Introduction: Advanced bioengineering in the generation of stem cell-derived islets (SC-islets) enabled clinical testing in patients. Due to planar (2D) or combined suspension (3D) culturing, optimized methods still have limitations in terms of preserving cell loss, minimizing heterogeneity (quality), and scalability (quantity). We developed a fully scalable suspension SC-islets biomanufacturing protocol using patients’ induced pluripotent stem cells (iPSC).

Methods: Combining optimal protocols, we performed modifications to generate iPSC- islets in complete suspension differentiation using a vertical-wheel® bioreactor system. Generated SC-islets were assessed for molecular (flow, histology, gene expression), electrophysiological, and functional (insulin secretion) characterization. In-vivo functional assessment for glucose control was evaluated post-transplantation in SCID-beige mice.

Results: We created a protocol for directed differentiation of patients’ iPSCs that resulted in functional, metabolically active, and transcriptionally mature SC-islets. In a single batch of small 0.1L and large 0.5L bioreactors, ~35,000 to 110,000 IEQ SC-islets are produced. Without modifying the cytoarchitecture of SC-islets, entire suspension method reliably differentiated cells and controlled dramatic cell loss (<50%) at all stages. Cell composition in SC-islets indicated a mixture of functional endocrine cells, including α cells (10% glucagon), β cells (42% insulin), γ cells (8% somatostatin). Single-cell visualization of SC-islet mass flowcytometry with viSNE and FlowSOM dimensional reductionality revealed >90% ChgA+NKx6.1+ cells, ~63% Cpep+ISL+NKX6.1+ β-like cells with fewer SLC18A1+ EC-like cells. Gene-array profiling reflected transcriptional maturity for islet-specific genes (INS, UCN3, NKX6.1, GP2, CHGB, MAFB, PAX4) and upregulated functional secretory and exocytotic genes (PCSK1/2, G6PC2, CHGB, CPE, GCK, ZnT8, TSPAN1). Dynamic insulin perifusion traces from SC-islets show a 2-fold increase in insulin secretion in response to glucose, a stimulator (exendin-4), and membrane depolarization. Fully mature S6-like cells exhibited Na+/Ca2+ current and exocytosis electrophysiological properties similar to donor islets. Finally, in diabetic mice (n=20), suspension-generated SC-islets provided physiological glucose control and functional regulation. Eight weeks post-transplantation, mice have improved glucose tolerance, graft maturity, and stimulated human C-peptide secretion. Maturation-associated genes like INS, GCG, MAFB, ZnT8, ISL1, PCSK1, ABCC8, ITGA1, and NEUROD1 were significantly upregulated in graft-harvested cells (120 days) compared to SC-islets pre-transplantation.

Conclusion: The development of a complete suspension differentiation protocol and further optimization to scale up SC-islet biomanufacturing has resulted in the large-scale generation of functional and mature SC-islets. Vertical-wheel® bioreactors provide a reliable source for clinical grade SC-islet cell production with high precision at large scale.