Abstract: Hematopoietic stem cells (HSCs) are pivotal for off-the-shelf allogeneic immune cell therapies, including natural killer (NK) cells. While HSC expansion molecules like IBR403 enable CD34+ cell growth, conventional 2D cell culture methods are inefficient, costly, labor-intensive, and lack scalability, limiting large-scale clinical and commercial production. To overcome these challenges, we developed a robust, scalable, and high-throughput platform using automated stirred-tank bioreactors to expand cord blood derived HSCs (CBUs) and differentiate them into NK cells. Using a Design of Experiments (DoE) approach with MODDE® software, we optimized culture conditions by comparing GMP-grade media with RUO media, supplemented with IBR403 and key cytokines. 72 bioreactor conditions were tested evaluating perfusion rates (0.5-2 VVD) and cytokine concentrations (0-100 ng/mL) across multiple donors over 28 days. CD34+ cell expansion and differentiation were performed in the Ambr® 15 bioreactor system, with functionality and purity assessed for clinical relevance. HSCs cultured at 2 VVD perfusion rate produced an average of 5 × 10⁷ CD34+ cells per mL of input cord blood in GMP-grade media, compared to 1.4 × 10⁶ in RUO media. The DoE identified an optimal formulation that reduced costs by 50% while achieving >6 × 10⁷ CD34+ cells/mL of input cord blood. A high (2 VVD) perfusion rate was critical for maximizing CD34+ yields per mL of input CBU. Variations in CO2 (5% vs. 10%) and dissolved oxygen setpoints (15% vs. 30%) had no impact on CD34+ expansion. NK cells differentiated in bioreactors under optimal conditions showed >95% CD56+ expression (55-98% on Day 28) and >75% cytotoxicity against A549 cells at a 1:1 effector-to-target ratio, with donor variability impacting HSC expansion and NK differentiation. This study successfully optimized CBU-derived HSCs expansion and NK cell differentiation in GMP-grade media using the Ambr® 15 bioreactor system. By refining key process parameters, we achieved high-yield, cost-effective CD34+ cell expansion and scalable NK cell production. These findings establish a robust platform that transforms cord blood into a valuable resource for advancing cell therapies.
