Effects of Single-Agent, Low Dose Exogenous Erythropoietin In a Long-Term In Vitro serum-Free 3D Culture of Human Cord Blood Mononuclear Cells for Directed Erythropoiesis

Abstract 341

Ex vivo expansion of cord blood mononuclear cells (CBMNCs) could provide a safe, flexible and ample supply of blood components for cellular therapies. Traditionally, hematopoietic cell expansion has been performed in 2D tissue culture flask or well-plate static cultures using abnormally high concentrations of cytokines which is expensive, reduces the self-renewal capacity, and skews normal differentiation. We have previously developed a 3D bone marrow biomimicry through the use of a synthetic scaffold made of polyurethane (PU) coated with collagen type I which could expand CBMNCs in a cytokine-free environment for at least 28 days ex vivo, with or without the addition of serum to the media. We hypothesised that the addition of near physiological concentrations (0.2U/mL and 1.845U/mL) of exogenous erythropoietin (EPO) to these established 3D CBMNC ex vivo cultures at day 14 in a serum-free and cytokine-free environment would be sufficient to enhance erythropoiesis. CBMNCs were separated by Ficoll-Paque density gradient and seeded onto collagen-coated PU 3D scaffolds at a cell density of 2.5×10⁶cells per scaffold (5×5×5mm³). Cultures were established in serum-free conditions and only EPO was added at days 14–28, with full-medium exchange every 2 days. Culture output was evaluated at days 14, 21 and 28 both by physically extracting cells from the scaffolds and by in situ analysis. Over 28 days, most stages of maturation, from erythroid progenitors to enucleated erythrocytes were observed by light microscopy of cytospins and by immunophenotypic analysis of extracted cells (CD45⁻/CD71⁺/CD235⁺), with more maturation occurring by day 28 of culture, after the addition of EPO. Although both concentrations of EPO produced comparable erythroid differentiation of cells, even by CFU assay, the viability (75% vs. 61%, p<0.05) and proliferative capacity at day 28 of culture was enhanced in the higher concentration of EPO compared with that in the lower concentration (p<0.05). In contrast, standard 2D control cultures (without serum or cytokines) collapsed within 5 days. In situ, scanning electron microscopy showed maturation of erythrocytes within central sections of the scaffolds to enucleation by day 28 and multiphoton microscopy confirmed the presence of structures resembling erythroid islands as early as day 14 of culture, prior to the addition of EPO. In conclusion, 3D PU-collagen scaffolds may provide a good model to study erythropoiesis ex vivo, using physiological concentrations of EPO, and has the potential to expand red cells in response to higher levels of exogenous EPO in a culture system that would be suitable for clinical applications.