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Cryopreserved cell banks are fundamental for off-the-shelf mesenchymal stromal cell (MSC) therapies. However, cryopreserved MSCs are functionally impaired after thaw. Since metabolic fitness is linked to the functional potency of MSCs, we tested whether cultured (fresh) and thawed MSCs exhibit metabolic differences. Donor- and age-matched cultured and cryopreserved human umbilical cord or bone marrow MSCs were lifted or thawed respectively, suspended in xeno-free media, and cultured at 37C, 5% CO2. Mitochondrial morphology was assessed by live cell imaging of Mitotracker-stained cultures. Cellular energetics were quantified using a Seahorse metabolic analyzer after 6-, 24-, and 48-hours post-thaw. Thawed cells exhibited mitochondrial fusion and reduced networking, accompanied by reduced metabolic flux. These effects lasted 24-48 hours, depending on the donor. MSCs exposed to IFN-γ and TNF-α have increased potency, so we tested whether this strategy enhances MSC metabolism. Indeed, cytokine preconditioning shifted MSCs into glycolysis, independent of dose or time, and this effect was retained during cryopreservation and thaw. We next tested whether the glycolytic state of preconditioned MSCs improves their function. Fresh and thawed MSCs were challenged with IFN-γ and TNF-α and changes in functional biomarkers including IDO-1 quantified using RT-qPCR. Six and 24 hours after thaw, the preconditioned MSCs produced equivalent IDO-1 to fresh cells, suggesting that cytokine preconditioning may be a useful strategy to accelerate metabolic and functional recovery of cryopreserved MSCs. [ABSTRACT FROM AUTHOR]