Ex vivo manufacture of human neutrophils: optimizing the cost of production
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Abstract
Neutrophils are the most abundant leukocyte in human bloodstream. Nevertheless when neutrophils absolute count (ANC) values are inferior to 1.5x106 cells/mL, an immunocompromising medical condition known as neutropenia occurs. The aim of this research is to evaluate the impact the quantity of ex vivo produced neutrophils, from CD34+ cells isolated from umbilical cord blood, have in the cost of goods (CoG) for stem cell factor in the production process. To set a cornerstone for the development of cell therapy to treat neutropenia. To achieve this, CD34+ cells were isolated from umbilical cord blood (UCB). To obtain CD34+ cells, mononuclear cells (MNCs) were isolated by Ficoll density gradient separation. Posteriorly CD34+ cells were isolated either from fresh or thawed MNCs, using 1 or 2 successive MS columns during the CD34 enrichment process. Isolated CD34+ cells were cultured in Stemline II medium supplemented with stem cell factor (SCF), granulocyte colony stimulating factor (G-CSF), and thrombopoietin peptide mimetic (Tpo), 100 ng/mL each one. Finally the impact of using immobilized stem cell factor in ex vivo neutrophil production was in silico evaluated. Freshly isolated CD34+ cells resulted in both higher purity (94.5%) and 254,280 CD34+ cells than thawed cells. The cultured cells successfully produced mature neutrophils, exhibiting a CD11b+/CD15+ phenotype, with functional ROS production capacity. Additionally freshly processed cells produced higher expansion in the final cell number, 3,264.88 ± 545.57 fold increase. Regarding the in silico evaluation, the concentration of immobilized SCF and the number of cells produced resulted in the most critical factors to be optimized to effectively reduce the production costs associated to ex vivo produce neutrophils. According to the results of this research purer CD34+ cell fractions and the usage iSCF at low concentrations could considerably reduce the CoG associated to SCF in the ex vivo production of neutrophils. Thus, the panorama to produce a technical and economically feasible cell therapy is clearer.