Micropocket-Based Differentiation System to Streamline and Scale Stem Cell-Derived Pancreatic Islet Production

Stem-cell-derived islets (SC-islets) offer an alternative to cadaveric islets for Type 1 diabetes treatment. Controlling aggregate size during differentiation can improve reproducibility and performance, but current protocols are challenging to scale while maintaining control over this important parameter. We first establish that due to stochastic fusion, SC-islet sizes produced with conventional protocols can create chronic oxygen limitations during differentiation. We then propose and demonstrate the use of a micropocket hydrogel platform to facilitate the handling and long-term culture of separated cellular aggregates during SC-islet production. Micropockets are formed with an overhanging lip-and-funnel geometry, which allows single cells to easily enter the aggregation chamber while preventing aggregates from escaping during media exchanges. We design and fabricate these micropockets in inert polyacrylamide hydrogels and demonstrate that this design protects the aggregates from shear stress (∼50× reduction) during media exchanges. We also compare pyramidal, conical, and spherical micropocket chamber geometries to optimize the aggregate formation. Pancreatic progenitor cells were aggregated and differentiated into SC-islets over 23 days, during which aggregate sizes consistent with human islets were maintained (136 μm, ±31 μm SD). In contrast, aggregates produced in suspension culture increased in size throughout differentiation (from 114 μm ± 8 μm SD to 275 μm ± 62 μm SD) due to stochastic aggregate fusion. Furthermore, no aggregate losses during daily media exchanges were incurred in micropockets, suggesting a highly scalable approach to produce appropriately sized islets while maintaining precise control over microenvironmental conditions. Overall, this work demonstrates the utility of a single-step culture system to form and maintain aggregates during lengthy differentiation processes, which can ultimately be scaled for therapeutic production applications.