There is a constant worldwide need for blood products, traditionally obtained from donations. In vitro red blood cell (RBC) production could supplement this demand and offer benefits such as thorough screening for improved safety, the possibility of genetic manipulation to restore genetic deficiencies, and therapeutic loading. Induced pluripotent stem cells (iPSCs) are a promising cell source for transfusable RBCs due to their immortality and independence from donors. However, current iPSC differentiation protocols—including both monolayer and embryoid body-based systems—have failed to produce sufficient erythroid cells (1011-12 per unit) for therapeutic application, primarily due to developmental immaturity, inefficient enucleation (5–25%), and suboptimal, static culture conditions lacking physiological relevance. This study describes the optimization of an iPSC to RBC differentiation platform and its step-by-step translation process to dynamic culture conditions, allowing scalability and eventual bioreactor application. The optimized dynamic culture yields ≈4.6 × 103 RBC/iPSC, requiring an estimated ≈4.9 × 107 iPSCs to produce a minitransfusion unit, achieving a consistent 40–70% enucleation rate and bona fide function, demonstrated by both in vitro and in vivo assays. Our feeder-free, GMP-compatible system accomplishes an enucleated RBC production rate sufficient for large-scale application and serves as a bridge to large-scale bioreactor RBC production, facilitating clinical application. ... Read more

Cell therapies based on inducible pluripotent stem cells offer promising new treatments for a variety of different illnesses. However, the sensitivity of stem cells to hydrodynamic stress makes developing reliable stem cell production processes challenging. Understanding hydrodynamic stress conditions experienced by stem cells during early-stage process development is important to guide scale-up and design scale-down experiments. We characterize the hydrodynamic stresses in a 125 mL shake flask using Lattice-Boltzmann implicit large eddy simulations (LB-ILES). First, we validated the LB-ILES shake flask simulations using volumetric power input measurements and experimental liquid distribution data showing good overall agreement, while also numerical challenges of the LB-ILES method regarding grid and time step dependencies are discussed. The mean shear stress in the shake flask increases from 0.01 to 0.24 Pa when increasing the shaking frequency from 55 to 250 rpm, and the mean Kolmogorov length scale decreases from 185 to 51 μm. Furthermore, time-averaged distributions of the shear stress and Kolmogorov length scales were evaluated and compared to reported stress thresholds for stem cells. Based on the shear stress and Kolmogorov length scale distributions, our developed shake flask CFD model can help to design small-scale experiments to characterize stem cell cultures in terms of their hydrodynamic stress tolerance, and ultimately guide scale-up stem cell cultures to larger cultivation systems. ... Read more