Figure 2. Cryopreserved MSC maintain immunosuppressive potential.

(A) Representative Western blot of IDO protein in fresh and cryo-MSC after exposure to IFN-γ for 24, 48, or 72 hours. β-actin provided as a loading control. (B) Representative Western blot of IDO in fresh and cryo-MSC after exposure to IFN-γ or TNF-α/IFN-γ for 48 hours. β-actin provided as a loading control. (C) IDO activity as measured by the concentration of kynurenine in the conditioned media collected from fresh or cryo-MSC exposed to IFN-γ or TNF-α/IFN-γ for 48 hours (mean ± SD, One-way ANOVA with Sidak correction for multiple comparisons, p < 0.05 considered significant, n = 6). (D) Example unstimulated and stimulated PBMC controls used for gating and setting the activation threshold. PBMCs stained with CFSE remain as a single population in unstimulated conditions but upon stimulation with CD3/CD28 dynabeads become activated and proliferate. (E) Example flow cytometry histograms of stimulated CFSE stained PBMCs co-cultured with fresh or cryo-MSC at MSC:PBMC ratios of 1:3, 1:6 or 1:12. (F) Quantification of the percent of activated PBMCs in each co-culture condition compared to unstimulated and stimulated controls. No statistical differences between fresh MSC and cryo-MSC at each ratio. (mean ± SD, One-way ANOVA with Sidak correction for multiple comparisons, p < 0.05 considered significant, 1:3 and 1:6 n = 4, 1:12 n = 3). All experiments performed with MSC from donors 8002L and 7083 at passages P3–P5.