Low calcium levels in serum-free media maintain chondrocyte phenotype in monolayer culture and reduce chondrocyte aggregation in suspension culture.

OBJECTIVE Extracellular calcium influences chondrocyte differentiation and synthesis of extracellular matrix. Previously, calcium concentrations ranging from 0.1 mM to 2 mM have been used in vitro and these studies indicated that low calcium concentrations were generally favorable for chondrocyte culture. Our objective was to extend these findings to yet lower calcium concentrations and to comprehensively examine effects on morphology and phenotype in two culture systems. METHODS Serum-free media containing 1 mM, 50 microM or 15 microM of calcium and a serum-containing medium were used to culture chondrocytes in suspension and in monolayer, at high and low inoculation density. RESULTS In monolayer, at low and high density, removing serum and decreasing calcium concentration decreased cell spreading and lowered collagen type I expression whereas collagen type II expression remained stable. In suspension, cells aggregated for all media tested; however, aggregates were smaller and looser in the absence of serum. CONCLUSION The serum-free 50 microM and 1 mM calcium media provide good alternatives to classical media for monolayer culture since both growth and chondrocyte phenotype were maintained. In suspension culture, the serum-free 1mM calcium medium also possesses the beneficial properties of limiting aggregate size while maintaining growth and phenotype.