Many human tissues are comprised of multilayered tissue structures in which spatial organization is essential to provide biological tissue functions.

Recently, strategies such as 3D bioprinting, photolithography, 3D auto-assembly, molding or bulk acoustic cells manipulation have been developed to fabricate layered tissue mimics. These methods have broad applications in tissue engineering for the bioengineering of multilayered structures, and for the fundamental understanding of many microphysiological and pathological process like cell differentiation. Each method relies on the use of a special scaffold structure made of natural or artificially created biopolymers, and of specific cell types.

In the field of neuronal 3D constructs fabrication, where ex-vivo samples are difficult to get, different strategies have been developed going from rat neurons culture to embryonic stem cells culture and differentiation into neurons after their encapsulation in 3D scaffolds.

All those possibilities open new perspectives for the future, aiming to the development of different types of tissues composed of different multilayer structures.