Multi-array formation of hepatocyte hetero-spheroids on micro-fabricated PEG-brush surface

New high-throughput techniques, based on tissue and cell microarrays, will facilitate clinical and pharmaceutical analysis of molecular targets, because living cells can monitor the targets through their physiological changes that are induced by exposure to drugs and environmental perturbations, such as toxicants, pathogens or other agents[1, 2]. In this regard, primary hepatocytes are the most useful candidate to construct tissue and cell-based biosensors (TBB and CBB). Here, a two-dimensional microarray of ten thousand (100×100) hepatocyte hetero-spheroids, underlaid with endothelial cells, was successfully constructed with a 100 μm spacing in an active area of 20×20 mm on micro-fabricated glass substrates coated with poly(ethylene glycol) (PEG) brushes (Fig. 1A). Co-cultivation of hepatocytes with endothelial cells was essential to stabilize hepatocyte viability and liver-specific functions, 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 TBB and CBB to detect a wide variety of clinically, pharmacologically, and toxicologically active compounds through a cellular physiological response.