Culturing Leukemic Blasts with Therapeutics

Thawed primary human AML blasts were cultured on irradiated stromal feeder cell layers with cytokine- and SR1- (1uM) supplemented media. Live blast purification was achieved by magnetic bead-based dead cell removal prior to culture (Miltenyi # 130-090-101). Fresh media was added every 2–3 days and cells were re-plated prior to reaching 70–80% confluence. After 12 h of preconditioning, IACS-010759, diluted in DMSO, was added to a final concentration of 30 nM. Similarly, Cytarabine, diluted in PBS, was added to a final concentration of 100 ng/ml. Equivalent volumes of PBS and DMSO were added to the mock-treated conditions. Cells were harvested at Day 5 post-culture and analyzed by flow cytometry and transplanted into recipient-irradiated female NSG mice.