Evaluating AMG 330's Effects on Immature AML Cells

To test the effect of AMG 330 on immature AML cell populations, unsorted cells from thawed AML specimens were cultured in IMDM supplemented with 20% FBS and 10 ng/mL each of IL-3, SCF, G-CSF, and GM-CSF containing no AMG 330 or AMG 330 at either 100 pg/mL or 500 pg/mL (because of limited cell numbers available, experiments could only be conducted with 2 different concentrations of AMG 330) together with CellVue Burgundy dye-labeled T-cells at an E:T = 3:1 cell ratio. After 48 hours, CellVue Burgundy dye-negative primary AML cells were separated using a FACSAria flow cytometer (BD Biosciences), and cells were cultured in IMDM with 20% FBS containing the recombinant human cytokines SCF, interleukin-6, FLT3 ligand, and thrombopoietin (all from Invitrogen), StemRegenin-1 (SR1, Cellagen Technology, San Diego, CA), and penicillin-streptomycin [20 (link)]. Cultures were replenished with fresh medium weekly and kept at 37°C in 3% O₂ and 5% CO₂ for 4 weeks. Defined fractions of the cultures were removed after 2 and 4 weeks and subjected to CFC assays. After 10–14 days at 37°C in 3% O₂ and 5% CO₂, colony forming units-granulocyte and/or monocyte (CFU-GM) of at least 30–50 cells were quantified [20 (link)].