In recent years, scientists have used tissue engineering in attempts to create 3D neuronal cultures that emulate the high cell density and connectivity seen in vivo
by using two main approaches to biomaterials: polymer gels and solid porous matrices17–22
. Polymer gels molded to form tubes have been used successfully to provide 2D growth surfaces for peripheral neurons and to protect their processes from the inflammatory response of surrounding scar tissue18,19
. When cast as uniform blocks with cells embedded in them, they have provided supports for 3D cultures of neurospheres (neuronal stem cell aggregates)22
. Polymer gels have not been applied to isolated primary neurons. Moreover, to create layered 3D cultures from polymer gels would require gel microprinting, and it would be difficult to place cells of different types in desired 3D relations to one another. Although the use of solid porous matrices is attractive because of their rigid mechanical properties20,21
, these matrices have limited porosity, and thus they do not allow for deep cell migration within the matrix. This could be overcome by grinding or slicing the matrix into ‘unit’ modules, but the diversity of size and shape of the particles would be expected to interfere with packing into regular layered arrays.
In contrast to other methods, our approach makes it possible to grow dissociated neurons on moveable surfaces that allow for genetic and mechanical manipulation and assembly into ordered 3D networks. We worked with silica beads that are large enough to provide an adhesion surface for neuronal cell bodies, and for growth and differentiation of axons and dendrites. The neurons can be moved by displacing the beads without disrupting cell adhesion or damaging the delicate processes. They can be transfected before transfer either into culture dishes containing conventional 2D neuronal cultures, with which they form contacts, or in such a way that they spontaneously organize into regular ordered 2D layers. Layers can be added in succession to make 3D hexagonal arrays. Some of the layers can contain beads coated with chemical guidance cues that direct process growth to create synaptic layers. Finally, the ability to combine groups of cells that are differentially transfected makes it possible to express light-gated channels exclusively in one layer of neurons and use functional imaging to determine the development of functional connections between the light-activated layer and other neuronal layers. These properties of the colloid method make it possible to do what the earlier methods could not, namely, to build artificial layered networks out of genetically modified cells, under conditions that provide for cell densities that approach those found in the brain, and allowing synaptic connections to be formed and their spatial distribution and strength to be assessed.
Our method should pave the way for studying synapse formation and modification in the context of specific molecular interactions, chemical cues and activity patterns in designed 3D networks. Because the approach is amenable to long-term culture, it affords a unique system for cell-based assays of neurally targeted drugs and could prove useful for developmental studies of interactions between neurons in a controlled environment.