Transfusion-ready red blood cells (RBC) can be cultured ex vivo from hematopoietic progenitors. Despite its promising outlook, a cultured RBC transfusion unit cannot be produced at competitive costs. Large volumes of medium are required to maintain a maximum erythroblast cell density of 1-2.106 cells/mL during the proliferation stage of the culture. To identify the origin of the cell density limitation, we compared the growth support and changes in the cellular metabolomic signature while using different media formulations and feeding regimens. Media that were exposed to an increasing density of erythroblasts (here called spent media) displayed a proportional decrease in their ability to support erythroblast proliferation. A 1:1 combination of spent media with fresh medium (not previously exposed to the cells) restored growth for all tested conditions. Filtering both fresh and spent media with a 3 kDa cut-off filter, and subsequent recombination of the two fractions, indicated that exhaustion of the small molecular weight fraction (<3 kDa) was primarily responsible for growth limitation. We performed targeted and untargeted metabolomics analysis, for both the intra- and extracellular compartments, following seeding in fresh medium (12, 24, 36 h). We observed degradation of nucleosides, depletion of amino acids, and a decrease in intermediates of the glutathione-ascorbate, gamma-glutamyl and cysteine-methionine cycles. The latter compounds suggested an increase in oxidative stress in high density erythroblast cultures. Elimination of nucleosides from the medium led to a lower accumulation of purine salvage intermediates, and a 30% increase in cell productivity. In conclusion, we demonstrate that high-density erythroid cultures are subject to metabolic stress, defining critical constraints for scalable culture expansion.
Gallego-Murillo, J. S., van Lakwijk, I., Yagci, N., Reisz, J. A., Pozo Garcia, V., D'Alessandro, A., van der Wielen, L. A. M., von Lindern, M., Wahl, S. A., Van den akker, E.
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