Microfluidic Hydroporation Platform for Effective Allogeneic CAR‐T Cell Production and Subsequent Functional Cytotoxicity Analysis

ABSTRACT Chimeric antigen receptor (CAR)‐T cell therapy demonstrates high efficacy in hematologic malignancies; however, its clinical utility is constrained by patient T cell quality and manufacturing complexities. Allogeneic donor‐derived CAR‐T cells offer a scalable alternative, yet safe and efficient T cell engineering, extension to solid tumors, and identification of highly cytotoxic donors remain challenging. To address these limitations, we developed a microfluidic hydroporation platform to advance allogeneic CAR‐T cell‐based immunotherapy. Using this system, CRISPR/Cas9 ribonucleoproteins targeting TRAC and B2M achieved ∼49% knockout efficiency, and mRNA delivery resulted in >70% anti‐CD19 CAR expression. Hydroporation‐engineered CAR‐T cells exhibited potent antigen‐specific cytotoxicity and IFN‐γ secretion against CD19 ⁺ leukemia cells. Extending the platform to solid tumors, anti‐HER2 CAR‐T cells infiltrated and disrupted HER2 ⁺ breast cancer spheroids, demonstrating efficacy in a solid tumor model. Functional testing across multiple donors revealed substantial variability in cytotoxicity despite comparable CAR expression, indicating that receptor density alone is insufficient to predict potency. Machine learning analysis identified expansion rate, CD8 ⁺ enrichment, and viability as stronger determinants of functional efficacy. These findings establish hydroporation as a safe, efficient, and scalable platform for ex vivo CAR‐T cell engineering, and combined with predictive analytics, provide a foundation for effective allogeneic CAR‐T cell therapies.