(104b) A Physiologically Relevant Microenvironments for the Scalable Biomanufacturing Human Pluripotent Stem Cells

Qiang Li, Haishuang Lin, Ou Wang and Yuguo Lei

Department of Chemical and Biomolecular Engineering, University of Nebraska, Lincoln, Nebraska, USA

Introduction: Human pluripotent stem cells (hPSCs) are required in large numbers for various biomedical applications including cell therapies, tissue biofabrication, drug screening and toxicity tests. These applications require large numbers of high-quality cells. However, the scalable and cost-effective culturing of high-quality hPSCs and their derivatives remains very challenging. Here, we report a novel and physiologically relevant 3D culture system (called the AlgTube cell culture system) for hPSC expansion and differentiation. With this technology, hPSCs are processed into and cultured in microscale alginate hydrogel tubes (or AlgTubes) that are suspended in the cell culture medium in a culture vessel. The hydrogel tubes create cell-friendly microspaces that allow cells to interact with each other and expand. Meanwhile, they protect cells from hydrodynamic stresses in the culture vessel and confine the cell mass less than 400 µm (in radial diameter) to ensure efficient mass transport during the entire culture. Additionally, this technology is simple, scalable, defined and compatible with the current Good Manufacturing Practices that make it commercially viable. This system can be readily scaled to support research from basic biological study to clinical development and the future industry-scale production.

Materials and Methods: A custom-made micro-extruder was used to process AlgTubes. A hyaluronic acid (HA) or methylcellulose (MC) solution containing single cells and an alginate solution was pumped into the central and side channel of the home-made micro-extruder, respectively, and extruded into a CaCl₂ buffer (100 mM) to make AlgTubes. Subsequently, the CaCl₂ buffer was replaced by cell culture medium. For a typical cell culture, 20 µL of cell solution in AlgTubes were suspended in 2 mL E8 medium in a 6-well plate and cultured in an incubator with 5% CO₂, 21% O₂ at 37 °C. Medium was changed daily. To passage cells, medium was removed and alginate hydrogels were dissolved in 0.5 mM EDTA for 5 minutes. Cell mass was collected by centrifuging at 100 g for 5 minutes, treated with Accutase at 37 ºC for 12 minutes and dissociated into single cells for the following culture.

Results and Discussion: We demonstrated long-term culturing (>10 passages) of multiple hPSC lines without uncontrolled differentiation and chromosomal abnormalities. Cultures between batches and cell lines were very consistent. hPSCs in AlgTubes had high viability, growth rate (1000-fold/10 days/passage in general) and yield (~5x10⁸ cells/mL microspace). The expansion per passage (e.g. up to 4200-fold/passage was achieved) and volumetric yield are much higher than current 3D suspension culturing. In vitro embryoid body (EB) differentiation and in vivo teratoma formation confirmed their pluripotency after the long-term culture. All hPSCs were successfully differentiated into endodermal, mesodermal and ectodermal cells in the EB assay. All hPSCs formed teratomas containing the three germ layer tissues in immune-deficient mice in the teratoma assay. In addition, after long-term culture, all hPSCs retained normal karyotypes. These results show that AlgTubes can support long-term culturing of hPSCs. The conceptual and technical innovations of AlgTubes lead to high culture efficiency. In addition, AlgTubes-based scalable bioreactors could be readily built. Our comparative study showed the AlgTubes did not significantly alter hPSCs’ gene expression profiles, but significantly reduced cell death, resulting in high cell expansion and yield.

Conclusion: The AlgTube technology combines physiologically relevant culture microenvironments, high performance, high scalability, cGMP compliance and commercial viability, and has potential to address the hPSC manufacturing challenge. Advances in biology have developed protocols for efficiently differentiating hPSCs into many human cell types. Future research can explore integrating these protocols into AlgTubes to produce various human cell types. It will be valuable to systematically study culturing other cell types, such as human adult stem cells and T cells in AlgTubes.