Cryopreservation and Culture of Primary Hematopoietic Cells

Primary cells were cultured as previously done in Pei et al.^{66 (link)}. Primary human AML cells, normal cord/peripheral blood mononuclear cells (CBMCs/PBMCs) were resuspended at about 100–200 e⁶ cells per ml in freezing media composed of 50% FBS (GE Healthcare), 10% DMSO (Sigma) and 40% IMDM media (Gibco) and then cryo-preserved in liquid nitrogen. Cells were thawed in 37 °C water bath, washed twice in thawing media composed of IMDM (Gibco), 2.5% FBS (GE Healthcare) and 10 ug/ml DNAse (Sigma). Normal CD34+ CBMCs/PBMCs were enriched from CBMCs/PBMCs using the CD34 MicroBead kit (Miltenyi Biotec). Cells were cultured in normal or complete serum-free media (SFM) in 37 °C, 5% CO₂ incubator. Normal SFM is composed of IMDM (Gibco), 20% BIT 9500 (STEMCELL Technologies), 10ug/ml LDL (Low Density Lipoprotein, Millipore), 55uM 2-Mercaptoethanol (Gibco) and 1% Pen/Strep (Gibco). Complete SFM were made by supplementing the normal SFM with FLT-3, IL-3 and SCF cytokines (PeproTech), each at 10 ng/ml. MDS-L cells were a generous gift from Daniel Starczynowski and grown in RPMI-1640 medium with 10% FBS, 1% penicillin-streptomycin and supplemented with 10 ng/ml IL-3^{67 (link)}.