Abstract
Introduction and Objective
Intervertebral disc (IVD) degeneration accompanying with low back pain is a serious worldwide problem. Even though, surgical treatments are available for pain relief, there is an urgent need to establish enduring cell-based remedies. Notochordal (NC) cells as the ancestor of nucleus pulposus (NP) cells in human IVD are a promising therapeutic target. It has been reported that the loss of NC cells after childhood could promote the onset of disc degeneration. Thus, we firstly, aimed to optimise the culture of NC cells in vitro without using the FCS in alginate (3D) culture systems, secondly, investigate their behaviour in healthy and degenerate niche and lastly, co-culture these cells with degenerated NP cells to assess their regeneration potentials.
Materials and Methods
Porcine NC cells were extracted using pronase treatment followed by overnight digestion in 0.01% collagenase II. After extraction, cells were culture in 1.2% alginate beads (gold standard 3D culture) in either low glucose DMEM or αMEM medium. Cells were harvested after 24 hours, 1 week and 2 weeks for gene expression analysis and formalin fixed paraffin embedding. Quantitative Real-Time PCR and Immuno-staining were performed for analysis of NC markers (KRT18, FOXA2 and T) and COL I as a negative marker. Next, NC cells were cultured in healthy and degenerate medium to assess their viability and behaviour.
Results
A mixed phenotype of NC and NP cells was observed in alginate bead cultures. NC phenotype was observed within all culture conditions with production of GAGs and maintenance of vacuolated phenotype. Gene expression analysis showed no significant difference between the culture of NC cells in low glucose DMEM and αMEM medium. Interestingly, NC cell viability was maintained in both healthy and degenerate media, despite observing more dead cells in degenerate conditions. Current investigations are comparing the behaviour of NC cells in healthy and degenerate niche.
Conclusions
Investigating the preservation of NC phenotype in alginate culture and studying their behaviour between healthy and degenerate conditions would lead us to better understand their characteristics in different niches and how we can further use them in therapeutic purposes for disc degeneration.
