Early degenerative changes in the nucleus pulposus (NP) are observed after the disappearance of notochordal cells (NCs). Thus, it has been suggested that NCs play an important role in maintaining the NP and may have a regenerative potential on other cells of the NP. As the number of resident NP cells (NPCs) decreases in a degenerating disc, mesenchymal stromal (stem) cells (MSCs) may be used for cell supplementation. In this study, using cells of one species, the regenerative potential of canine NCs was assessed in long-term 3D co-culture with canine NPCs or MSCs. Canine NCs and canine NPCs or MSCs were co-cultured in alginate beads for 28 days under hypoxic and high osmolarity conditions. The cell viability, cell morphology and DNA content, the extracellular matrix production, and the expression of genes related to NC markers (Brachyury, KRT18) and NP matrix production (ACAN, COL2A1, COL1A1) were assessed after 1, 15, and 28 days of culture. NCs did not completely maintain their phenotype (morphology, matrix production, gene expression) during 28 days of culture. In co-cultures of NPCs and NCs, both extracellular matrix content and anabolic gene expression remained unchanged compared to monoculture groups, while co-cultures of MSCs and NCs showed increased glycosaminoglycan (GAG)/DNA. However, the deposition of these proteoglycans was observed near the NCs and not the MSCs. Brachyury expression in the MSC and NC co-culture group increased in time. The latter two findings indicate a trophic effect of MSCs on NCs rather than vice versa. No regenerative potential of canine NCs on canine NPCs or MSCs was observed in this study. However, significant changes in NC phenotype in long-term culture may have resulted in a suboptimal regenerative potential of these NCs. In this respect, NC-conditioned medium (NCCM) may be better than co-culture for future studies to the regenerative potential of NCs.