Fig. 5

Comparison of lyophilized and cryopreserved CM on T-cell subset polarization and on Tregs. Allogeneic PBMC were stimulated with anti-CD3 mAb or in mixed T lymphocyte reaction (MLR-T) in the presence of three different concentrations of cryopreserved or lyophilized CM, 100 µL (50% of final volume), 50 µL (25%) or 10 µL (5%), from hAMSC at passage 0 (CM-hAMSC p0) or passage 2 (CM-hAMSC p2), or CM from hAM collected at 5 days after culture (CM hAM D5). A Different Th subsets were analyzed by flow cytometry at day 7 and expressed as a percentage of CD4+CD45RA− cells gated for Th1 (CD183+CD196−), Th1/Th17 (CD183+CD196+) and Th2 (CD183−CD196−CD194+) (in presence of two different concentrations of cryopreserved or lyophilized CM, 100 µL (50% of final volume) or 50 µL (25% of final volume), from hAMSC at passage 0 (CM-hAMSC p0) or passage 2 (CM-hAMSC p2), or CM from hAM collected at 5 days after culture (CM hAM D5). B Induction of Treg was evaluated by flow cytometry after six days of co-culture in allogeneic PBMC stimulated with anti-CD3 mAb (upper panel) or in mixed T lymphocyte reaction (bottom panel), in presence of 3 concentrations of cryopreserved or lyophilized CM, 100 µL (50% of final volume) or 50 µL (25% of final volume), or 10 µL (5% of final volume), and displayed as a percentage of CD45RA − FoxP3⁺CD25^hi cells. Results are represented as violin plots showing median (thick line), 25th and 75th quartiles (*p < 0.05, **p < 0.01, ****p < 0.0001 versus control PBMC + antiCD3 or T cells + PBMC*), N ≥ 3 individual experiments