Understanding the physiological behaviour of disc cells in an in vitro imitation of the healthy and degenerated disc niche

Abstract

INTRODUCTIONThe natural environment within the nucleus pulposus (NP) is hypoxic, acidic, low in nutrition and exerts a high osmotic-pressure. NP cells have adapted to this harsh environment, however, in vitro conditions often fail to recapitulate this environment. Hence, this study aimed to develop media to mimic the conditions of the native NP, with regards to pH, osmolality, glucose, combined with culture in physioxia (5% O2), and determine their effects upon human NP cells and tissue. METHODSAll media utilised low glucose DMEM/serum free conditions with supplements supporting matrix synthesis, based on media recommended for alginate culture (standard media). Healthy media modulated osmolarity (425mOsm/Kg), and pH7.2, degenerate media consisted of 325mOsm/kg and pH6.8. The latter was further supplemented with 100pg/ml IL-1{beta} (degenerate+IL-1{beta} media). NP cells in 3D alginate and NP tissue explants were cultured in these media for up to 2 weeks in physioxia (5% O2) to determine effects on viability, mitochondrial activity, protein expression and secretome. RESULTSMedia osmolarity and pH remained stable and cell viability was not altered by any media composition. Mitochondrial activity was increased during short term cultures, whilst a decrease was seen following 14 days in degenerate media. The secretome of NP cells was differentially affected in healthy or degenerate media, with most increases in catabolic cytokines observed following the addition of IL-1{beta}. Tissue explants showed stability of protein expression of matrix components in both healthy and degenerate+IL-1{beta} media, with limited effects seen on the secretome. DISCUSSIONThe media formulations developed here can provide more appropriate environmental conditions in vitro, mimicking more closely the in vivo conditions observed within healthy and degenerate IVDs. The application of which can provide more appropriate culture conditions to test potential therapeutic approaches and understand more fully the pathogenesis of disease using in vitro and ex vivo models.