Defined Serum-Free Medium for Bioreactor Culture of an Immortalized Human Erythroblast Cell Line

Anticipated shortages in donated blood supply have prompted investigation of alternative approaches for in vitro production of red blood cells (RBCs), such as expansion of conditional immortalization erythroid progenitors. However, there is a bioprocessing challenge wherein factors promoting maximal cell expansion and growth-limiting inhibitory factors are yet to be investigated. The authors use an erythroblast cell line (ImEry) derived from immortalizing CD71+CD235a+ erythroblast from adult peripheral blood for optimization of expansion culture conditions. Design of experiments (DOE) is used in media formulation to explore relationships and interactive effects between factors which affect cell expansion. Our in-house optimized medium formulation produced significantly higher cell densities (3.62 ± 0.055) × 10⁶  cells mL^-1 , n = 3) compared to commercial formulations (2.07 ± 0.055) × 10⁶  cells mL^-1 , n = 3; at 209 h culture). Culture media costs per unit of blood is shown to have a 2.96-3.09 times cost reduction. As a proof of principle for scale up, ImEry are expanded in a half-liter stirred-bioreactor under controlled settings. Growth characteristics, metabolic, and molecular profile of the cells are evaluated. ImEry has identical O₂ binding capacity to adult erythroblasts. Amino acid supplementation results in further yield improvements. The study serves as a first step for scaling up erythroblast expansion in controlled bioreactors.