Biosynthesis of proteoglycans and hyaluronate in rabbit corneal fibroblast cultures. Variation with age of the cell line and effect of foetal calf serum

Abstract

A cell line of rabbit corneal fibroblasts, established by subcultivation every week, was investigated over as many as 141 passages. Biosynthesis of hyaluronate and proteoglycans was followed by incorporation of [3H]glucosamine. When cells were grown in medium supplemented with 10% newborn calf serum the average molecular size of the hyaluronate and proteoglycans secreted to the culture medium, as indicated by gel chromatography, appeared to decrease after about 10 passages of the cells. This phenomenon could be related to a decrease in the proportion of large molecular size hyaluronate. It could be reversed by replacement of newborn calf serum in the culture medium with foetal calf serum. No activity which degraded the fraction containing hyaluronate and proteoglycans was observed in ‘old cultures grown in newborn calf serum. No factors were detected in foetal calf serum which affected the macromolecular properties of this fraction in the absence of cells. The proportion of hyaluronate in the total glycosaminoglycans synthesized also decreased with the age of the cell line, while the proportion of dermatan sulphate increased, but this was independent of the serum conditions. No change in the relative proportions of the remaining glycosaminoglycans was observed. In another cell line from rabbit corneal stroma, the culture medium was supplemented with 10% foetal calf serum throughout. No change in the average molecular size of hyaluronate or in the relative proportions of the glycosaminoglycans could be observed. These cells showed no signs of spontaneous transformation and died after about 30 passages.