J.S. Gallego Murillo
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6 records found
1
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.
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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.
Iron, supplemented as iron-loaded transferrin (holotransferrin), is an essential nutrient in mammalian cell cultures, particularly for erythroid cultures. The high cost of human transferrin represents a challenge for large scale production of red blood cells (RBCs) and for cell therapies in general. We evaluated the use of deferiprone, a cell membrane-permeable drug for iron chelation therapy, as an iron carrier for erythroid cultures. Iron-loaded deferiprone (Def3·Fe3+, at 52 µmol/L) could eliminate the need for holotransferrin supplementation during in vitro expansion and differentiation of erythroblast cultures to produce large numbers of enucleated RBC. Only the first stage, when hematopoietic stem cells committed to erythroblasts, required holotransferrin supplementation. RBCs cultured in presence of Def3·Fe3+ or holotransferrin (1000 µg/mL) were similar with respect to differentiation kinetics, expression of cell-surface markers CD235a and CD49d, hemoglobin content, and oxygen association/dissociation. Replacement of holotransferrin supplementation by Def3·Fe3+ was also successful in cultures of myeloid cell lines (MOLM13, NB4, EOL1, K562, HL60, ML2). Thus, iron-loaded deferiprone can partially replace holotransferrin as a supplement in chemically defined cell culture medium. This holds promise for a significant decrease in medium cost and improved economic perspectives of the large scale production of red blood cells for transfusion purposes.
Transfusion of donor-derived red blood cells (RBCs) is the most common form of cell therapy. Production of transfusion-ready cultured RBCs (cRBCs) is a promising replacement for the current, fully donor-dependent therapy. A single transfusion unit, however, contains 2 × 1012 RBC, which requires large scale production. Here, we report on the scale-up of cRBC production from static cultures of erythroblasts to 3 L stirred tank bioreactors, and identify the effect of operating conditions on the efficiency of the process. Oxygen requirement of proliferating erythroblasts (0.55–2.01 pg/cell/h) required sparging of air to maintain the dissolved oxygen concentration at the tested setpoint (2.88 mg O2/L). Erythroblasts could be cultured at dissolved oxygen concentrations as low as 0.7 O2 mg/ml without negative impact on proliferation, viability or differentiation dynamics. Stirring speeds of up to 600 rpm supported erythroblast proliferation, while 1800 rpm led to a transient halt in growth and accelerated differentiation followed by a recovery after 5 days of culture. Erythroblasts differentiated in bioreactors, with final enucleation levels and hemoglobin content similar to parallel cultures under static conditions.
Hypoxia is associated with increased erythropoietin (EPO) release to drive erythropoiesis. At high altitude, EPO levels first increase and then decrease, although erythropoiesis remains elevated at a stable level. The roles of hypoxia and related EPO adjustments are not fully understood, which has contributed to the formulation of the theory of neocytolysis. We aimed to evaluate the role of oxygen exclusively on erythropoiesis, comparing in vitro erythroid differentiation performed at atmospheric oxygen, a lower oxygen concentration (three percent oxygen) and with cultures of erythroid precursors isolated from peripheral blood after a 19-day sojourn at high altitude (3450 m). Results highlight an accelerated erythroid maturation at low oxygen and more concave morphology of reticulocytes. No differences in deformability were observed in the formed reticulocytes in the tested conditions. Moreover, hematopoietic stem and progenitor cells isolated from blood affected by hypoxia at high altitude did not result in different erythroid development, suggesting no retention of a high-altitude signature but rather an immediate adaptation to oxygen concentration. This adaptation was observed during in vitro erythropoiesis at three percent oxygen by a significantly increased glycolytic metabolic profile. These hypoxia-induced effects on in vitro erythropoiesis fail to provide an intrinsic explanation of the concept of neocytolysis.
Saccharomyces pastorianus lager brewing yeasts are domesticated hybrids of Saccharomyces cerevisiae and cold-tolerant Saccharomyces eubayanus. To understand the contribution of both parental genomes to maltose metabolism in brewing wort, this study focuses on maltose transport in the S. eubayanus type strain CBS 12357T/FM1318. To obtain complete sequences of the MAL loci of this strain, a near-complete genome assembly was generated using the Oxford Nanopore Technology MinION sequencing platform. Except for CHRXII, all sixteen chromosomes were assembled as single contigs. Four loci harboring putative maltose transporter genes (SeMALT1-4), located in subtelomeric regions of CHRII, CHRV, CHRXIII, and CHRXVI, were completely resolved. The near-identical loci on CHRV and CHRXVI strongly resembled canonical S. cerevisiae MAL loci, while those on CHRII and CHRXIII showed different structures suggestive of gene loss. Overexpression of SeMALT1-4 in a maltose-transport-deficient S. cerevisiae strain restored growth on maltose, but not on maltotriose, indicating maltose-specific transport functionality of all four transporters. Simultaneous CRISPR-Cas9-assisted deletion of only SeMALT2 and SeMALT4, which shared 99.7% sequence identity, eliminated growth of S. eubayanus CBS 12357T on maltose. Transcriptome analysis of S. eubayanus CBS 12357T established that SeMALT1 and SeMALT3, are poorly expressed in maltose-grown cultures, while SeMALT2 and SeMALT4 were expressed at much higher levels than SeMALT1 and SeMALT3, indicating that only SeMALT2/4 are responsible for maltose consumption in CBS 12357T. These results represent a first genomic and physiological characterization of maltose transport in S. eubayanus CBS 12357T and provides a valuable resource for further industrial exploitation of this yeast.