3D Expansion of Induced Pluripotent Stem Cells

Abstract

Human induced pluripotent stem cells (iPSCs) have numerous applications in drug discovery, drug safety assays, in vitro disease modeling, and cell-based regenerative therapy since iPSCs are capable of becoming specialized cells. Therefore, there is a great need in industry to generate a cost-effective, scalable, and robust process to increase the number of iPSCs while maintaining their undifferentiated state. The current standard of growing iPSCs in 2D culture is space, labor, and cost intensive; 3D suspension culture is a viable alternative to grow iPSCs, with benefits such as homogenous culture conditions, automation, monitoring, and feedback-based control. The two principal ways of growing iPSCs in suspension are as aggregates or on microcarriers (MCs). For aggregates, medium, seeding density, agitation and passaging method were evaluated: in a 125 mL shaker vessel, 1-3e5 cells/mL at 70 RPM was found to be best for STEMCELL Technologies 3D media. Three methods of aggregate passaging –strain, settle and centrifuge– were assessed with the goal of reducing cell loss while maintaining characteristic morphology. Strain method lost ~15-20% of cells while settle lost <10% of cells with each passage; centrifuge method was discontinued since keeping free cells in culture had a negative impact on morphology. For the iPSCs tested, it was found that it may be necessary to coat MCs with a matrix for improved cell attachment, however, preparation of these MCs sometimes resulted in shattering or improper hydration, leading them to float. Furthermore, challenges were encountered when counting the cells on MCs. The aggregate expansion system described has the potential to grow iPSCs to clinically relevant quantities.