Culturing Primary Human ALL Cells with MSCs

Primary human ALL cells were obtained after informed consent at West Virginia University Cancer Center; Cincinnati Children’s Hospital Medical Center Respiration Core; and Pittsburgh Biospecimen Core under the approved Institutional Review Broad (IRB) protocols: #1310105737; STUDY19030357 and # 2011-3023. Primary human ALL cells were cultured on hTERT-immortalized primary BM mesenchymal stromal cells (MSCs)^{39 ,40 (link)}. MSCs were seeded at a density of 10⁴ cells/cm² in MSC medium 48 h prior to adding to ALL. ALL cells were seeded onto MSC at a density of 2 × 10⁶ cells/ml in SFEM II medium (StemCell Technologies, Vancouver, BC, Canada) supplemented with 20% fetal calf serum (GIBCO, Life Technologies), 20 ng/ml recombinant IL-3 (R&D Systems, Abingdon, UK) and 10 ng/ml recombinant IL-7 (R&D Systems, Minneapolis, MN). ALL cells were harvested every 7 days. Non-adherent cells present in supernatant medium were washed with PBS and passed through a 15 μm filter (pluriSelect Life Science, Leipzig, Germany). ALL cells were separated from MSCs by magnetic cell separation using hCD45 microbeads (Miltenyi Biotec, Auburn CA). Viable ALL cells were counted by Trypan blue exclusion, re-suspended in fresh ALL medium, and seeded onto fresh MSC.