Physioxic Culture of Chondrogenic Cells

Cartilage resides under a low oxygen tension within articulating joints. The oxygen tension within cartilage of the knee joint has been measured to be between 2% and 5% oxygen. Although the literature has historically termed this level of oxygen as hypoxia, particularly when doing experiments in vitro in this range, this is actually the physiological oxygen tension experienced in vivo and is more accurately termed physioxia. In general, culture of chondrogenic cells under physioxia has demonstrated a donor-dependent beneficial effect on chondrogenesis, with an upregulation in cartilage genes (SOX9, COL2A1, ACAN) and matrix deposition (sulfated glycosaminoglycans (sGAGs), collagen II). Physioxia also reduces the expression of hypertrophic markers (COL10A1, MMP13). This chapter will outline the methods for the expansion and differentiation of chondrogenic cells under physioxia using oxygen-controlled incubators and glove box environments, with the typical assays used for qualitative and quantitative assessment of chondrogenesis.