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Life sciences · Journal article
Frontiers in Immunology · September 16, 2026
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Introduction Natural killer cells, as critical components of the innate immune system, have shown a promising potential in cancer immunotherapy. Currently, irradiated genetically modified feeder cells are widely used to produce large quantities of highly cytotoxic NK cells. However, it is frequently overlooked that NK cell products prepared in this way tend to form non-dissociable and macroscopic aggregates during storage and transportation, posing a significant challenge in clinical applications. The formation of cell aggregates not only compromises cell yield and quality but also raises concerns for vascular occlusion or unexpected immune-related toxicity upon infusion. Methods To address this issue, we systematically investigated key factors contributing to aggregate formation throughout the NK cell manufacturing process. Primary NK cells isolated from cord blood or peripheral blood were expanded using feeder cells. We evaluated the impact of harvesting parameters (centrifugal force, final cell concentration, filtration pore size, and human serum albumin concentration), anticoagulant supplementation, feeder cells, and plasma fibrinogen content on aggregate formation. Aggregate formation was monitored and quantified microscopically post-formulation. Meanwhile, NK cell quality attributes, including cell apoptosis, purity, proliferation and cytotoxic function, were assessed by cell counter and flow cytometry to confirm the quality of NK cell products. Results Our findings suggest that the coexistence of feeder cells and fibrinogen in the culture medium jointly induces aggregation in the final NK cell product. Replacing autologous plasma with a fibrinogen-free serum substitute can effectively prevent the aggregation, without impairing NK cell expansion fold, phenotypic stability, viability, or cytotoxic potency. Conclusions In summary, our study unveils the phenomenon and mechanism of cell aggregation in feeder-derived NK cell products, and establishes an optimized protocol for NK cell manufacturing, which supports the clinical feasibility of feeder cell-derived NK cell therapies.