Abstract: The advent of reprogramming technologies has revolutionized stem cell research, offering unique opportunities in regenerative medicine and immunotherapy. Among the most promising applications is the scalable generation of functional human iPSC-derived CAR-macrophages (CAR-iMacs). However, achieving reliable, reproducible, and scalable production of iPSCs and their derivatives remains a significant challenge. Critical factors include accurate cell counting and analysis, as well as smooth and efficient processes for the expansion and differentiation of various stem cell populations including iPSCs. For instance, dissociating stem cells—from mild, cluster-preserving methods to more singularized and harsher treatments—poses significant challenges in consistently counting and analyzing cells using a unified method. Addressing these challenges is essential to ensure reproducibility and standardization in research applications. To tackle these issues, we developed a scalable workflow that integrates an advanced cell counting and analysis method, utilizing cell volume and size measurements, with a parallel bioreactor-based, three-dimensional suspension system for the continuous generation of CAR-iMac with a reproducible phenotype. This platform enables smooth handling of cells regardless of their origin or shape, allowing for efficient management of both homogeneous and heterogeneous populations. The advanced cell counting and analysis method provides a label-free approach to regularly monitor cell quality, such as population homogeneity, without requiring additional sample treatments. This significantly reduces workflow complexity, improves process efficiency, and enhances the overall reproducibility of experiments. This streamlined two-step workflow supports the reliable and reproducible continuous generation of CAR-macrophages using an easy-to-use bioreactor. By addressing critical bottlenecks in stem cell analysis and differentiation, our approach provides an innovative, scalable solution for advancing research in immunotherapy and regenerative medicine.
