Application of extracellular-matrix-supported human embryonic stem cells for cytotoxicity testing

Objectives: Human embryonic stem cells (hESCs) hold great promise to future dental applications. This study aims to establish a novel feeder-free, xeno-free and serum-free culture system to support long-term undifferentiated growth of hESCs. This study explored the application of this novel culture system for cytotoxicity testing of sodium fluoride (NaF). Methods: Previously, we established an efficient method to derive autologous fibroblasts (H9 ebF) from H9 hESCs (Fu et al., 2009). H9 ebF cells were treated with ascorbic acid and dextran sulfate for 5 days to allow the deposition of extracellular matrix (ECM). Cells were lyzed by freeze at -80°C overnight and were removed by washing, leaving the ECM attached to the plate. The undifferentiated states, pluripotencies and genomic integrities of H9 hESCs cultured on ECM were characterized by immunofluorescence staining, reverse-transcriptase polymerase-chain-reaction and G banding, using Matrigel-supported hESCs as the control. To test the cytotoxic effect of NaF, H9 hESCs, H9 ebF and immortalized human cell line (CRL1486) were treated with varying concentrations (2mM, 4mM, 8mM and 12mM) of NaF for 2 hours, followed by the evaluation of MTS assay. Results: ECM supported undifferentiated growth of H9 hESCs for 20 passages, with supportive ability comparable to Matrigel™. H9 hESCs cultured on ECM and Matrigel™ exhibited significantly higher tolerance to the cytotoxicity of NaF than H9 ebF and CRL1486. Conclusion: ECM derived from autologous fibroblasts can support long-term culture of hESCs. This novel culture system suggests a potential model to study the embryotoxicity of NaF using hESCs.