Cell viability in human samples of herniated intervertebral disc: implications for biological therapies

Purpose: The intervertebral disc is dependent on the extracellular matrix for its function, which is produced and maintained by the resident cells. However, disc degeneration is common and may present early on in life, being associated with major clinical problems such as herniation and back pain. Previous work suggests that cells isolated from the degenerate intervertebral disc have a very limited potential for regeneration, with a number of the cells necrotic and/or senescent. Never-the-less cells are currently being used in the clinic for cell therapy which have been isolated from routine discectomy patients. Since regulatory authorities are now placing more emphasis on cell therapies being classed as pharmaceutical products, a deeper knowledge of the source and quality of cells used is required. Thus, this study was designed to assess the viability of cells present in surgical samples of degenerate intervertebral disc tissue.Methods: The technique for live/dead differentiation of cells used in this study utilises the fluorescent probes chloromethyl fluoresce in diacetate (CMFDA) and ethidium homodimer (EH). Samples of herniated discs were obtained from patients undergoing routine discectomy and processed within 2 hours of excision. A small piece of each sample, approximately5mm2, was incubated in 1 ml of 25 mM CMFDA/2mM EH solution in the dark for 2 hours at 37°C. The tissue was then blotted dry and snap frozen. Cryo sections were taken at various points throughout the thickness of the tissue sample, mounted with Vectashield containing DAPI and visualised with a fluorescent microscope. Cell viability was analysed by counting at least 200 cells from a minimum of five fields of view.Results: To date 21 surgical disc samples from patients, aged 17–57 years,have been examined. Preliminary results show that greater than 80%of cells are alive after excision. Cells in these discs commonly occur in clusters (approximately 54% of disc cells are clustered). Clustered cells exhibited a higher proportion of live cells compared to single cells with in the same tissue section, e.g. 93% of clustered cells were viable compared to 82% of single cells. Cell viability appeared consistent across all locations of the disc samples. Dead cells, when they did occur, were predominantly seen around the periphery of the disc sections which may be derived from trauma during dissection.Conclusions: In contrast to reports in the literature, these results indicate that the viability of cells from the herniated disc may be adequate to consider for utilising in biological therapies. Interestingly, a greater proportion of clustered cells appear to be viable compared to single cells.Additionally we plan to investigate whether there is any relationship between cell viability and the pathology, duration of symptoms or age of patient, as this may serve as an indicator of the suitability of degenerate discs for cell therapy.