FIGURE 2.

Optimizing nTreg cell viability and recovery post thawing. (A,B) Different readout parameters before freezing (white) and at 0 h (blue bars) or 24 h post thawing (gray bars) are shown for the six freezing media as listed in Table 1 (n = 2 cell products produced under GMP-like conditions). (A) Viability (%): There is only a small difference in viability between fresh cells and all the other preparations directly after thawing, but strong decreases are found at 24 h depending on the approach, freezing medium with 10% DMSO content performed the worst, while addition of PEG brings only minimal improvement in viability at 24 h compared to the freezing medium with 5% DMSO content; (B) Cell Recovery (%): Analysis at 24 h time point gives a more informative readout on quantitative differences and best performance with 5% DMSO; and (C) Phenotypic and Functional Analysis: Shown are the results of phenotyping as well as cytokine secreting cells before freezing (fresh) and after cryopreservation (thawed) for the investigated 6 different freezing media for the two initial nTreg cell products. The green bars show the percentage of Treg cells (% CD4+CD25+Foxp3+), the red bars represent the percentage of interleukin 2 (IL2)-producing cells, and the yellow bars show the percentage of interferon gamma (IFN-ɣ)-secreting cells both expressed as % positive cells.