Grouping of UVCB Substances with Dose-Response Transcriptomics Data from Human Cell-Based Assays

The application of in vitro biological assays as new approach methodologies (NAMs) to support grouping of UVCB (unknown or variable composition, complex reaction products, and biological materials) substances has recently been demonstrated. In addition to cell-based phenotyping as NAMs, in vitro transcriptomic profiling is used to gain deeper mechanistic understanding of biological responses to chemicals and to support grouping and read-across. However, the value of gene expression profiling for characterizing complex substances like UVCBs has not been explored. Using 141 petroleum substance extracts, we performed dose-response transcriptomic profiling in human induced pluripotent stem cell (iPSC)-derived hepatocytes, cardiomyocytes, neurons, and endothelial cells, as well as cell lines MCF7 and A375. The goal was to determine whether transcriptomic data can be used to group these UVCBs and to further characterize the molecular basis for in vitro biological responses. We found distinct transcriptional responses for petroleum substances by manufacturing class. Pathway enrichment informed interpretation of effects of substances and UVCB petroleum-class. Transcriptional activity was strongly correlated with concentration of polycyclic aromatic compounds (PAC), especially in iPSC-derived hepatocytes. Supervised analysis using transcriptomics, alone or in combination with bioactivity data collected on these same substances/cells, suggest that transcriptomics data provide useful mechanistic information, but only modest additional value for grouping. Overall, these results further demonstrate the value of NAMs for grouping of UVCBs, identify informative cell lines, and provide data that could be used for justifying selection of substances for further testing that may be required for registration.

Supplementary description of cell culture conditions iCell Hepatocytes 2.0 Vials of hepatocytes were thawed for 3 min at 37°C in a water bath and subsequently resuspended in RPMI medium containing 2% (v/v) iCell hepatocyte medium supplement, 0.1 μM dexamethasone, 2% (v/v) B27 supplement, 25 μg/mL gentamicin, and 20 ng/mL oncostatin-M. Following microscopic evaluation of the cell density, the suspension was further diluted to a final concentration of 6.72 × 10 5 cells/mL. 25 μL of this suspension was then added to each well on collagen I coated 384-well plates (Corning, Product# 354664), yielding a final cell density of 16,800 cells per well. Plates were initially kept at room temperature (RT) for 30 min and then transferred to an incubator set at 37°C and 5% CO2. After 4 h of incubation, the plating medium was replaced with 25 μL fresh medium, a step that was repeated daily for 4 days. On day five, the plating medium was exchanged with 25 μL per well maintenance medium, consisting of RPMI containing 2% (v/v) iCell hepatocyte medium supplement, 0.1 μM dexamethasone, 2% (v/v) B27 supplement, and 25 μg/mL gentamicin. Maintenance medium was exchanged daily for the duration of the experiment. See additional details in Grimm et al. (2015).

iCell Neurons
Cryopreserved cells were thawed and plated according to the protocol provided by Cellular Dynamics International. Briefly, cells were plated on poly-D-lysine precoated 384-well plates (Greiner-Bio, Ref#: 781946) with iCell Neural Base Medium (Catalog#: M1010) added with iCell Neural Supplement A (Catalog#: M1032) and 3.3 mg/mL of laminin. Cells were plated at densities of 7,500 cells/well. Plates were initially kept at RT for 30 min before transferring to an incubator set at 37°C and 5% CO2 for 48 h until assay day. See additional details in Grimm et al. (2015).
iCell Cardiomyocytes 384-well microplates were precoated with 25 μL 0.1% (w/v) gelatin solution per well for 2 h at 37°C and 5% CO2. Cryopreserved cells were thawed according to the manufacturer's instruction using iCell cardiomyocyte plating medium with 1:500 (v/v) penicillin/streptomycin. Cell suspension was diluted in plate medium to provide a final cell concentration of 2 × 10 5 cells/mL. Subsequently, the gelatin solution was aspirated from the plates and 25 μL cell suspension was added to each well, making the final cell plating density 5,000 viable cells/well. Plates were kept at room temperature for 30 min before they were incubated at 37°C and 5% CO2. 48 h following cell seeding, the plating medium was exchanged with 40 μL of maintenance medium containing 1:500 penicillin/streptomycin. Maintenance medium was subsequently changed every other day for another 12 days until assay day. See additional details in Grimm et al. (2016).
iCell Endothelial cells Endothelial cells were plated and expanded on T-75 tissue culture flasks coated with human fibronectin solution at 3 μg/cm 2 . Cells were cultured with maintenance medium containing the VascuLife VEGF Medium Complete Kit (SKU: LL-0003), with FBS, and iCell Endothelial cells medium supplement. Cell density was determined using Trypan Blue exclusion test, and a cell suspension was prepared that resulted in 1.0 × 10 4 cells/cm 2 . The fibronectin solution was aspirated and cells were seeded in a T-75 flask. Cells were incubated at 37°C and 5% CO2 with media changes every 2 days and passaged every 3-4 days by TrypLE Express. Experiments were conducted with cells between passages 1 and 5. Cells were transferred into 384-well plates with 50 μL maintenance medium at a density of 750 cells/well for cytotoxicity assay and 7,500 cells for angiogenesis assay. Cells were kept in microplates for 2-3 days until a monolayer formed before adding chemicals for cytotoxicity assays. See additional details in Iwata et al. (2017) A375 and MCF7 Cell lines were obtained from ECACC. A375 were maintained in DMEM HG (Gibco) without phenol red, 10% HI FBS (1050064 South America Origin), 2 mM L-glutamine, pen/strep 100 µg/mL / 100 U/mL, and split every 5-7 days 1:6. MCF7 were maintained in EMEM (Gibco), 10% HI FBS (1050064 South America Origin), 2 mM NEAA (5 mL), 2 mM L-glutamine, pen/strep 100 µg/mL / 100 U/mL, and split every 7-10 days 1:3. All cell lines were used through maximum 20 passages and then replaced from frozen stock.
For use, the harvest cell suspension was counted using a haemocytometer and diluted for seeding in 384-well plates for treatment. 12,000-14,000 cells/well gave 90% confluence within 2-3 days of seeding in 50µl of the 10% FBS growth medium.
For assay, the medium for growth and maintenance, 50 µL/well, was removed on the morning of the day of treatment and replaced with to 20 µL/well fresh medium without FBS. 200X stock plates for log10 dilutions of the UVCB substances where pure extracted substance is 1X and then dilutions from there of 10X, 100X and 1000X were set up. Positive controls at 200X final concentration were also included in this plate. The UVCB substance 200X with positive control plate was first diluted 40-fold by diluting 4 µL stock chemical with 156 µL fresh medium without FBS. After mixing by trituration and microplate spinning, 5 µL of treatment solution from these diluted plates was added to the 20 µL medial on the cells to give a final 200-fold dilution from the 200X stock. Thus, final concentrations of the UVCB substance extract were 200X, 2000X, 20,000X and 200,000X. Treated plates were incubated for total 24 h and processed according to the individual assay requirements according to the manufacturer's protocols.