M. Rook

Scalable stirred suspension culture for the generation of billions of human induced pluripotent stem cells using single-use bioreactors.

The production of human induced pluripotent stem cells (hiPSCs) in quantities that are relevant for cell‐based therapies and cell‐loaded implants through standard adherent culture is hardly achievable and lacks process scalability. A promising approach to overcoming these hurdles is the culture of hiPSCs in suspension. In this study, stirred suspension culture vessels were investigated for their suitability in the expansion of two hiPSC lines inoculated as a single cell suspension, with a free scalability between volumes of 50 and 2400 ml. The simple and robust two‐step process reported here first generates hiPSC aggregates of 324 ± 71 μm diameter in 7 days in 125 ml spinner flasks (100 ml volume). These are subsequently dissociated into a single cell suspension for inoculation in 3000 ml bioreactors (1000 ml volume), finally yielding hiPSC aggregates of 198 ± 58 μm after 7 additional days. In both spinner flasks and bioreactors, hiPSCs can be cultured as aggregates for more than 40 days in suspension, maintain an undifferentiated state as confirmed by the expression of pluripotency markers TRA‐1‐60, TRA‐1‐81, SSEA‐4, OCT4, and SOX2, can differentiate into cells of all three germ layers, and can be directed to differentiate into specific lineages such as cardiomyocytes. Up to a 16‐fold increase in hiPSC quantity at the 100 ml volume was achieved, corresponding to a fold increase per day of 2.28; at the 1000 ml scale, an additional 10‐fold increase was achieved. Taken together, 16 × 106 hiPSCs were expanded into 2 × 109 hiPSCs in 14 days for a fold increase per day of 8.93. This quantity of hiPSCs readily meets the requirements of cell‐based therapies and brings their clinical potential closer to fruition.

Media and microcarrier surface must be optimized when transitioning mesenchymal stem/stromal cell expansion to stirred tank bioreactors

Background The long-term outlook for regenerative medicine predicts an increased need for high quality materials that are compatible with the limited number of downstream processing steps required for cell-based therapies. Large scale manufacturing of adherent-dependent cell types necessitates movement away from planar culture and toward technologies such as stirred tank bioreactors where suspension culture using microcarriers is enabled [1]. Microcarriers are available in a variety of base materials including glass, polystyrene or dextran, and have been coated or derivatized to carry charge, peptides or extracellular matrix proteins such as collagen that may be animal-derived. Cell culture medium may also contain animal-derived components. Fetal bovine serum (FBS) in particular is associated with regulatory, supply, and consistency challenges [2]. Eliminating this commonly-used reagent will require thorough evaluation of animal originfree materials for compatibility with cell therapy applications. Here, we evaluated growth of human mesenchymal stem/stromal cells (MSCs) with a variety of microcarriers and cell culture media formulations. Not only was a wide range of performance observed between the microcarriers and media screened, but positive performance in static culture was not necessarily predictive of that under agitated conditions.

466. Manufacturing Solutions for Robust Cell Therapy Expansion and Harvest

The long-term view of regenerative medicine therapies predicts an increased need for expansion solutions that ease scalability, utilize animal origin-free materials and are compatible with limited downstream processing steps. As more cell therapeutics progress through clinical testing, current in vitro culture methods are proving cumbersome to scale and lack robustness. Moreover, high quality animal origin-free reagents and downstream processing devices support the future implementation of large scale manufacturing solutions that will be required following clinical success. Here, we describe the implementation of single use bioreactors and high quality media for expansion of cell therapies. We include examples from allogeneic mesenchymal stem cells and autologous T cells. The presentation will review solutions addressing animal origin-free expansion of cells within the context of different upstream process development steps as well as scaling and downstream processing with good cell quality, high recovery, high viability and good activity. Start to finish solutions for expansion and harvest, including high quality reagents, are key enabling technologies for success in commercializing cell therapies.