A clinical-scale expansion of mobilized CD 34+ hematopoietic stem and progenitor cells by use of a new serum-free medium.
BACKGROUND
The autologous transplantation of CD 34+ cells expanded ex vivo in serum-free conditions dramatically reduces post-myeloablative 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 HP 01 (Macopharma), was evaluated.


METHODS
Purified CD 34+ cells (Isolex 300i, Baxter) from mobilized peripheral blood samples of myeloma patients were thawed, washed, and cultured, as for previous clinical assays. Twenty million CD 34+ cells were resuspended per 1 L of SCF-, G-CSF-, and MGDF-supplemented medium (HP 01 or IS), introduced into 3-L culture bags (AFC), and cultured for 10 days in 5 percent CO(2), 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 HP 01 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 CD 34+ cells (3.5+/- 1.2-fold for HP 01 vs. 2.7+/- 1.5-fold for IS) was not significant. IS medium partially maintained SCID-repopulating cells (SRC), whereas the culture in HP 01 medium fully maintained the stem cell activity for 10 days. A higher frequency of CD 41+ cells after expansion in HP 01 than in IS medium was also observed.


CONCLUSIONS
Maco Biotech HP 01 medium is suitable for clinical-scale expansion of CD 34+ cells with the SCF, G-CSF, and MGDF cytokine cocktail, permitting an intensive amplification of CFCs and maintenance of SRCs.
Ivanovic
Zoran
Z
French Blood Establishment Aquitaine-Limousin, Bordeaux; CNRS UMR 5164, University of Bordeaux 2, Bordeaux, France.
Duchez
Pascale
P
Dazey
Bernard
B
Hermitte
Francis
F
Lamrissi-Garcia
Isabelle
I
Mazurier
Frédéric
F
Praloran
Vincent
V
Reiffers
Josy
J
Vezon
Gérard
G
Boiron
Jean-Michel
JM
D018952:Antigens, CD34; D018929:Cell Culture Techniques; D049109:Cell Proliferation / Q000187:drug effects; D016895:Culture Media, Serum-Free; D016207:Cytokines / Q000494:pharmacology; D019650:Hematopoietic Stem Cell Mobilization; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells; D006801:Humans; D009101:Multiple Myeloma / Q000150:complications / Q000628:therapy; D009503:Neutropenia / Q000209:etiology / Q000628:therapy; D014182:Transplantation, Autologous
D018952:Antigens, CD34; D016895:Culture Media, Serum-Free; D016207:Cytokines