In vitro evaluation of nucleus pulposus (NP) tissue regeneration would be useful, but current systems for NP culture are not ideal for injections. The aim of this study was to develop a long-term culture system for NP tissue that allows injections of regenerative agents. Bovine caudal NPs were harvested and placed in the newly designed culture system. After equilibration of the tissue to 0.3 MPa the volume was fixed and the tissue was cultured for 28 days. The cell viability and extracellular matrix composition remained unchanged during the culture period and gene expression profiles were similar to those obtained in earlier studies. Furthermore, to test the responsiveness of bovine caudal NPs in the system, samples were cultured for 4 days and injected twice (day 1 and 3) with (1) PBS, (2) Link-N, for regeneration, and (3) TNF-a, for degeneration. It was shown that TNF-a increased COX2 gene expression, whereas no effect of Link-N was detected. In conclusion, the newly designed system allows long-term culture of NP tissue, wherein tissue reactions to injected stimulants can be observed.