Background
- Profiling the cytotoxicity of test compounds on cell monolayers or 3D cell models involves measuring the viability of the cells using molecular probes which bind to dead cells. Cytotoxicity, determined by measuring decreases in cell viability, is an important endpoint in the evaluation of the safety and toxicity of drug compounds, and a critical measure of the effectiveness of antineoplastic agents on cancer cells.
- Utilizing High Content Screening to image molecular probes that label nonviable cells but are impermeable to viable cells furnishes an effective assay for cytotoxicity and cell viability in cell models [1].
- In conjunction with other assay endpoints, such as the assessment of cell proliferation or apoptosis, the measurement of cell viability is a useful tool for interrogating the effect of cancer drugs in vitro [1].
- In addition to High Content Screening, Visikol offers cell viability quantification via higher throughput endpoints such as MTT assays and CellTiterGlo
- 3D cell culture models effectively recapitulate the in vivo tumor environment, and are an effective tool for the evaluation of cytotoxic effects of promising anti-cancer drug compounds.
- Assessment of cell viability is critical for the evaluation of the safety and toxicity of test compounds. Assaying the cytotoxicity of compounds in 3D hepatocellular models is an excellent method for identifying and eliminating lead compounds that possess toxic liabilities early in the drug discovery process.
- This assay can be coupled with up to two other imaging-based endpoints (e.g. cell proliferation and apoptosis).
Protocol
| Instrument | Molecular Devices ImageExpress Micro Confocal |
| Analysis Method | High content screening |
| Markers | LIVE/DEAD fixable dead cell probe (Molecular Probes) DAPI (total cell count) Other dead cell probes available on request |
| Cell Model Type | 3D cell models (e.g. tumor spheroids, organoids, etc.) or adherent monolayer / cell suspension |
| Cell Types Available | HepG2, HepaRG, primary human hepatocytes ATCC cancer cell lines (e.g. A549, BT549, MCF-7) Primary cells Client provided cells iPSC derived neural organoid (Stemonix) Custom models available on request |
| Test Article Concentration | 8 point assay (0.05, 0.1, 0.5, 1, 5, 10, 50, 100 µM) (custom concentrations available) Single point assay |
| Number of Replicates | 3 replicates per concentration |
| Quality Controls | 0.5% DMSO (vehicle control) Staurosporin (positive control) |
| Test Article Requirements | 50 uL of 20 mM solution or equivalent amount of solid |
| Data Delivery | Dose response curves, LD50 values, total cell counts, Viability % for each test concentration Evaluation of statistical significance of results with respect to vehicle and positive control |
| Assay Reference Code | INV-2102 |
General Procedure
- For 3D cell models, grown to approximately 200 μm in diameter. Adherent monolayers cultured to confluence.
- Treatment with test compounds
- After 24 hours, cells are labeled with fixable dead cell probe.
- Nuclei are labeled with DAPI
- For 3D cell models, tissue clearing is applied to render 3D cell models transparent
- High content imaging is conducted on well plates
- Images are analyzed to quantify total number of cells and total number of dead cells
Data
Figure 1. Maximum Z-projection of Z-stack obtained from High Content Imaging of HepG2 tumor spheroids treated with cisplatin. Data was quantified by automated image analysis to count total number of cells, and number of dead cells
Figure 2. Representative data showing the observed fraction of dead cells detected in primary tumor spheroids treated with various cytotoxic agents
References
- Wiederschain, G.Y. Biochemistry Moscow (2011) 76: 1276. https://doi.org/10.1134/S0006297911110101