Microencapsulation system for scalable differentiation of peripheral motor neurons from human induced pluripotent stem cells.

Stem cell-derived neural cells hold great potential for treating neurological disorders, but clinical translation is limited by the need for scalable, consistent, and functionally robust production systems. To address these challenges, we developed a microfluidic alginate encapsulation chip (MAEC) system for the high-throughput production of mature peripheral motor neurons from human induced pluripotent stem cells. Alginate was selected for its biocompatibility, low immunogenicity, and calcium-triggered gelation, enabling precise size control. Encapsulation conditions were optimized to produce uniform microcapsules, each containing a single embryoid body of defined size. A refined two-step purification strategy, combining on-chip mineral oil flushing and off-chip medium washing, efficiently removed cytotoxic oleic acid residues and significantly improved post-encapsulation cell viability. Encapsulated cells showed enhanced spontaneous differentiation capacity, and upon exposure to defined patterning cues, upregulated both early and terminal motor neuron markers. Extended cultures, both encapsulated and decapsulated, exhibited characteristic morphological and molecular features of mature motor neurons. Functional maturation was confirmed by whole-cell patch-clamp recordings, revealing repetitive spike firing and large-amplitude action potentials. The MAEC platform provides a scalable and immunoprotective system that supports stable encapsulation for transplantation and capsule-free release for downstream applications, enabling functionally relevant regenerative therapies and high-throughput drug screening and disease modeling.