Physiomimetic Microwell Platform For Biomanufacturing Human Pancreatic Isletoids

Abstract

Diabetes mellitus is a chronic socioeconomic burden on a global scale, with 1 in 11 adults affected, and in response, efforts are underway to overcome this burden, utilizing isletoids (re-aggregated islet cells) and iPSCs for research or clinical transplantation. However, islets tend to be highly sensitive and vulnerable to stressors that occur in traditional culture. Additionally, due to their heterogeneous sizes upon isolation, larger islets tend to be susceptible to hypoxia and reduced compound diffusion, contributing to analytical inconsistencies. Thus, traditional culture often fails to maintain proper function and viability of these cells, while 3D microphysiological systems (MPS) provide a more natural environment, offering a potential avenue for optimal function and lifespan, as well as homogeneous sizing when used in conjunction with re-aggregation techniques. Therefore, the main objective is to provide a versatile, physiomimetic culture platform for generating and maintaining homogenously sized isletoids for long-term culture, reproducible analysis, and clinical use, expanding its use-case beyond disease modelling. A multilayer PMMA device incorporating a polystyrene through-pore microwell array created through a robust and reproducible workflow was utilized to aggregate dissociated primary human islets. Isletoid morphology was monitored via brightfield imaging, while viability was assessed with live/dead staining. Functional performance was evaluated through static glucose-stimulated insulin secretion (GSIS) assays. When seeded at 1,000 cells/well, the single cells aggregated into distinct, compact, spherical isletoids with a diameter of 148.4±1.6µm (similar to 1 IEQ (islet equivalent)). Viability and insulin stimulation index were both comparable to control islets on Day 5. By Day 10, isletoids had higher viability, while maintaining comparable function to controls. The physiomimetic aspect of this platform has also been enhanced with the addition of photomodulated ColMA hydrogel, validated to support higher viability and proliferation in both islets and iPSCs. These findings suggest that this platform is suitable for the aggregation and culture of homogeneously sized, spherical, viable, and functional isletoids and iPSC spheroids. With its capabilities, this platform should prove useful for the development, long-term maintenance, reproducible analysis, and experimentation with various cell types in addition to islets, especially for the generation of tissue-specific organoids, without the limitations of traditional culture.