Use of a New 3D Pluripotent Stem Cell Suspension Culture Medium for Rapid Scale-Up and Downstream Differentiation of Pluripotent Stem Cells

Date:  April 20, 2021

Time: 10:00am PDT

In order to generate sufficient numbers of pluripotent stem cells (PSCs) for downstream applications, three-dimensional (3D) suspension cultures offer key advantages over two-dimensional (2D) adherent cultures. Suspension cultures are more cost-efficient and consume less plastics to grow the same number of cells in adherent cultures. However, existing solutions may be unable to fully utilize the potential of a suspension culture system. The protocols can be cumbersome and result in low fold-expansion per passage. To address these deficiencies, we recently developed a new 3D suspension culture medium – Gibco™ StemScale™ PSC Suspension Medium. StemScale medium utilizes simplified media exchanges and passaging schedules for an easy-to-use protocol. Furthermore, it enables singularized PSCs (seeded at 150,000 cells/mL) to form self-aggregating spheroids with robust expansion. Spheroids remain viable and pluripotent when grown 4 – 5 days per passage. StemScale medium maintains a consistent growth rate from small-scale orbital shaker platform cultures (well plates and shake flasks) to large-scale bioreactor cultures (3L volumes). We also demonstrate that cells obtained from dissociated spheroids can be reseeded into 3D suspension cultures, cryopreserved as single cell suspensions, or taken through downstream differentiation protocols. To demonstrate the potential of expanded PSCs in suspension with downstream applications, we have developed protocols enabling efficient and scalable neural and cardiomyocyte differentiation in 3D culture using reagents originally designed for monolayer culture. Key parameters and considerations have been identified and optimized for suspension culture differentiation from StemScale PSC spheroids. This approach has yielded wide-ranging benefits including faster differentiation and expansion steps, scalable expansion of neural stem cells (NSCs) and neural progenitor cells, and earlier onset of neuronal maturation and functional activity.

Learning Objectives:

• Discuss and learn how to achieve a high pluripotent stem cells (PSCs) yield (5 – 10 fold expansion per passage) in suspension culture.
• Describe and understand how to optimize PSC spheroid growth in various suspension culture vessels and bioreactors.
• Discuss and learn how to prepare and use 3D spheroids in downstream differentiation applications.

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