Isolation and culture techniques of foetal calf chondrocytes

Abstract

Large quantities of differentiated mammalian chondrocytes from normal hyaline cartilage were isolated after digestion of foetal bovine tracheas with collagenase. Incubation of the newly isolated cells for 1 day in the presence of dextran sulphate inhibited formation of cell aggregates during subsequent subculture in the absence of dextran sulphate. After incubation with dextran sulphate, the cells were plated in Ham's F12 medium with or without foetal calf serum on hydrophilic or hydrophobic Petri dishes. Chondrocytes cultured on hydrophilic substrates in the presence of serum attached to the substrate and showed cytoplasmic spreading. The cells did not attach to hydrophobic substrates in the presence of serum, but remained in suspension as single cells. In the absence of serum the chondrocytes attached to either substrate, but did not show any cytoplasmic spreading. By using labelling with [35S]sulphate and [3H]-thymidine it was shown that glycosaminoglycan synthesis did not require the presence of serum, whereas DNA synthesis required serum factors. Extracellular glycosaminoglycans were recovered in two pools: the medium pool and the pericellular pool, the latter being isolated by proteolytic digestion. The kinetics of these pools differed, depending on the presence or absence of serum and the type of substrate used. The turnover of the pericellular pool was studied in a pulse-chase experiment. At the end of the chase (72 h), only 60% of the material in the pericellular pool had been metabolized.