Upscaling ad differentiating human induced pluripotent stem cell spheroids in 3D

Background & Aim: The efficacy of current treatments targeting neurological diseases are affected by their inability to cross the blood-brain barrier. Innovative study models to circumvent this is critical. Human induced pluripotent stem cells (hiPSCs) are capable of indefinite cultivation and differentiation into any cell, including traditionally inaccessible cells critical for brain barrier function like astrocytes. hiPSCs thus provide a path for developing novel therapeutic products. The challenge in implementation is the critical need for billions of high-quality cells. Thus, we aimed to develop methods for large-scale hiPSC culture for subsequent differentiation to astrocytes in 3D. To scale-up hiPSC culture we have utilized the SCINUS Cell Expansion System (SCES); a semi-automated bioreactor offering lower costs, labor and shear stress than other agitating systems. Upscaled hiPSCs in suspension were then tested for their capacity to differentiate into astrocytes.

Methods, Results & Conclusion: hiPSCs were cultured as self-assembled spheroids in 6-well plates at a density of 1.5x105 cells/ml, maintained on an orbital shaker agitating at 70 rpm. Scaling up required determining critical culture parameters by imposing variations in refreshing regimes, seeding densities and agitation regimes. Morphological analysis, cell viability, and fold change, were used to evaluate favorable parameter combinations. Spheroid homogeneity and differentiation capacity was tested by inducing differentiation to astrocytes. hiPSC-derived neural progenitor cells (NPCs) were characterized using Nestin, Pax6 and FOXP2 staining.

The balance in cell density, medium refreshment and agitation regimes were improved during this study to maintain the growth and proliferation of hiPSC spheroids (Figure 1). Rocker speeds upwards of 400°/s were critical to control spheroid size ensuring nutrient availability until harvest. Results demonstrate robust cells with three SCES passages for the first time via Accutase dissociation. Spheroids were also successfully differentiated to astrocytes in suspension (Figure 2). hiPSC-derived NPC spheroid stains showed homogenous marker expression and indicated that spheroid size was adequate to maintain nutrient availability.

Overall, we were able develop a method to upscale hiPSC spheroid culture in a large-scale bioreactor for subsequent differentiation. This work offers promising methods for generating large quantities of cells needed for neurological therapeutic applications.