eCM (Eur Cell Mater / e Cells & Materials) eCM Open Access Scientific Journal
 ISSN:1473-2262         NLM:100973416 (link)         DOI:10.22203/eCM

2023   Volume No 45 – pages 72-87

Title: Three-dimensional culture model to study the biology of vacuolated notochordal cells from mouse nucleus pulposus explants

Authors:  L Paillat, K Coutant, M Dutilleul, S Le Lay, A Camus

Address: Nantes Université, Oniris, CHU Nantes, INSERM, Regenerative Medicine and Skeleton, RMeS, UMR 1229, F-4400 Nantes, France

E-mail: anne.camus at

Abstract: Intervertebral disc degeneration (IDD) involves cellular changes in the nucleus pulposus (NP) characterised by a decline of the large vacuolated notochordal cells (vNCs) and a rise of smaller vacuole-free mature chondrocyte-like NP cells. An increasing number of studies demonstrate that notochordal cells (NCs) exert disease-modifying effects, establishing that NC-secreted factors are essential for the maintenance of a healthy intervertebral disc (IVD). However, understanding the role of the NCs is hampered by a restricted reserve of native cells and the lack of robust ex vivo cell model. A precise dissection enabled the isolation of NP cells from 4 d post-natal stage mouse spines and their culture into self-organised micromasses. The maintenance of cells’ phenotypic characteristics was demonstrated by the presence of intracytoplasmic vacuoles and the immuno-colocalisation of the NC-markers (brachyury; SOX9) after 9 d of culture both in hypoxic and normoxic conditions. A significant increase of the size of the micromass was observed under hypoxia, consistent with a higher level of Ki-67+ immunostained proliferative cells. Furthermore, several proteins of interest for the study of vNCs phenotype (CD44; caveolin-1; aquaporin 2; patched-1) were successfully detected at the plasma membrane of NP-cells cultured in micromasses under hypoxic condition. IHC was performed on mouse IVD sections as control staining. An innovative 3D culture model of vNCs derived from mouse postnatal NP is proposed, allowing future ex vivo exploration of their basic biology and of the signalling pathways involved in IVD homeostasis that may be relevant for disc repair.

Keywords: Intervertebral disc, nucleus pulposus cells, vacuolated notochordal cell, ex vivo mouse tissue, three-dimensional culture system, hypoxia, tissue engineering.

Publication date: March 3rd 2023

Article download: Pages 72-87 (PDF file)

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