This study reports a new perfusion-based, micro three-dimensional (3-D) cell culture platform for high-throughput cell culture using enabling microfluidic technologies. In this work, the micro 3-D cell culture platform is fabricated based on SU-8 lithography and polydimethylsiloxane replication processes. The micro cell culture platform can maintain homogenous and stable culture environments, as well as provide pumping of multiple mediums and efficient cell/agarose (scaffold) loading functions, which allows realization of more precise and high-throughput cell culture-based assays. In this study, the design of a high-throughput medium pumping mechanism was especially highlighted. A new serpentine-shaped pneumatic micropump was used to provide the required medium pumping mechanism. Pneumatic microchannels with a varied length and U-shape bending corners were designed to connect three rectangular pneumatic chambers such that one can fine-tune the pumping rate of the S-shape micropump by using the fluidic resistance. To achieve a high-throughput medium pumping function, a pneumatic tank was designed to simultaneously activate all of the 30 pneumatic micropumps with a uniform pumping rate. Results show that the pumping rates of the 30 integrated micropumps were statistically uniform with a flow rate ranging from 8.5 to 185.1 microl h(-1), indicating the present multiple medium pumping mechanism is feasible for high-throughput medium delivery purposes. Furthermore, as a demonstration case study, 3-D culture of oral cancer cell was successfully performed, showing that the cell viability remained as high as 95% - 98% during the 48 h cell culture. As the result of miniaturization, this perfusion-based 3-D cell culture platform not only provides a well-defined and stable culture condition, but also greatly reduces the sample/reagent consumption and the need for human intervention. Moreover, due to the integrated capability for multiple medium pumping, high-throughput research work can be achieved. These traits are found particularly useful for high-precision and high-throughput, 3-D cell culture-based assay.