Hyperosmolar expansion medium improves nucleus pulposus cell phenotype

Background:Repopulating the degenerated intervertebral disc (IVD) with tissue-spe-cific nucleus pulposus cells (NPCs) has already been shown to promote regenerationin various species. Yet the applicability of NPCs as cell-based therapy has been ham-pered by the low cell numbers that can be extracted from donor IVDs and theirpotentially limited regenerative capacity due to their degenerated phenotype. Tooptimize the expansion conditions, we investigated the effects of increasing culturemedium osmolarity during expansion on the phenotype of dog NPCs and their abilityto produce a healthy extracellular matrix (ECM) in a 3D culture model.Methods:Dog NPCs were expanded in expansion medium with a standard osmolar-ity of 300 mOsm/L or adjusted to 400 or 500 mOsm/L in both normoxic and hypoxicconditions. Following expansion, NPCs were cultured in a 3D culture model in chon-drogenic culture medium with a standard osmolarity. Read-out parameters includedcell proliferaton rate, morphology, phenotype and healthy ECM production.Results:Increasing the expansion medium osmolarity from 300 to 500 mOsm/Lresulted in NPCs with a more rounded morphology and a lower cell proliferation rateaccompanied by the expression of several healthy NPC and progenitor markers atgene (KRT18, ACAN, COL2, CD73, CD90) and protein (ACAN, PAX1, CD24, TEK,CD73) level. The NPCs expanded at 500 mOsm/L were able to retain most of theirphenotypic markers and produce healthy ECM during 3D culture independent of theoxygen level used during expansion. Conclusions:Altogether, our findings show that increasing medium osmolarity duringexpansion results in an NPC population with improved phenotype, which couldenhance the potential of cell-based therapies for IVD regeneration.