Organoids were dissociated into single cells and plated on a 384-well assay plate in 10% Matrigel. After a 48-hour recovery period, chemotherapeutics were administered using a semi-automated
D300e dispenser (Tecan, Männedorf, Switzerland), normalized to 0.5% DMSO. Chemotherapeutics were tested across a logarithmically designed curve: gemcitabine, paclitaxel and irinotecan (range: 8.0 × 10
−12 mol/L to 2.0 × 10
−6 mol/L), and 5-FU and oxaliplatin (range: 1.0 × 10
−8 to 1.0 × 10
−4 mol/L). Negative controls included wells with DMSO normalization alone. Cell viability was assessed at 5 days using
CellTiter Glo (Promega Corp, Madison, WI, USA)
21 . Viability curves were fitted using nonlinear logistic regression with
Prism v8 (GraphPad Software, La Jolla, CA, US). Normalized area under curve (AUC) was obtained by dividing the AUC value by the maximum area for each concentration range. A population distribution was illustrated by collective analysis of each chemotherapeutic using a violin plot (R studio version 1.2.5033). Pharmacotyping was performed in four biologic replicates, and at least two technical replicates with most performed at early and late passage.
Seppälä T.T., Zimmerman J.W., Sereni E., Plenker D., Suri R., Rozich N., Blair A., Thomas DL I.I., Teinor J., Javed A., Patel H., Cameron J.L., Burns W.R., He J., Tuveson D.A., Jaffee E.M., Eshleman J., Szabolcs A., Ryan D.P., Ting D.T., Wolfgang C.L, & Burkhart R.A. (2020). Patient-derived organoid pharmacotyping is a clinically tractable strategy for precision medicine in pancreatic cancer. Annals of surgery, 272(3), 427-435.
