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 univ-nantes.fr |
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) |
