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

Michael Smith Laboratories

Given the promising results from phase I/II clinical trials of regulatory T cell (Treg) therapy 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, we found that levels of expansion and viability varied with the cell density on the day of restimulation. Tregs restimulated at low cell densities (1x105 cells/cm2 ) initially had high growth rates, viability, and FOXP3 expression, but these parameters decreased with time and were less stable than in cultures of Tregs restimulated at high cell densities (5x105 cells/cm2 ), which had slower growth rates. High density expansion was associated with expression of inhibitory molecules, lower intracellular oxygen and extracellular nutrient concentrations, and extracellular lactate accumulation. Experiments to test the effect of low oxygen revealed that transient exposures to low oxygen levels had little impact on expansion, viability, or phenotype. Similarly, blockade of inhibitory molecules had little effect. In contrast, replenishing nutrients by increasing the feeding frequency between 2 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.

Article; Postprint

eng

Vancouver : University of British Columbia Library

10.14288/1.0447550

Applied Science, Faculty of; Medicine, Faculty of; Non UBC; Biomedical Engineering, School of; Chemical and Biological Engineering, Department of; Pathology and Laboratory Medicine, Department of; Surgery, Department of

10.1016/j.jcyt.2022.06.006

Faculty; Graduate

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