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Background Reproducible assessment of target-specific activation is necessary for a robust validation strategy for CAR-T products. Antigen-expressing cell lines are commonly used to assess target-specific activation, but are prone to phenotypic drift after multiple passages or selection events introducing or knocking out a target or reporter and can be time-consuming and expensive to maintain. TruCytes™ Potency cell mimics are a renewable, stable activation alternative for potency assay development and standardization.Methods Cell mimics were loaded with either CD19 or BCMA and an enhancer protein and lyophilized. Commercially purchased CD19- or BCMA- specific CAR-T and donor matched non-transduced T cells were co-cultured at defined E:T ratios and time points with mimics, cell lines, or target-coated microbeads. After indicated time points, supernatant was collected and assessed for interferon gamma (IFN-γ) or panel of pro-inflammatory cytokines by CBA to assess CAR-T activation.Results TruCytes Potency cell mimics loaded with CD19 or BCMA activate their respective CAR-T, inducing IFN-γ release as early as 4 hours and up to 48 hours after co-culture. Mimics induced activation superior to target-coated microbeads, and enhancer protein increased specific activation on par with commonly used cell lines, including Raji and Daudi for CD19 and H929 and MM1.S for BCMA. Unloaded mimics and enhancer-only mimics did not induce CAR-T activation above background. IFN-γ production was induced with E:T ratios between 5:1 to 1:5 for cognate CAR-T but not donor-matched non-transduced T cells, confirming specific activation. IFN-g quantities correlated to CD19-specific CAR-T+ frequencies between 20-60% CAR-T+ and to the level of CD19 loading. To demonstrate reproducibility, cryopreserved BCMA-specific CAR-T from one lot were cultured with BCMA-loaded mimics and H929 cell line in 5 independent experiments, generating inter-day%CVs for BCMA-loaded mimics of 5.0% (6 hours) and 11.2% (24 hours), compared to 19.5% (6 hours) and 16.5% (24 hours) for H929 cells.Conclusions TruCytes Potency cell mimics provide a robust, chemically-defined alternative to cell lines for potency assay development and implementation. They deliver target-specific activation across varied E:T ratios, CAR-T frequencies, and time points, with superior lot-to-lot and inter-day reproducibility. Their defined composition and renewable format make them a reliable tool for assessing specific activation of CAR-T cells for potency assay development and implementation.