Segment Technology Manager- Cell Therapy Sartorius Stedim North America, Michigan, United States
Abstract: Human induced pluripotent stem cells (iPSCs) are pivotal for advancing tissue engineering and require scalable methods to generate billions of cells, particularly for wholly cellular bioinks used in bioprinting. Conventional 2D cell culture methods face challenges such as high costs, space requirements, and handling inefficiencies. To address these limitations, we developed a robust, scalable pipeline to produce hiPSC aggregates (hAs) using an automated stirred-tank bioreactor system (250 mL-1 L) for bioprinting applications. We employed a Design of Experiments (DoE) approach to optimize hA culture in 250 mL bioreactors, testing 15 conditions with varying impeller speed, seeding density, and polyvinyl alcohol use. Key metrics such as cell expansion, aggregate morphology, and pluripotency marker expression were measured for robustness across serial passages. The optimized protocol was scaled to a 1 L Biostat® bioreactor with continuous perfusion feeding. Harvested hAs were compacted into cellular bioinks and 3D bioprinted into collagen-Matrigel matrices, followed by differentiation into ectodermal, mesodermal, and endodermal lineages. At the 250 mL scale, we achieved a 23-fold cell expansion in 5 days, producing 1 billion hiPSC hAs with optimal sizes and diameters. Over 72% of hAs from the SCVI-15 line and 90% from the WTC-11 line had diameters ≤ 300 µm and circularity ≥ 0.73. Pluripotency marker expression remained above 90% across three serial passages in both cell lines. Scaling up to a 1 L stirred-tank bioreactor, the optimized parameters remained effective, achieving a ~20.4-fold cell expansion by day 4, yielding about 4 billion hiPSC hAs per vessel. Pluripotency marker expression exceeded 94%, with high cell density, viability, and circularity maintained for both cell lines. The 3D bioprinted tissues exhibited high post-printing viability and differentiated into vascular and neuroepithelial tissues. We successfully established a scalable hiPSC-to-3D bioprinting pipeline, enabling large-scale expansion and differentiation of iPSCs. The optimized suspension culture process supports billion-cell-scale production, with significant potential for therapeutic-scale tissue production and regenerative medicine application.
