The articular cartilage is dissected from the condyles of the distal femur of a single 7-day-old male miniature pig for chondrocyte isolation and further expansion.
The cartilage pieces are washed 3 times and subsequently digested using a purified mixture of collagenase/neutral protease (Liberase Blendzyme) in HL-1 Complete Serum-free Medium (1.6WU/mL). This mixture is incubated at 37°C for no more than 9h, and dissociated chondrocytes are filtered through a 70μm nylon strainer.
To expand the primary population, a cell suspension (3.33 ×10^4 cells/cm2) is inoculated into T-150 culture flasks containing expansion medium formulated to retain chondrocyte hyaline phenotype after expansion. The expansion medium is comprised of HL-1 Complete Serum-Free Medium, containing gentamicin, L-glutamine, FGF-2, TGF beta-1, hyaluronic acid, and L-ascorbate. Culture medium is changed every 3–4 days and the cells are harvested at day 21 using a solution of Liberase Blenzyme. Chondrocytes were washed and counted before resuspension to a density of 50 ×106 cells/mL in CryoStor CS5 cryopreservation solution, containing 5% DMSO. Next, 0.5mL of the cell suspension is aliquoted into cryovials, frozen at a rate of −1°C/min with the use of a controlled rate freezer, and stored in liquid nitrogen for future use.
EMBRYONIC DEVELOPMENT & STEM CELL COMPENDIUM
