Immune cell separation for cell therapy: A comprehensive review of techniques, and challenges

Cell-based immunotherapies, such as CAR-T and TIL therapies, represent a significant shift in the treatment of cancers and immune disorders. A critical first step in manufacturing these therapeutics is the efficient and selective isolation of specific immune cell populations (e.g., T cells, NK cells, and monocytes) from complex biological sources like peripheral blood or leukapheresis products. This comprehensive review systematically examines the current landscape of immune cell separation technologies that are essential for cell therapy manufacturing. We detail established methods, including density gradient centrifugation, magnetic-activated cell sorting (MACS), and fluorescence-activated cell sorting (FACS), as well as emerging techniques like microfluidics, acoustophoresis, and affinity ligand-based platforms. The review evaluates each technique based on key parameters: purity, yield, viability, processing time, scalability, cost-effectiveness, and compatibility with Good Manufacturing Practice (GMP) requirements. Furthermore, we emphasize the crucial role of rigorous post-separation characterization, which includes phenotypic analysis (flow cytometry), functional assessment (proliferation, cytokine release, cytotoxicity), and sterility testing to ensure product quality and safety. We thoroughly analyze significant challenges, such as the need for gentle processing to preserve cell function, achieving sufficient scalability for clinical and commercial production, minimizing process-induced cellular stress, reducing reagent costs (especially for antibody-based methods), navigating complex regulatory pathways, and establishing robust release criteria. This synthesis underscores that while considerable progress has been made, advancements in separation technologies, integrated characterization strategies, and standardized approaches are vital for overcoming existing bottlenecks and fully realizing the transformative potential of cell-based immunotherapies.