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.