brain regions, brain spheroids have the potential to generate higher-order 3D structures that go beyond 2D cell culture in replicating the structural organization of the brain, thus providing support for elucidating animal models of human brain pathology. Many 3D spheroidal brain-disease models for cerebral ischemia have been reported. It has been shown that 3D spheroids can reproduce key features of ischemic injury and may serve as a useful model for therapeutic screening. This ischemic injury model provides an in vivo-relevant, high-throughput compatible, flexible platform to study the mechanisms of ischemic injury.

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• Custom 3D In Vitro Model of Ischemic Stroke

Limitations of 2D Cell Culture Models and Animal Models of Stroke

Many different animal models have been developed to replicate the human stroke condition. Despite the advantages of animals in maximizing the retention of physiological correlates, the relatively high cost and time-consuming experimental procedures of animal models greatly limit their applicability. In addition, the challenge of performing controlled experiments in animals complicates the process of elucidating the underlying molecular mechanisms of stroke due to the presence of unexplained intertwined factors in the organism.

In addition, a variety of various in vitro cellular models have been used to mimic cerebral ischemia, ranging from 2D cell line culture and 2D primary cell culture to organotypic brain slide culture. In vitro modeling of stroke relies heavily on the induction of an ischemia-like condition through oxygen-glucose deprivation (OGD) or chemical or enzymatic disruption of cellular metabolism. While 2D cell culture platforms are the most convenient in vitro models to use, they often fail to reproduce important pathophysiological features of 3D in vivo brain tissue.

3D Cortical Spheroid Model

Scientists have reported a self-assembled, scaffold-free, three-dimensional in vitro cortical spheroid model.

• These spherical microtissues are derived from the primary postnatal rodent cortex and have a cellular composition, tissue stiffness, and cell density similar to what is found in the in vivo cortex. Neurons within the spheroids become electrically active and form synaptic connections within two weeks.
• This model enables the creation of hundreds of spheroids from each rodent, allowing for extensive condition testing while minimizing animal use.
• This model has the ability to study brain pathologies and treatments in both female- and male-derived microtissues.
• The spheroids contain various brain cell types and feature spontaneously formed capillary-like networks, a distinctive aspect not replicated in other in vitro models like organoids. Their reproducibility in size and composition makes them suitable for high-throughput assays.

Fig.1. Spheroids contained reactive astrocytes following oxygen and oxygen-glucose deprivation. (McLaughlin, et al., 2023)

3D Cortical Spheroid Model for Studying the Mechanisms of Stroke

In this model, to examine the cellular and molecular processes involved in stroke, spheroids can be starved of glucose, oxygen, or oxygen and glucose for 24 h. Certain molecular and cellular processes associated with brain injury due to ischemia can also be investigated.

Ace Therapeutics use 3D cortex spheroids model to help clients studying the mechanisms of stroke and develop novel therapeutics. We can conduct research in the following areas:

• Confirmation of cell survival in oxygen and oxygen-glucose starvation.
• Analysis of the neural cytoskeletal structure following oxygen and oxygen-glucose deprivation.
• Investigating whether the astrocytes in cortical microtissues show signs of astrogliosis after oxygen and oxygen-glucose deprivation.
• Observing whether there are structural changes in the capillary-like networks (CLNs) after oxygen and oxygen-glucose deprivation.
• Oxygen and oxygen-glucose deprivation calcium dynamics experiments.

1. McLaughlin, R. M., et al. (2023). Cortical Spheroid Model for Studying the Effects of Ischemic Brain Injury. In vitro models, 2(1), 25-41.