AbstractAutologous chondrocytes (C cells) are effective sources of cell therapy for engineering cartilage tissue to repair chondral defects, such as degenerative arthritis. The expansion of cells with C cell characteristics has become a major challenge due to inadequate donor sites and poor proliferation of mature C cells. The perichondrial progenitor cells (P cells) from the cambium layer of the perichondrium possessed significantly higher mesenchymal stem cell markers than C cells. In the transwell co-culture system, P cells increased the passaging capacity of C cells from P6 to P9, and the cell number increased 128 times. This system increased the percentage of Alcian blue-positive C cells from 40% in P6 to 62% in P9, contributing about 198 times more Alcian blue-positive C cells than the control group. C cells co-cultured with P cells also exhibited higher proliferation than C cells cultured with P cell-conditioned medium. Similar results were obtained in nude mice that were subcutaneously implanted with C cells, P cells or a mixture of the two cell types, in which the presence of both cells enhanced neocartilage formation in vivo. In aggregate, P cells enhanced the proliferation of C cells in a dose–dependent manner and prolonged the longevity of mature C cells for clinical applications.