Hematopoietic growth medium hpgm

Bone marrow samples were collected from 11 cases of newly diagnosed CML patients at the Department of Hematology of the Second Hospital of Dalian Medical University. Patients were diagnosed according to French–American–British classification. Informed consent was obtained from all patients in accordance with the Declaration of Helsinki, and all manipulations were approved by the Medical Science Ethic Committee of Dalian Medical University. Mononuclear cells were isolated by density gradient centrifugation using Lymphoprep, and cryopreserved. In addition, 5 potential donors for allogeneic bone marrow transplantation were used to purify healthy hematopoietic cells. Human CD34⁺ cells were enriched from bone marrow mononuclear cells using MiniMACS (Miltenyi Biotech, Bergisch Gladbach, Germany) following the manufacturer’s instructions [27 (link)]. Confirmation of CD34⁺ cells phenotype and purity was assessed by flow cytometry analysis using CD34-PE-Cy7 (BD Biosciences, San Diego, CA). Purified CD34⁺ cells were grown in serum-free hematopoietic growth medium (HPGM; Lonza) supplemented with 10 ng/mL recombinant human interleukin 3 (rhIL-3), 10 ng/mL rhIL-6, and 50 ng/mL recombinant human stem cell factor (PeproTech) in a humidified incubator at 37 °C and 5% CO₂/95% air (v/v).

Horse serum was purchased from Biochrom AG (Berlin, Germany). The protein-assay dye reagent concentrate was from Bio-Rad (Munich, Germany) and the GelGreen nucleic acid gel stain was from Biotium (Hayward, CA, USA). Hematopoietic Growth Medium (HPGM) was from Lonza (Cologne, Germany). Recombinant human fms-related tyrosine kinase 3 ligand (Flt3), purified recombinant human stem cell factor (SCF) and recombinant human thrombopoietin (TPO) were from MACS Miltenyi Biotec (Gladbach, Germany). Mitomycin C was from Medac (Hamburg, Germany) and methanol was from Carl Roth (Karlsruhe, Germany). 1,4-Diazabicyclo[2.2.2]octane (DABCO), albumin from human serum, cytochalasin B, L-glutamine solution, methyl methanesulfonate, penicillin-streptomycin (10,000 units penicillin and 10 mg streptomycin per ml), RPMI 1640 medium (HEPES modification) and sodium pyruvate solution were purchased from Sigma-Aldrich (Munich, Germany). Doxorubicin hydrochloride, Mitomycin C and vinblastine sulfate were from Teva (Ulm, Germany).