Developmental Toxicity Assay Based on Real-Time Monitoring of Fibroblast Growth Factor Signal Disruption in Human Induced Pluripotent Stem Cells

This study explores an alternative to animal testing, that employs culture cell to assess chemical toxicity. The central question is whether disruption in signaling pathway of human iPS cells can serve as a level indicator of developmental toxicity. We found that regardless of the mechanism, developmental toxicant disrupt the signaling pathway, and the temporal sums serves as an indicator.

This iPSC based assay enables comprehensive evaluation of developmental toxicant, with high accuracy and therapeute, and without the use of animals. To begin, seed cells into an ECM coated 96-well plate containing 200 microliters of fresh maintenance medium supplemented with 10 micromolar ROCK inhibitor. Seal the plate with a gas-permeable film.

After 24 hours, replace the medium in each well with 200 microliters of fresh maintenance medium, and reseal the plate. Next, dissolve the test chemical in assay medium to its maximum solubility. Prepare a five-point five-fold serial dilution of the test chemical in assay medium using the previously prepared solution.

Add 11 microliters per well of the prepared chemical solutions. For the vehicle control, add 11 microliters per well of assay medium containing the same concentration of DMSO. After 48 hours of treatment, incubate the plates at room temperature for 30 minutes.

Then, add 111 microliters per well of pre-equilibrated ATP-based luminescent cell viability assay reagent at 25 degrees Celsius. For the blank well, add 111 microliters of assay medium and 111 microliters of the reagent. Using a micro-plate shaker, shake the plate at 300 revolutions per minute for two minutes.

Transfer 200 microliters of the mixture from each well to a black plate for luminescence measurement, and incubate the black plate at 25 degrees Celsius for 10 minutes. Then, measure the luminescence intensity. For the determination test, after incubating the cells for 96 hours, replace the medium in each well with 100 microliters of assay medium.

Dissolve the test chemical in assay medium based on the results obtained from the preliminary test. Prepare a nine-point two-fold serial dilution of the test chemical in assay medium using the previously prepared solution. Add 11 microliters per well of the prepared chemical solutions.

For the vehicle control, add 11 microliters per well of assay medium containing the same concentration of DMSO. After incubating the cells for 96 hours, replace the medium in each well with 100 microliters of assay medium containing 1%luminescent substrate. Dissolve the test chemical in assay medium at the concentration determined from the previous section.

Prepare an eight-point two-fold serial dilution of the test chemical in assay medium using the previously prepared solution. Two hours after the medium exchange, add 11 microliters per well of the serially diluted chemical solutions. For the vehicle control, add 11 microliters per well of assay medium containing the same concentration of DMSO.

One hour after starting chemical exposure, add 12 microliters per well of assay medium containing basic fibroblast growth factor. Measure luminescence continuously for 48 hours using a real-time luminescence measurement system under 5%carbon dioxide at 37 degrees Celsius. Phase contrast imaging confirmed a confluent cell layer covering nearly the entire surface of the wells, both before and after chemical exposure.

Live and dead staining showed that while the number of propidium iodide stained dead cells increased after chemical exposure, the majority of cells remained viable and were stained with calcein-AM. In the absence of test chemicals, luminescence intensity showed two distinct peaks across all six replicate wells, demonstrating high reproducibility and temporal resolution. Automated real-time monitoring revealed that valproic acid disrupted fibroblast growth factor signaling in a concentration dependent manner, with stronger suppression at higher concentrations.

Manual luminescence measurements captured a general suppression pattern by valproic acid, but lacked the temporal resolution seen in automated detection.