The nucleus pulposus (NP) is composed of NP and notochord cell. It is a paucicellular tissue and if it is to be used as a source of cells for tissue engineering the cell number will have to be expanded by cell passaging. The hypothesis of this study is that passaged NP and notochordal cells grown in three-dimensional (3D) culture in the presence of transforming growth factor β (TGFβ) will show enhanced NP tissue formation compared with cells grown in the absence of this growth factor. Bovine NP cells isolated by sequential enzymatic digestion from caudal intervertebral discs were either placed directly in 3D culture (P0) or serially passaged up to passage 3 (P3) prior to placement in 3D culture. Serial cell passage in monolayer culture led to de-differentiation, increased senescence and oxidative stress and decreases in the gene expression of NP and notochordal associated markers and increases in de-differentiation markers. The NP tissue regeneration capacity of cells in 3D culture decreases with passaging as indicated by diminished tissue thickness and total collagen content when compared with tissues formed by P0 cells. Immunohistochemical studies showed that type II collagen accumulation appeared to decrease. TGFβ1 or TGFβ3 treatment enhanced the ability of cells at each passage to form tissue, in part by decreasing cell death. However, neither TGFβ1 nor TGFβ3 were able to restore the notochordal phenotype. Although TGFβ1/3 recovered NP tissue formation by passaged cells, to generate NP in vitro that resembles the native tissue will require identification of conditions facilitating retention of notochordal cell differentiation. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:438-449, 2020., (© 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)