Preparation of Highly Functional Spheroid of Endocrine Cells Based on Thermosensitive Glycol Chitosan.

BACKGROUND

Pancreatic islet transplantation holds great potential as a therapeutic approach for treating type 1 diabetes mellitus (T1D). However, large islets suffer from hypoxia due to the limited diffusion distance of oxygen, leading to cell loss. Therefore, smaller spheroids are needed for better transplantation outcomes. This study aims to develop a method for forming highly functional islet spheroids using glycol chitosan (GC) derivatives, such as N-acetylated glycol chitosan (AGC) and N-hexanoyl glycol chitosan (HGC).

METHODS

Thermogelling polymers were produced by performing N-acylation of GC using the correspondingly carboxylic anhydrides. Islet spheroids were formed using a dual application with AGC-coated plates and HGC gelation. The AGC solution was applied to the plate for coating and evenly distributed using a 1 mL syringe. Then, the HGC encapsulated with islet single cells was cultured on top of it. Spheroid viability and functionality were evaluated using CCK-8 assay and glucose-stimulated insulin secretion assay.

RESULTS

The aqueous solutions of AGC (4%, w/v) and HGC (36% hexanoylation) (2%, w/v) demonstrated a sol-gel transition temperature around 37 °C, suitable for the physiological environment. These polymers also showed no cytotoxicity to intact islets. Islet single cells were cultured on HGC gels with varying degrees of hexanoylation (DH) values, where higher DH values led to smaller and more uniform spheroids. The resulting spheroids formed on AGC-coated plates and HGC36 gelation were smaller and more uniform than those formed on untreated plates. These spheroids exhibited significantly improved glucose responsiveness, with superior insulin secretion.

CONCLUSION

The optimized method using AGC and HGC offers a more efficient way to produce smaller, uniform, and functional spheroids.