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This study reports a cell culture platform consisting of 48 microbioreactors for high-throughput perfusion three-dimensional (3-D) cell culture-based assays. Its main characteristics are the capability of conducting multiplex backflow-free medium delivery, and the function for both efficient and high-throughput micro-scale 3-D cell culture construct loading. In addition, a micro-scale perfusion 3-D cell culture-based assay was successfully demonstrated using the proposed cell culture chip. The experimental results were also compared with the same evaluation based on conventional static 3-D cell culture with a larger culture scale. It can be concluded that the choice of the cell culture format can bring different assay results. Therefore, because of the inherent natures of miniaturized perfusion 3-D cell culture, the cell culture chip not only can provide stable, well-defined and more biologically-relevant culture environments, but it also features in low consumption of research resource.