Consequences of adjusting cell density and feed frequency on serum-free expansion of thymic regulatory T cells

Given the promising results from phase 1/2 clinical trials of therapy involving regulatory T cells (Tregs), it is critical to develop Treg manufacturing methods that use well-defined reagents.Seeking to maximize expansion of human thymic Tregs activated with anti-CD3/CD28 antibody-coated beads and cultured in serum-free medium, the authors investigated the effect of adjusting process parameters including cell density and cell concentration, and feeding strategy on Treg yield and quality.The authors found that levels of expansion and viability varied with cell density on the day of restimulation. Tregs restimulated at low cell densities (1 × 105 cells/cm2) initially had high growth rates, viability and FOXP3 expression, but these parameters decreased with time and were less stable than those observed in cultures of Tregs restimulated at high cell densities (5 × 105 cells/cm2), which had slower growth rates. High-density expansion was associated with expression of inhibitory molecules and lower intracellular oxygen and extracellular nutrient concentrations as well as extracellular lactate accumulation. Experiments to test the effect of low oxygen revealed that transient exposure to low oxygen levels had little impact on expansion, viability or phenotype. Similarly, blockade of inhibitory molecules had little effect. By contrast, replenishing nutrients by increasing the feeding frequency between 2 days and 4 days after restimulation increased FOXP3, viability and expansion in high-density cultures.These data show the previously undescribed consequences of adjusting cell density on Treg expansion and establish a Good Manufacturing Practice-relevant protocol using non-cell-based activation reagents and serum-free media that supports sustained expansion without loss of viability or phenotype.