Authenticated Lung and Gingival Fibroblasts Cell Models for In Vitro Toxicity Testing

There is an increasing demand for in vitro models to replace animal models in toxicity testing. Drivers for this change include decreased overall cost for cell-based models and the ability to do more high-throughput screening. The development of cell-based in vitro models for toxicity testing is a challenging task. Primary cells can best represent the in vivo situation; however, donor variability and replicative senescence restrict the potential usefulness of this cell model in the study of toxicity. Conversely, continuous cell lines often have altered genomes and do not fully represent the parental cells as a result of their altered genomic state. Human telomerase reverse transcriptase (hTERT)-immortalized primary cells provide a better solution—these cells can be continuously cultured while retaining the physiological characteristics of the parental primary cell.

In this study, we established two clonal hTERT-immortalized cell lines by expressing hTERT in lung fibroblasts and gingival fibroblasts isolated from normal donors. Both lines have been cultured continuously for more than 35 population doublings without any signs of replicative senescence. Further, both hTERT-immortalized fibroblasts retained a normal diploid karyotype over extended culture period and maintained typical fibroblastic characteristics, including positive staining for a fibroblast marker and negative staining for an epithelial cell marker. Additionally, the lung fibroblast cells responded to TGF-beta treatment with elevated smooth muscle actin expression as did the parental cells. Notably, both cell lines are sensitive to the toxicological agent chlorhexidine in a dose-dependent manner, similarly to the primary cells. These results show that the hTERT-immortalized lung fibroblasts and gingival fibroblasts retain the important physiological characteristics of the primary cells from which they were derived and provide a very useful in vitro cell model for toxicity screening.