Fractionation and recovery of secretions of synovial cells synthesized in culture with radioactive precursors.

The synthesis of many constituents of connective tissues, including glycosaminoglycans (Grossfeld, Meyer, Godman, and Linker, 1957), collagen (Jackson and Smith, 1957; Porter and Pappas, 1959), collagenase (Evanson, Jeffrey, and Krane, 1967) and a protein immunologically cross-reactive with chondromucoprotein (Janis, Sandson, Smith, and Hamerman, 1967), has been widely studied in cell cultures from synovium and related tissues. For all but brief intervals, the culture medium for these cells must contain natural nutrients such as serum, which sets a problem in distinguishing the macromolecules secreted by the cells from those added with the medium. This can be overcome in many ways, ranging from simple comparison of cellexposed and control medium with the mucin clot test, to electron microscopy, isotopic tracing, immunochemistry, and complicated chemical extractions. In identification or measurement of the secretions, some minor degradation might not matter; but their separation in the native state from the great excess of extraneous material in culture medium is quite a different exercise, especially in the case of hyaluronic acid which is singularly prone to physical and chemical degradation (Ogston and Stanier, 1952). For the production of undegraded radioactive hyaluronic acid by cell culture, we have preferred chromatography in the cross-linked dextran, Sephadex G200, as the initial step in the treatment of the culture medium (Fraser, 1963). The introduction of agarose gels for fractionation of synovial fluid (Barker and Young, 1966) has made the purification of cell-secreted hyaluronic acid virtually a one-stage process. However, Sephadex G200 also separates other classes of secretion of potential interest. It is the purpose of this paper to report experience accumulated with these methods of analyzing cell-culture secretions formed from glucose and other simple substrates.