Reconstitution of a functional neural network using a nanoscale thin-film-based 3D template - new biological interface for regenerative medicine technology -

Background of the research

Recent advances in medical technology and information processing technology have brought prospects for biological interface (bio-interface) that allow direct access to and monitoring of living biological tissue. Since the bio-interfaces should be durable, soft, and, compatible to the tissues, the search is on all over the world for new materials that can meet these requirements.
Graphene, which is essentially a single layer of carbon atoms, is one of the most promising two-dimensional (2D) functional materials. This extremely thin sheet-like material has high transparency, strength, chemical resistance, heat resistance, flexibility, and biocompatibility. Thanks to these characteristics, it is being examined as a potential alternative to using silicon and rare metals in transistors, batteries, and sensors. However, because graphene is an extremely thin 2D sheet, it has proven difficult to assemble and refine it into a three-dimensional (3D) structure with sufficient definition to be practically used in the above devices.
A method for processing and bending graphene freely and easily has not been established, and there are still technical problems that cause fracture and exfoliation. If graphene can be formed into 3D structures, it could be used as an interface that fits the surface of biological samples. As such, it could be used to make in vivo implantable electrodes and microscale cell-culture substrates. Therefore, graphene is potentially useful in regenerative medicine and for making medical/therapeutic devices.

Research results

We developed an easy method to assemble graphene into a fine three-dimensional structure, by transferring it from a glass substrate to a parylene polymer thin film. The transferred graphene adheres strongly to the parylene film and spontaneously deforms it into a 3D structure when it is released from the substrate due to the difference in the elastic moduli of the materials (this phenomenon is called self-folding). This method enables us to finely bend graphene with a curvature radius of 4µm at a minimum by changing the thickness of the polymer thin film (Figure 1, Video 1). In addition, all of the materials used in this technology are cell-friendly and highly flexible. We used self-folding 3D graphene formed into a cylinder as an interface with biological samples (cells). This self-folding graphene scaffold encapsulated the cells (neurons in this case), and the cells were stably cultured inside its hollow center it for a long time. In particular, we succeeded in artificially growing a minute and tissue-like nerve structure along the scaffold. We formed a network between it and other neurons outside the scaffold and demonstrated cell-cell interactions both inside and outside the structure.

Technical point: Self-assembly of 3D graphene structure that is compatible with cells

1.Three-dimensional structure of graphene using polymer thin film

The final self-folded three-dimensional shape depends on the original two-dimensional micropattern and the thicknesses of the thin film. For example, a finer structure can be produced by thinning the thin film; thin films with rectangular, parallelogram, and radial micropatterns can be spontaneously assembled into cylinders, coils, and spheres, respectively (Figure 2). In addition, it is possible to increase the permeability of the structure to molecules from the outside by making tiny holes in a rectangular two-dimensional pattern. These tiny holes allow nutrients and oxygen to be supplied to the encapsulated cells for a long period and thereby provide a means of stable culture.

2.Application of 3D graphene structure to interfaces with cells

Since the materials constituting the 3D graphene structures are not cytotoxic, it is possible to encapsulate cells inside them (Figure 3, Video 2). In addition, their high transparency enables us to observe and analyze the cells using various microscopes. The encapsulated cells can be from cell lines or primary cells that are isolated from other living tissues. In addition, by controlling the density of the encapsulated cells and the shape of the three-dimensional structure, we can produce cell aggregates with any shape after culturing for about a week. For example, when the hippocampal neurons were cultured in the cylindrical structure, the cells aggregated and formed fiber-like artificial nerve tissue. The excellent robustness and stiffness of the graphene and parylene led to a stable and long-term cell culture without any disruption of the cells.

3.Long-term culture of neurons and creation of neural network

Nutrients and oxygen in a culture solution can easily flow through a 3D graphene structure with many pores. This enables a more stable and longer culture compared with the one in which the graphene did not have pores (Figure 4, Video 3). Furthermore, we observed that the neurons inside the structure never passed through the pores, but extended their neurites through them (Figure 4). We could thus connect the neurons inside the structure to ones outside it via these extended neurites. The resulting neuronal network exhibited synchronization of spontaneous neural activities. This result indicates that by extending axons through the pores, functional synaptic connections can be formed and neurons in the scaffold can be functionally integrated into surrounding neuronal networks (Video 4).

Future prospects

We developed a technology to spontaneously form graphene into any desired shape and produce transparent biocompatible 3D structures. We experimentally encapsulated and stably cultured cells inside a self-folded 3D structure. Our method can be used to make scaffold structures for regenerative medicine and examine the behaviors of single cells; it can also be used to make new bio-interfaces, for example, in vivo implantable electrodes that utilize the high conductivity of graphene.

Publication details

Description of the experiments

Two types of materials were used to make the bi-layer thin film: monolayer graphene sheets and polymeric parylene with thicknesses ranging from 50 nm to 500 nm. In addition, a calcium alginate hydrogel layer was placed underneath the graphene/parylene bilayer ⁴ as a sacrificial layer. We could control the film thickness of both the graphene and parylene and process them into any two-dimensional pattern by using lithography technology.
Calcium alginate gel can be dissolved instantly by adding chelating agents such as ethylenediaminetetraacetic acid (EDTA ⁵) or sodium citrate solution. This non-cytotoxic dissolution of a sacrificial layer allows the release of the bilayer membrane from the substrate. In the experiment, the calcium alginate sacrificial layer gradually dissolved from the outer periphery of the thin film, which led to the thin films deforming into a 3D shape. In particular, rectangular films were deformed into a cylindrical structure such that the graphene was on the outside surface. This transformation of graphene-laden parylene films encapsulated the cells without damaging them.

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