Supplementary file 1_Fibrinogen depletion prevents non-dissociable cell aggregates of feeder-derived NK cell products.docx

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.</p>Results<p>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.</p>Conclusions<p>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.</p>