Brain in vitro models, devices, systems, and methods of use thereof

An engineered functionally connected trineural pathway, comprising:

• a base layer including a first region, a second region, and a third region, wherein the first region, the second region, and the third region are spatially separated from each other;
• a first independent plurality of cells comprising neurons and astrocytes obtained from a first brain region, the first independent plurality of cells adhered to the first region of the base layer;
• a second independent plurality of cells comprising neurons and astrocytes obtained from a second brain region, the second independent plurality of cells adhered to the second region of the base layer;
• a third independent plurality of cells comprising neurons and astrocytes obtained from a third brain region, the third independent plurality of cells adhered to the third region of the base layer;
• a first independent plurality of axons adhered to the base layer and functionally connecting the first plurality of cells to the second plurality of cells;
• a second independent plurality of axons adhered to the base layer and functionally connecting the first plurality of cells to the third plurality of cells;
• and
• a third independent plurality of axons adhered to the base layer and functionally connecting the second plurality of cells to the third plurality of cells.

A method of forming an engineered functionally connected trineural pathway, comprising:

• providing a base layer including a first region, a second region, and a third region, wherein the first region, the second region, and the third region are spatially separated from each other;
• depositing a pattern of extracellular matrix protein onto the base layer, wherein the pattern comprises a first plurality of lines connecting the first region with the second region, a second plurality of lines connecting the first region with the third region, and a third plurality of lines connecting the second region with the third region,
• placing a patterned mask onto the base layer, wherein the patterned mask includes a first opening, a second opening, and a third opening corresponding to the first, second and third regions, respectively, seeding neural cells and astrocytes obtained from a first area of the brain onto the first region through the first opening;
• seeding neural cells and astrocytes obtained from a second area of the brain onto the second region through the second opening;
• and seeding neural cells and astrocytes obtained from a third area of the brain onto the third region through the third opening;
• culturing the cells for a time sufficient for the cells in each region to adhere to the base layer;
• removing the patterned mask and further culturing the cells for a time sufficient for the cells from the first and second areas of the brain to functionally connect, for the cells from the first and third areas of the brain to functionally connect, and for the cells from the second and third areas of the brain to functionally connect, thereby generating the engineered functionally connected trineural pathway.