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bioRxiv · 10.1101/2025.11.14.688556

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

Pham, D. L.·Kalluri, M.·Weaver, C.·Hsu, A.·Gillette, A.·Zhao, W.·Kearl, T.·Hematti, P.·Shah, N.·Skala, M. C.
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Abstract

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 hours upon thawing. In cryopreserved peripheral T cells from lymphoma patients in our bispecific CD19/CD20 CAR T therapy 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, only metabolically-fit T cells from complete responders exhibited metabolic responses to activating stimuli within the first 4.5 hours post-thaw. Overall, our findings suggest that 4-5 hours post-thaw is a critical time window to assess the impact of cryopreservation and thawing, and support the potential of OMI to optimize cryopreservation protocols and evaluate patient T cell quality for cell therapy.

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Pham, D. L., Kalluri, M., Weaver, C., Hsu, A., Gillette, A., Zhao, W., Kearl, T., Hematti, P., Shah, N., Skala, M. C.. 2025-11-16. Autofluorescence imaging reveals the impact of cryopreservation on T cell metabolism and activation response. https://doi.org/10.1101/2025.11.14.688556

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