Phenotypic stability of bovine articular chondrocytes after long-term culture in alginate beads

Articular chondrocytes embedded in alginate gel produce de novo a matrix rich in collagens and proteoglycans. A major advantage of this culture system is that the cells can be recovered by chelating the calcium, which otherwise maintains the alginate in its gel state. Chondrocytes thus released are surrounded by tightly bound cell-associated matrix, which seems to correspond to the pericellular and territorial matrices identified in cartilage by electron microscopy. The cells and their associated matrix can be easily separated by mild centrifugation from more soluble matrix components derived principally from the 'interterritorial' matrix. This new cell culture system thus makes it possible to study the assembly and turnover of molecules present in two distinct matrix pools. Importantly, a significant proportion of the aggrecan molecules in each of these two pools can be extracted using a non-denaturing solvent, thereby making possible studies of the metabolism and turnover of native proteoglycan aggregates. We show in this report that chondrocytes isolated from the full depth of adult bovine articular cartilage and maintained for 8 months in alginate gel are still metabolically active and continue to synthesize cartilage-specific type II collagen and aggrecan. The cells did not synthesize large amounts of type I collagen or of the small nonaggregating proteoglycans as usually occurs when chondrocytes lose their phenotypic stability. After this extended period of time in culture, the cells were present as two populations exhibiting differences in size, shape and amount of extracellular matrix surrounding them. The first population was found only near the surface of the bead: these cells were flattened and surrounded by a matrix sparse in proteoglycans and collagen fibrils. The second population was found throughout the remaining depth of the bead: the cells were more round and almost always surrounded by a basket-like meshwork consisting of densely packed fibrils running tangential to the surface.