Two-dimensional cell manipulation technology. An artificial neural circuit based on surface microphotoprocessing.

Abstract

The precise regional control of cell adhesion and growth on a substrate may create two-dimensional tissue formation, as in a neural network. To prepare a neural circuit, micropositioning of neural cells and guidance of extending axons in a given region are required. In general, the adhesion of neural cells and their axonal extension are mediated by adhesive proteins found in the extracellular matrix. This paper describes a novel surface photoprocessing that enables the creation and guidance of regionally selective cell adhesion, leading to a neural network. The non-adherent region was created by chemical fixation of a photoreactive hydrophilic co-polymer of azidostyrene and N, N-dimethylacrylamide on a hydrophobic substrate. Ultraviolet irradiation with the use of a photomask placed on a substrate hydrophilically modified the irradiated regions, which was evident in ESCA and contact angle measurements. The addition of a collagen buffer solution resulted in collagen adsorption only on the non-irradiated hydrophobic portions. Seeded neuroblastoma cells adhered only on collagen-adsorbed pathways 130 microns in width. One day after seeding, nerve growth factor was added to the culture medium, resulting in cell differentiation from growth to axonal extension. The axons grew along the collagen-adsorbed pathways. Sooner or later, cells were interconnected with extended axons, which was clearly visible microscopically. Further culturing completed the honeycomb-like patterning, as designed. The surface processing developed here can manipulate fundamental cellular behavior, leading to two-dimensional patterned tissue, which may provide information on the morphogenesis of the neural network and neurotransmission.