Plasma substitute combination enhances in vitro expansion of blood cells by modulating redox status and signaling pathways.

Plasma is the primary microenvironment, where red blood cells (RBCs) survive and function, with its components playing crucial roles in erythroid expansion and RBC functionality. This study aims to elucidate the relationship between the combination of critical components in plasma and the expansion and cell state of erythroid cells. Using Design of Experiment (DOE) methods, we screened and optimized the concentrations of plasma components that significantly impact the in vitro expansion of TF-1 cells. We identified a plasma substitute combination composed of hypoxanthine, dexamethasone, and vitamin B complex and, significantly enhancing TF-1 cell expansion in the serum-free medium supplemented with bovine serum albumin by 1012.41 folds, compared to 327.50 folds in the negative control. In addition, the proportion of CD34⁺ cells in the medium supplemented with this combination was 54.77%, comparable to the negative control, while hemoglobin expression was 0.64 pg/cell, significantly higher than that of the negative control. Given that various components of this formulation affect intracellular redox status and signaling pathway activation, we further investigated these aspects. Cells cultured with this combination showed improved mitochondrial membrane potential, lower intracellular reactive oxygen species (ROS) levels, reduced apoptosis rates, and enhanced STAT5 phosphorylation. These results indicated that the plasma substitute combination improves intracellular redox status and activates the JAK/STAT signaling pathway in TF-1 cells. This study provides valuable insights for developing serum-free media for the in vitro expansion of erythroid cells.