Development of Low- and High-serum Culture Conditions for Use of Human Oral Fibroblasts in Toxicity Testing of Dental Materials

Abstract

With the aim of establishing conditions applicable to the testing of dental materials in human target cells, fibroblastic cell lines have been derived and grown from explants of human oral mucosa. Both a high-serum medium (termed "HSM") (CMRL 1066 supplemented with 10% fetal bovine serum) and a low-serum medium (termed "LSM") (a 1:1 mixture of M 199:MCDB 153 supplemented with 1.25% serum) supported radial outgrowths of cells from oral explants, as well as the subsequent transfer and growth of the cells in mass culture and at clonal density. Cells were typically fibroblastic in that they expressed vimentin uniformly, but did not express immunocytochemical markers of epithelial or endothelial cells. Cells derived in either LSM or HSM showed significantly higher colony-forming efficiency and clonal growth rate when transferred in LSM, as compared with HSM. Because cell migration occurred to a lesser extent in LSM, microscopic scoring of colony formation was also markedly facilitated. In both LSM and HSM, cellular low-molecular-weight thiols constituted about 30% of the total amount of sulfhydryls. Glutathione was present in about six- to seven-fold-higher amounts than cysteine-glutathione primarily in its reduced form and cysteine primarily in its oxidized form. A corrosion product of dental amalgam, i.e., Hg2+, decreased cell survival measured as colony-forming efficiency in a dose-dependent manner following either an acute (one h) exposure or continuous exposure (seven days). These studies demonstrated that human oral fibroblasts could be cultured at about one-tenth of the serum content that is commonly used. It is concluded that human oral fibroblasts grown at low-serum conditions provide a highly sensitive and easily applicable in vitro model for toxicity studies of dental materials.