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Steady and useful culture for chondrocytes is essential for cartilage regenerative medicine. However, in conventional plate culture, the chondrocytes become dedifferentiated and lose their ability to make cartilage matrices. Three-dimensional culture mimicking the physiological environment in native chondrocytes is useful to maintain the chondrocyte properties during the proliferation culture. However, the three-dimensional culture is practically a hard task due to difficult harvest of the cells. Thus, we attempted to apply porous materials, hollow fibers for the three-dimensional culture, and developed their module to realize the effective harvest of the cells. Polyethersulfone-based hollow fibers, whose safety and cell affinity were confirmed by the experiment of the coculture with human chondrocytes, were collected to fabricate a module. The hollow fiber module was installed with screw ends, and enabled the easy removal of chondrocytes from the inner unit. Cultured human chondrocytes embedded within collagen hydrogel were put into the outer lumen of the hollow fiber module, while chondrocyte prolfieration medium was perfused through the inner lumen at 0 to 30 mL/min. After 2 weeks’ culture, the flow rate of 3 to 10 mL/min effectively supported the chondrocyte proliferation. Then, long-term culture using the hollow fiber module at flow rate of 5 mL/min was performed, revealing that the cell growth in this module at 3 weeks was approximately twice larger than that in static culture. The numbers of viable cells could be maintained by week 7. The hollow fiber module installed with screw ends can effectively culture and harvest the chondrocytes.