Autofluorescence imaging reveals the impact of cryopreservation on T cell metabolism and activation response.

Cryopreservation or the process of freezing cells is a cornerstone of most cell therapy protocols. Optimization of cryopreservation protocols and cryoprotectant agents to improve cell viability and functionality is under further investigation. However, the impact of cryopreservation on cellular metabolism and function immediately post-thaw is not fully understood. Here, we used label-free, non-invasive optical metabolic imaging (OMI) of NAD(P)H and FAD to characterize the activation response of frozen T cells from healthy donors and lymphoma patients post-thaw. Using OMI, we identified significant metabolic shift, along with delayed and diminished activation response, in healthy donor T cells throughout the first 4.5 h upon thawing. In cryopreserved peripheral T cells from lymphoma patients in our bispecific CD20/CD19 CAR T clinical trial, OMI could identify early metabolic stress and allowed gating of metabolically fit cells associated with post-thaw viability. Notably, in our pilot study involving four patients, metabolically fit T cells from complete responders exhibited metabolic responses to activating stimuli within the first 4.5 h post-thaw. Overall, our findings suggest that 4-5 h post-thaw is a critical time window to assess the impact of cryopreservation and thawing, supporting the potential of OMI to optimize cryopreservation protocols and evaluate patient T cell quality for cell therapy.