Production of cultured red blood cells (cRBCs) hold the promise of being a potentially unlimited source of cells that could cover the increasing demand of RBCs for transfusion purposes, while having more control on the quality and safety of the cells compared to the current donor-dependent system. cRBCs could also be used for novel therapies in which cells are used as carriers of therapeutic molecules. Scaling up cRBC manufacture is essential to produce the large number of cells needed for such applications. However, scaling up the current static culture systems for the production of erythroblasts (RBC precursor cells) would be prohibitively labor-intensive, requiring large volumes of medium and a high footprint. The work presented in this thesis aims to develop solutions to some of the key challenges in the scaling up of cRBC manufacture. Stirred tank bioreactors (STRs) are the standard for the large-scale production of biopharma therapeutics, including monoclonal antibodies and vaccines. Agitation in this type of reactors can reduce the concentration gradients of essential nutrients compared to static culture systems such as culture dishes. STRs also offer active control of critical operating parameters in the culture, such as dissolved oxygen concentration, pH and temperature. We therefore developed a culture protocol for the proliferation and differentiation of erythroblasts in STRs (Chapter 2). To define the operating conditions that sustain erythroblast proliferation in STRs, the effect of agitation, aeration strategy, and dissolved oxygen concentration was evaluated using 0.5 L STRs. Using this knowledge, the cultivation process could then be scaled up to 3 L bioreactors. Erythroblasts lose their replication capacity when transitioning from proliferation to differentiation culture conditions. Thus, efficient proliferation of erythroblasts is essential to produce the large number of cells required for cRBC manufacture. Growing erythroblasts under proliferative conditions is typically performed following a repeated-batch cultivation strategy, in which the culture is diluted every 24 hours with fresh medium to a fixed lower cell concentration. To reduce culture volumes, it is desirable to use higher cell concentrations. However, at increasing cell densities we observe a decrease in growth (Chapter 3). The observed growth limitations of erythroblast cultures at high cell densities appeared to be caused by depletion of low molecular weight nutrients (molecular mass <3 kDa) in the spent medium. We quantified consumption rates of amino acids, major contributors to biomass synthesis in proliferating mammalian cell cultures. Although the concentration of some amino acids decreases considerably over time, supplementation with additional amino acids did not improve growth. Following an untargeted metabolomics approach, we identified multiple pathways that indicate an excess of oxidative stress in erythroblast proliferation cultures. Perfusion proved to be a successful alternative cultivation strategy to overcome growth limitations due to depletion of nutrient components (Chapter 3). Increasing the maximum cell concentration in erythroblast cultures leads to an increase in the volumetric productivity (number of cells produced per reactor volume per culture time), which decreases the reactor volume needed to produce the same amount of cRBCs. However, large volumes of medium would still be required to sustain those cultures. Currently, the cost of culture medium for erythroid cultures makes cRBC manufacture economically unfeasible. Growth factors and proteins added to the medium are major contributors to the cost of the medium. Holotransferrin, an iron-carrying protein, is the main cost driver in erythroblast differentiation medium. We show that holotransferrin in erythroblast cultures can be replaced by a GMP-compatible iron chelator (deferiprone; Def), bound to ferric ion (Def3⋅Fe3+; Chapter 4) . Addition of Def3⋅Fe3+ to the culture medium resulted in similar final cRBC yields of cRBCs during proliferation and differentiation of erythroblast cultures compared to optimal holotransferrin concentrations. During differentiation, Def3⋅Fe3+ fully supported enucleation and hemoglobinization. We did not observe toxic effects of Def3⋅Fe3+. Finally, the main conclusions of this thesis are discussed, providing also an overview of the next developments that are required to make the production of cRBCs at large scale technically and economically feasible (Chapter 5). A multidisciplinary approach is needed to further reduce media cost, optimize medium composition to improve cell yields, and to improve the bioreactor culture system developed in this work. @en