After the embryonic period, notochord remnants persist inside the intervertebral disc (IVD), where they give rise to the nucleus pulposus. Notochordal cells (NTCs) gradually disappear during maturation. This phenomenon is correlated with onset of disc degeneration. The objective of this study was to design a protocol for the isolation of NTCs to study his role in IVD regeneration.

Lumbar IVDs from immature rats were either enzymatically dissociated or mechanically taken out or cells isolation. Cells RNA extraction for PCR analysis was performed to assay Sonic and Indian Hedgehog (Ihh and Shh) and his receptor Patched (Ptc) expression.

NTCs were readily detectable in culture as large vacuolated “physalipherous” cells, with the enzymatic method. The cells isolated mechanically were enable to grow in monolayer while grown 2 weeks in a 3-D pellet culture. Ihh and Ptc was expressed in the cells isolated with both method, while Shh was expressed only in the cells isolated through the mechanical method.

Our findings show that the better way to isolate a pure population of NTCs is a mechanical extraction from a immature IVD. This is a first step in order to study his role for the regeneration of IVD.