Collagen biosynthesis by human skin fibroblasts. I. Optimization of the culture conditions for synthesis of type I and type III procollagens

Abstract

Skin fibroblasts in culture can provide a convenient means to study aberrations of collagen metabolism in a variety of clinical conditions. In the present study, the culture conditions for the synthesis of procollagen by cultured human skin fibroblasts were optimized by independently varying parameters in the cell culture environment. To study the synthesis of procollagen the cell cultures were labeled with [3H]proline and the collagenous polypeptides were determined either by measuring the synthesis of hydroxy[3H]proline or by assaying the 3H-labeled proteins digested into dialyzable 3H-labeled peptides by bacterial collagenase. On the basis of the experimental results, the following culture conditions are suggested for optimal synthesis of procollagen: (a) cell culture medium should be supplemented with ascorbic acid (25--50 micrograms/ml) and fetal calf serum (20%); (b) the pH of the culture medium should be kept above 7.2 and preferably in the pH range 7.5--7.8; (c) the cell cultures should be used one to two days after reaching visual confluency. Under these conditions the synthesis and secretion of [3H]procollagen was found to be linear through a 24 h incubation period, and procollagen was demonstrated to be a major gene product of the fibroblasts. The relative synthesis of type I and type III procollagens was also monitored by isolating these genetically distinct procollagens by DEAE-cellulose chromatography or by measuring type I and III collagens by sodium dodecyl sulfate polyacrylamide gel electrophoresis after limited pepsin proteolysis. No marked changes were observed in type I/III procollagen ratios in situations where the total formation of hydroxy[3H]proline was significantly affected. The average coefficient of variance for procollagen synthesis between replicate cultures was found to be relatively small (14%), and the optimization of the culture conditions for the control cells has, therefore, created a reliable and reproducible basis for employing human skin fibroblasts to study collagen metabolism in acquired and inherited diseases.