Chaotropic etching for fabricating microwells for cell culture

Formation of simple neuronal networks in vitro is one of the promising methods to study biological information processing. Agarose microchambers have several advantages to form and maintain simple network structures. Here, in this work, a novel method for fabricating microwells in an agarose-layer is reported. Chaotropic effects of sodium iodide (NaI) is applied for etching agarose films. A conventional glass micropipette filled with NaI solution was aligned and a small amount of NaI was ejected to surface of the film. The agarose was denatured by the soaked NaI. The denatured agarose was washed out by distilled water. The size of the well was determined by the quantity of ejected NaI solution and its diffusion time. Conditions for fabricating wells of 100 to 600 μm diameters were established. Multiple wells up to 100 were formed on a single surface sequentially by programmed movement of the microscope stage. Rat hippocampal neurons were successfully cultured in the wells. Combining this method with microelectrode-array substrates will enable us of recording neuronal activity from simple neuronal networks as well as co-culture systems of heterogeneous tissues.