Are Non-animal Systemic Safety Assessments Protective: A Toolbox and Workflow
Are Non-animal Systemic Safety Assessments Protective: A Toolbox and Workflow
Following treatment, cell media was aspirated and cells washed with PBS. After removal of all residual PBS, 2X TempO-Seq Enhanced lysis buffer (BioSpyder Technologies, proprietary kit) was diluted to 1X with PBS and added at a volume of 1µL per 1000 cells with a minimum of 10 µL per well and incubated for 10 min at room temperature. Following lysis, the samples were frozen at -80°C prior to sequencing The cell line HepG2 (human hepatoblastoma) was obtained from Public Health England European Collection of Cell Cultures (ECACC, Salisbury, UK). Cells were cultured in complete Minimal Essential Medium (EMEM) supplemented with 10% foetal bovine serum (FBS), 2 mM L-GlutaMAX, 1% non-essential amino acids (NEAA), 53 U/mL penicillin and 53 µg/mL streptomycin in 75 cm2 cell culture flasks. Cells were maintained in a humidified atmosphere with 5% CO2 at 37°C. Cells were kept at a confluence below 85% and not maintained in culture more than 4 weeks (8 passages). MCF-7 cells (Human Caucasian breast adenocarcinoma) was obtained from ECACC (Salisbury, UK). Cells were cultured in complete RPMI supplemented with 10% foetal bovine serum (FBS), 2 mM L-GlutaMAX, 53 U/mL penicillin and 53 µg/mL streptomycin. HepaRG cells (Cryopreserved differentiated) were obtained from Caltag Medsystems (UK). HepaRG cells were cultured in Williams E medium supplemented with 2 mM L-glutamine and HPRG670 supplement (Lonza, UK),. HepaRG cells were changed in to serum free medium following the initial 24 hour seeding procedure (Williams E medium supplemented with 2 mM L-glutamine, 100 units/ml penicillin, 100 Î¼g/ml streptomycin and HPRG640 supplement), for six days prior to dosing, with media replenishment every second day. HepG2 and MCF-7 cells were seeded onto 384 well, clear bottom black walled tissue culture plates at a density of 3,000 cell/well and left overnight to attach. HepaRG cells were seeded in collagen coated 384 well, clear bottom black walled tissue culture plates, at a density of 20,000 cells Compound was prepared as a stock solution at 200x higher concentration than the desired top concentration (solvent concentration maintained at 0.5% DMSO). Serial dilutions were performed to generate seven different concentrations of the compound with 0.5% DMSO used as a vehicle control. 5 biological replicates were generated at independent times with the appropriate dose of compound for 24 hours in a humidified atmosphere with 5% CO2 at 37°C. RNA was prepared according to the standard TempO-Seq protocol (Yeakley et al 2017) which generates a cell lysate without further purification Library construction was performed at Bioclavis according to standard protocols. In brief RNA in cell lysate solution was hybridized with the provided detector oligo (DO) pool. TempO-Seq DO pairs were designed to target a 50 nt sequence per gene, with each DO targeting 25 bases (Yeakley et al 2017) The detector pool used was Human Whole genome version 2.1 containing approximately 21000 oligos covering 19300 genes. Subsequently the mix was treated as follows, a nuclease digestion step, a ligation step, followed by heat denaturation. The ligation product was then transferred to an amplification microplate containing PCR mix per well. Ligation products were uniquely labeled during product amplification, with well-specific, â€œbarcodedâ€ primer pairs. Sample amplicons from each well was subsequently pooled into a single sequencing library. The TempO-Seq library was further processed using a PCR clean-up kit. TempO-Seq Human whole genome attenuation panel v 2.1 was used to selectively attenuate highly expressed transcripts