Small-molecule consumption drives metabolic stress and restricts erythroblast expansion in high-density cultures

Transfusion-ready red blood cells can be cultured ex vivo from hematopoietic progenitors. Despite its promising outlook, a cultured transfusion unit cannot be produced at competitive costs. Large media volumes are required to maintain a maximum erythroblast cell density of 1-2.10 ⁶ cells/mL during the erythroblast proliferation stage. To identify the origin of the cell density limitation, we investigated the cellular support and metabolomic phenotype using different media formulations and feeding regimens. Media that were exposed to an increasing density of erythroblasts (termed spent media) displayed a proportional decrease in erythroblast proliferation support. A 1:1 combination of spent media with fresh media (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, γ-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.