Orderly arrangement of hepatocyte spheroids on a microfabricated chip

This article describes a novel method for preparing several spherical multicellular aggregates (spheroids) that have almost the same diameter on a microfabricated chip. The chip, fabricated by a simple method that uses a micromilling system, consisted of several thousand cavities, 100-500 microm in diameter, in a triangular arrangement on a polystyrene plate. Although no spheroid was formed on any chip when cultured under stationary conditions, hepatocytes formed spheroids in the cavities when turning force was applied with a rotary shaker. Especially on the chip with cavities 300 microm in diameter, one spheroid formed in each cavity; an orderly array of spheroids (1100 spheroids/cm(2)) of almost the same diameter was constructed. Ammonia removal and albumin secretion by the spheroids on the chip continued to occur at initial levels for at least 14 days of culture. Enzyme P-450 activity of the spheroids was also maintained and detected on this transparent chip by fluorescence of resorufin converted from ethoxyresorufin. The spheroid microarray chip seems to be a promising cellular platform for various biomedical applications such as in cell-based biosensors for toxicological and pharmacological examinations, and in bioartificial livers.