title: The ex vivo expansion capacity of normal human bone marrow cells is dependent on experimental conditions: role of the cell concentration, serum and CD34+ cell selection in stroma-free cultures.
abstract: The present study was conducted to establish defined culture conditions for ex vivo expansion of normal human bone marrow cells. We investigated the role of three experimental expansion parameters: the cell concentration in the initial culture medium, the role of animal serum, human plasma and serum-free substitute, and the expansion potential of mononucleated cells (MNC) versus CD34+ cells. Cells were cultured in suspension with stem cell factor (SCF), IL3, IL6 and Erythropoietin (Epo) for 10 days. 1) Reducing the cell concentration from 3 x 10(4) to 1.5 x 10(3)/ml increased total cell expansion almost 20 fold, progenitor expansion more than 3 fold, and the maintenance of long term culture-initiating cells (LTC-IC). 2) In medium containing a serum-free substitute, total and CD34+ cell expansion was 3 times greater than in medium containing 1-10% human AB plasma or 25% animal serum. 3) The expansion potential of selected CD34+ cells was significantly greater than that of the total MNC population. However, taking into account the cell loss due to CD34+ selection, the overall results for quantitative expansion in relation to the initial number of MNCS favor the use of non-selected MNCS. 4) SCF + IL3 + IL6 was clearly the best combination of early cytokines for LTC-IC maintenance, with or without lineage-restricted cytokines, whereas the presence of IL1 beta in any combination augmented the decrease in LTC-IC. Addition of G-CSF to the medium resulted in 1 log increase in total cell expansion and a 2-fold increase in CFU-GM expansion. Addition of Epo always induced a dramatic proliferation of erythroid cells (up to 2000 fold) as well as of CFU-GM (up to 4 fold), without exhausting the LTC-IC pool. We concluded that the expansion of hemopoietic cells for clinical purposes needs establishment of controlled, reproducible and reliable culture conditions.
authors.affiliation: Centre Claude Bernard, CHU Saint-Antoine, Paris, France.
authors.forename: A
authors.initials: A
authors.lastname: Poloni
authors.forename: M C
authors.initials: MC
authors.lastname: Giarratana
authors.forename: H
authors.initials: H
authors.lastname: Firat
authors.forename: L
authors.initials: L
authors.lastname: Kobari
authors.forename: N C
authors.initials: NC
authors.lastname: Gorin
authors.forename: L
authors.initials: L
authors.lastname: Douay
mesh_terms: D000818:Animals; D018952:Antigens, CD34 / Q000378:metabolism* / Q000502:physiology; D001853:Bone Marrow / Q000378:metabolism*; D001854:Bone Marrow Cells; D002417:Cattle; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods*; D002455:Cell Division; D003470:Culture Media / Q000592:standards; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D005312:Fetal Blood / Q000502:physiology; D016298:Hematopoietic Cell Growth Factors / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000166:cytology; D006801:Humans; D017154:Stromal Cells / Q000166:cytology
chemical_list: D018952:Antigens, CD34; D003470:Culture Media; D016895:Culture Media, Serum-Free; D016298:Hematopoietic Cell Growth Factors