Feeder-cell-free system for

Introduction: Natural Killer (NK) cells hold significant promise as therapeutic agents in immuno-oncology due to their ability to target and eliminate cancerous and infected cells without causing graft-versus-host disease or cytokine release syndrome. However, the limited availability of robust, scalable methods for generating clinical-grade NK cells remains a limiting factor to broader clinical application. Methods: Here we report the development of a novel feeder-cell-free culture system optimized for producing NK cells from cord blood-derived CD34 Results: Flow and mass cytometric analysis confirmed the expression of activating receptors, transcription factors (ID2, T-bet) and cytotoxic molecules (perforin, granzyme A/B), all essential for ProT-NK cell functionality. These cells are in an immature state, indicated by the absence of maturation markers (CD16, KIRs). Functional assays demonstrated that these ProT-NK cells are capable of degranulation and cytokines production (TNFα) upon stimulation with K562 target cells and showed cytotoxicity against K562 cells superior to that of Peripheral Blood (PB)-NK. In NSG-Tg(hIL-15) mice, ProT-NK cells colonize bone marrow, the liver, and the spleen and persist and mature in bone marrow for at least 9 days post-injection. Compared to ProT-NK D21, ProT-NK D14 was superior in functional and homing potential. Discussion: Our ex vivo culture process supports scalable ProT-NK cell production in high yields, reducing dependency on feeder cells and mitigating contamination risks. Our findings demonstrate the feasibility of generating large, functional NK cell populations from HSPCs isolated from readily available cord blood sources and offer an efficient alternative to PB-NK cell therapies.