Optimized Media and Workflow for the Expansion of Human Pluripotent Stem Cells As Aggregates in Suspension Cultures

3D suspension culture enables the efficient and cost-effective scale-up of human pluripotent stem cell (hPSCs) manufacturing. However, the current practice of using media optimized for 2D adherent cultures directly in 3D suspension culture systems, can lead to low volumetric productivity and inefficient workflow. To overcome these limitations we developed mTeSR^TM3D, a defined medium based on mTeSR^TM1, and novel protocols for fed-batch culture of hPSC aggregates. Human embryonic stem cell (hESC) lines (H1 or H9) or human induced pluripotent stem cell (hiPSC) lines (WLS-1C or STiPS-M001) that were previously maintained in 2D mTeSR^TM1 culture were seeded into multiple suspension culture vessels (e. g. shaker bottles, spinner flasks and bioreactors) containing mTeSR^TM3D Seed Medium plus 10 μM Y-27632 ROCK inhibitor. 3D cultures were maintained using either daily 50% mTeSR^TM1 medium exchanges (control) or using a fed-batch protocol whereby the culture medium was supplemented daily with mTeSR^TM3D Feed Medium. After 3 or 4 days in suspension culture, aggregates were harvested, dissociated into small clumps with Gentle Cell Dissociation Reagent (GCDR) or single cell suspensions enzymatically, and re-seeded in mTeSR^TM3D Seed Medium plus 10 μM Y-27632. Passaging and feeding cycles were repeated for at least 5 passages. 3D cultures were assessed for growth, viability, hPSC marker expression, in vitro differentiation potential, and karyotype. In addition, media was analyzed for molar glucose to lactate yield to characterize metabolism. By day 4, aggregates cultured in mTeSR^TM3D typically grew to a mean diameter of 350 μm, with a 5-fold increase in cell number. Image analysis was routinely performed to estimate aggregate size during growth. Using mTeSR^TM3D, up to 10⁹ cells can be produced starting from a single 6-well plate within 2-3 weeks representing a greater than 500-fold expansion. hPSC cultures maintained in mTeSR^TM3D differentiated into all 3 germ layers with high efficiency. The average volumetric productivities were 0.7, 3.1 and 6.9 (x10⁵) viable cells / mL in 2D, with daily 50% media exchange, and mTeSR^TM3D fed-batch protocols, respectively. Using the GCDR clump passaging protocol, mTeSR^TM3D cultured hPSCs retained normal karyotypes. Culture performance evaluated in shaker bottles, spinner flasks and bioreactors was comparable, withtypical growth rates in the order of 1.5-fold expansion per day, confirming straightforward widespread scale-up and applicability. Metabolic activity as assessed by the moles lactate produced to glucose consumed was 1.7, consistent with a primarily glycolytic metabolism. Adaptation times for hPSCs transitioning from 2D to 3D aggregate culture varied with different hPSC lines with typically one passage in 3D suspension culture conditions required before a consistent and high expansion passage over passage was obtained. Additionally, protocols were developed for use on a Hamilton® robotic platform for reproducible, matrix-free, high-throughput hPSC suspension culture at a small scale (96-well format). mTeSR^TM3D enables efficient scale-up and scale-down of hPSC cultures with a greatly simplified workflow.