Functional proteins like antibody, cytokine and growth factor have been widely used for basic biological research, diagnosis and cancer therapy. Particularly, antibody drugs as attractive biopharmaceuticals will be expected to create an enormous new market. Chinese hamster ovay (CHO) cells are being increasingly used in industry for the production of recombinant therapeutic proteins including antibody drugs. Although three-dimensional culture is preferred to two-dimensional monolayer culture for the efficient large scale culture of CHO cells and subsequent mass production of recombinant proteins, it has the limitation of low protein production. Therefore, a new cell culture em essentially required for an efficient protein production. Here we report on a new three-dimensional cell culture system as a spheroid cell culture on the micropattern array for efficient production of protein in CHO cells. Furthermore, cocultivation of CHO spheroids with feeder cells including bovine aortic endothelial cells (BAEC) and NIH 3T3 was essential to more increase a protein production. The results indicated that CHO heterospheroids cocultured with BAECs were much superior to either CHO monolayers or CHO homospheroids in protein production. Significantly, the above cocultured spheroids in the serum-free medium drastically enhanced protein expression level up to 3-fold compared with CHO spheroids in serum medium, suggesting that a coculture of spheroid system with feeder layer cells is a promising method to enhance protein production under serum-free condition. The spheroid array constructed here is highly usuful as a platform of biopharmaceutical manufacturing as well as tissue and cell based biosensors to detect a wide variety of clinically active compounds through a cellular physiological response.