INTRODUCTION: Relatively little is known about cellular subpopulations in the mature nucleus pulposus. Detailed understanding of ontogenetic, cellular and molecular characteristics of the functional IVD cell populations is pivotal for successful development of cell replacement therapies and IVD regeneration. With this study we aimed to investigate whether phenotypically distinct clonal cell lines representing different subpopulations in the human nucleus pulposus could be generated using immortalization strategies. METHODS: Non-degenerate healthy disc material (age range 8 to 15 years) was obtained as surplus surgical material. Early passage nucleus pulposus (NP) monolayer cell cultures were initially characterized using a recently established NP marker set. NP cells were immortalized by simian virus Large T antigen (SV40LTAg) and human telomerase (hTERT) expression. Immortalized cells were clonally expanded and characterized based on Collagen types I, II and SRY-box 9 (SOX9) protein expression profiles, in addition to expression of a subset of established in vivo NP cell-lineage markers. RESULTS: A total of 54 immortal clones were generated. Profiling of a set of novel NP markers (CD24, CA12, PAX1, PTN, FoxF1 and KRT19 mRNA), in a representative set of subclones, substantiated successful immortalization of multiple cellular subpopulations from primary isolates and confirmed their NP origin/phenotype. We were able to identify two predominant clonal NP subtypes based on morphological characteristics and their ability to induce SOX9 and COL2A1 under conventional differentiation conditions. CD24-negative NP responder clones, in addition, formed spheroid structures in various culture systems, suggesting preservation of a more immature phenotype compared to CD24- positive non-responder clones. CONCLUSIONS: We here report the generation of clonal NP cell lines from non-degenerate human intervertebral disc tissue and present a detailed characterization of NP cellular subpopulations. Differential cell surface marker expression and divergent responses to differentiation conditions suggest the NP subtypes may correspond to distinct maturation stages and represent distinct NP cell subpopulations. Hence, we provide evidence that the herein applied immortalization strategy is capable of detecting cell heterogeneity in the NP. Our cell lines yield novel insights into nucleus pulposus biology and provide promising new tools for studies of IVD development, cell function and disease.