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In this chapter, we describe a strategy to expand adult human articular chondrocytes in monolayer while maintaining high post-expansion chondrogenic capacity. This method relies on the use of a specific growth factor cocktail during the two-dimensional (2D) propagation of chondrocytes. In addition, a protocol to isolate and grow single-colony-derived strains of chondrocytes is presented as a model to study the biology of different chondrocyte subpopulations.