In vitro 3D regeneration-like growth of human patient brain tissue.

In vitro culture of primary neurons is widely adapted with embryonic but not mature brain tissue. Here, we extended a previously developed bioengineered three-dimensional (3D) embryonic brain tissue model to resected normal patient brain tissue in an attempt to regenerate human neurons in vitro. Single cells and small sized (diameter < 100 µm) spheroids from dissociated brain tissue were seeded into 3D silk fibroin-based scaffolds, with or without collagen or Matrigel, and compared with two-dimensional cultures and scaffold-free suspension cultures. Changes of cell phenotypes (neuronal, astroglial, neural progenitor, and neuroepithelial) were quantified with flow cytometry and analyzed with a new method of statistical analysis specifically designed for percentage comparison. Compared with a complete lack of viable cells in conventional neuronal cell culture condition, supplements of vascular endothelial growth factor-containing pro-endothelial cell condition led to regenerative growth of neurons and astroglial cells from "normal" human brain tissue of epilepsy surgical patients. This process involved delayed expansion of Nestin+ neural progenitor cells, emergence of TUJ1+ immature neurons, and Vimentin+ neuroepithelium-like cell sheet formation in prolonged cultures (14 weeks). Micro-tissue spheroids, but not single cells, supported the brain tissue growth, suggesting importance of preserving native cell-cell interactions. The presence of 3D scaffold, but not hydrogel, allowed for Vimentin+ cell expansion, indicating a different growth mechanism than pluripotent cell-based brain organoid formation. The slow and delayed process implied an origin of quiescent neural precursors in the neocortex tissue. Further optimization of the 3D tissue model with primary human brain cells could provide personalized brain disease models.

A biofidelic 3D culture model to study the development of brain cellular systems.

Little is known about how cells assemble as systems during corticogenesis to generate collective functions. We built a neurobiology platform that consists of fetal rat cerebral cortical cells grown within 3D silk scaffolds (SF). Ivermectin (Ivm), a glycine receptor (GLR) agonist, was used to modulate cell resting membrane potential (Vmem) according to methods described in a previous work that implicated Ivm in the arrangement and connectivity of cortical cell assemblies. The cells developed into distinct populations of neuroglial stem/progenitor cells, mature neurons or epithelial-mesenchymal cells. Importantly, the synchronized electrical activity in the newly developed cortical assemblies could be recorded as local field potential (LFP) measurements. This study therefore describes the first example of the development of a biologically relevant cortical plate assembly outside of the body. This model provides i) a preclinical basis for engineering cerebral cortex tissue autografts and ii) a biofidelic 3D culture model for investigating biologically relevant processes during the functional development of cerebral cortical cellular systems.