Long-term Culture of Multicellular Spheroids Applicable to an Alternative to Animal Experiments.

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We report here for the first time a simple and effective way to form a two-dimensional micro-array of ten thousand (100x100) hepatocyte hetero-spheroids. The hepatocyte hetero-spheroids were underlain with endothelial cells in an area of 20x20.mm, by using micro-fabricated glass substrates, coated with poly (ethylene glycol) (PEG) brushes. Co-cultivation of hepatocytes with endothelial cells was essential to stabilize hepatocyte viability and liver specific function, allowing us to obtain hepatocyte spheroids with a diameter of 100-μm, functioning as a miniaturized liver to secret albumin for at least three weeks. The spheroid array constructed here is highly useful as a platform of tissue and cell-based biosensors (TBB and CBB), detecting a wide variety of clinically, pharmacologically, and toxicologically active compounds through a cellular physiological response. This system can be used as an alternative to animal experiments in a variety of situations in which the reliance on animals would prove impractical. Furthermore, the isolated spheroids from the array may have potential utility in constructing a tissue engineered liver by seeding spheroids into three-dimensional scaffolds.