Growth plate lesions or resections may cause severe growth arrest because of the bony bridge between the epiphysis and metaphysis. Actual treatments for epiphysiodesis include resecting the bone bar and setting an interpositional material. Growth plate cultures may provide the appropriate cartilage necessary to restore growth potential when implanted in a growth plate defect. The aim of this work was to determine certain cell culture parameters in order to optimize in vitro cultures to obtain abundantly mature and functional chondrocytes. We studied the manner in which enzymatic digestion, carried out by various enzymes, obtained chondrocytes. Treatment with trypsin (0.2%) during 30 minutes at 37°C and then collagenase (200 U/mL) during 6 hours was chosen. Under these conditions, 40 ± 16 106 chondrocytes per gram of growth plate were obtained, and cellular viability was 79 ± 12%. The density of the cellular seeding, the nature of the culture substrate, and the culture medium composition were determined to optimize the growth of differentiated cells. Seeding at 20,000 or 30,000/cm2 on a type I substrate and Ham F-12 medium not supplemented with either glucose or growth factors was demonstrated to be the best choice for this purpose.