Optimization of in vitro erythropoiesis from CD34+ cord blood cells using design of experiments (DOE)

In vitro culture studies of hematopoietic cells (HCs) have traditionally been limited to dose-response, empirical methods which can be uninformative, misleading and suboptimal. Herein, we use design of experiments methodology to quantitatively define and optimize in vitro erythropoiesis from human cord blood CD34+ cells to enucleation. DOE was used to theoretically define the effects of 7 cytokines known to affect red cell maturation in humans. Of these, stem cell factor (SCF) and erythropoietin (EPO) were found to be the most significant factors in red cell production (p < 0.01). DOE defined process characteristics and interactions of SCF and EPO on total cell expansion and maturation of cord blood HCs towards the erythroid lineage and delineated optimal in vitro concentrations of each cytokine (75 ng/mL and 4.5 IU/mL, respectively). The DOE-optimized cytokine cocktail achieved an average of 26,460-fold total cell expansion and accelerated erythroid maturation yielding over 90% glycophorin-A-expressing cells by day 7. Maturation of red cells was confirmed by morphology, immunophenotype, clonogenic capacity, gene and protein expression of adult and neonatal hemoglobins (α, β, and γ) and by progressive expression of GATA-1 with maturation. The use of DOE was effective and efficient for the analysis, characterization and optimization of in vitro erythropoiesis to the stage of enucleation and provides a systematic platform for the use of other growth factors in vitro for the study of hematopoiesis and expansion of cell products.

► Effective and efficient use of DOE for analysis, characterisation, and optimization.
► In vitro erythropoiesis to the stage of enucleation.
► Systematic platform for stem cell bioprocessing.