A clinical-scale expansion of mobilized CD34+hematopoietic stem and progenitor cells by use of a new serum-free medium

BACKGROUND: The autologous transplantation of CD34+ cells expanded ex vivo in serum-free conditions dramatically reduces postmyeloablative neutropenia in myeloma patients. In our cell therapy unit, cells for this clinical assay have been expanded under GMP with serum-free Irvine Scientific (IS) medium with stem cell factor (SCF), granulocyte-colony-stimulating factor (G-CSF), and megakaryocyte growth and development factor (MGDF; 100 ng/mL, respectively). Because this clinical-grade IS medium is no longer available, a new serum-free medium, Maco Biotech HP01 (Macopharma), was evaluated. STUDY DESIGN AND METHODS: Purified CD34+ cells (Isolex 300i, Baxter) from mobilized peripheral blood samples of myeloma patients were thawed, washed, and cultured, as for previous clinical assays. Twenty million CD34+ cells were resuspended per 1 L of SCF-, G-CSF-, and MGDF-supplemented medium (HP01 or IS), introduced into 3-L culture bags (AFC), and cultured for 10 days in 5 percent CO2, at 37 degrees C, and at 100 percent humidity. RESULTS: A higher amplification of total nucleated cells (NCs) and colony-forming cells (CFCs) was obtained with HP01 medium than with IS medium (42 +/- 16.6-fold vs. 20.5 +/- 5.9-fold for NCs and 26.7 +/- 7.4-fold vs. 15.5 +/- 2.5-fold for CFCs, respectively), whereas an increase in CD34+ cells (3.5 +/- 1.2-fold for HP01 vs. 2.7 +/- 1.5-fold for IS) was not significant. IS medium partially maintained SCID-repopulating cells (SRC), whereas the culture in HP01 medium fully maintained the stem cell activity for 10 days. A higher frequency of CD41+ cells after expansion in HP01 than in IS medium was also observed. CONCLUSION: Maco Biotech HP01 medium is suitable for clinical-scale expansion of CD34+ cells with the SCF, G-CSF, and MGDF cytokine cocktail, permitting an intensive amplification of CFCs and maintenance of SRCs.