Despite recent progress in the preparation of feeder cells for human induced pluripotent stem cells (hiPSCs), there remain issues which limit the acquisition of feeder cells in large scale. Approaches for obtaining feeder cells quickly on a large scale are in immediate need. To reach this goal, we established suspension-adhesion method (SAM) and three-dimensional (3D) suspension method (3DSM). In SAM, mouse embryonic fibroblast (MEF) growth were fully inhibited by 10 μg/ml mitomycin-C (MMC) in 0.5 hours, and the feeder cells generated display higher adherent and recovery rates as well as longer survival time compared to conventional method (CM). 3DSM, an optimized method of SAM in which MEFs were cultured and MMC treated in suspension, was developed to lower the costs and workload using CELLSPIN System. The yield of feeder cells is several times the yield of SAM while the adherent and recovery rates and the capacity of supporting hiPSCs growth were not sacrificed. Hence, 3DSM is an economical and easy way to generate large-scale feeder cells for hiPSCs cultures.