Prolonged expression of differentiated phenotype by chondrocytes cultured at low density on a composite substrate of collagen and agarose that restricts cell spreading.

The dedifferentiation of chondrocytes in culture is frequently associated with transition from a rounded to a spread morphology. A number of culture methods which prevent cell spreading have been described; however, all have disadvantages that limit their widespread use. In this paper we describe a new technique which allows prolonged cultivation of attached chondrocytes at low density while inhibiting spreading: the cells are grown on a composite substrate of agarose and collagen. By varying the ratio of agarose to collagen in the gel, the degree of spreading can be varied. The cultures are suitable for ultrastructural and immunofluorescence analysis and for studies of the synthesis and secretion of macromolecules. In order to determine whether the differentiated phenotype was maintained on composite gels, we compared the levels of messenger RNAs for cartilage-specific proteoglycan, link protein, alpha 1 (II) and alpha 1 (I) collagens in chondrocytes grown at low density on composite gels or at high or low density on tissue culture plastic for up to 21 days. The rate of decline in the level of mRNAs encoding the cartilage-specific products and the rate of increase in the level of alpha 1 (I) collagen mRNA were slower in the composite cultures than in the cultures on plastic. This culture technique may, therefore, prolong expression of the differentiated phenotype of chondrocytes relative to cultivation on plastic and will be useful for further studies on the role of cell shape in regulating differentiated gene expression.