Modulating Microenvironment for Improved In vitro Brain Models: a Study on Cell Fate Determination and Function

With the rising need for reducing animal use in research, in vitro models such as 3D cultures, spheroids and organoids have become an indisputable alternative. Brain organoids have achieved a high level of similarity to the native brain areas in terms of molecular machinery, however, there remains a huge difference in tissue patterning and functionality when it comes to recapitulating complex brain structures like the hippocampus. Taking this as an issue to address, we manipulated the non-cellular components of the microenvironment i.e., the extracellular matrix and signaling factors, for guiding neural stem cell differentiation and hippocampal formation. To this purpose, we first evaluated the neuroprotective and neurotrophic action of a novel viscoelastic biomaterial, i.e., magnesium alginate. This demonstrated to form 3D, soft yet stable, hydrogels promoting functional maturation of neuronal networks from primary neurons. Next, we used magnesium alginate as biomimetic extracellular matrix, along with hippocampal morphogens, to study the differentiation, tissue patterning, cytoarchitecture, and functionality of neural stem cell-derived hippocampal tissuoids. Microenvironmental cues were fundamental to address polarization and protein regionalization in the tissuoids, however, these did not achieve full functional maturation. Lastly, we observed the functional development of hippocampal primary neuron-derived spheroids which resulted to be reliable in vitro models of spontaneous epilepsy. Overall, the presented models provided valuable solutions to a wide range of applications for brain regenerative medicine, from studying the molecular basis of tissue development and disease to enabling new therapeutic design and testing.