In vitro drug screening for type 1 diabetes therapies has largely been conducted on human organ donor islets for proof of efficacy. While native islets are the ultimate target of these drugs (either in situ or for transplantation), significant benefit can be difficult to ascertain due to the highly heterogeneous nature of individual donors and the overall scarcity of human islets for research. We present an in vitro coculture model based on immortalized insulin-producing beta-cell lines with human endothelial cells in 3D spheroids that aims to recapitulate the islet morphology in an effort towards developing a standardized cell model for in vitro diabetes research. Human insulin-producing immortalized EndoC-βH5 cells are cocultured with human endothelial cells in varying ratios to evaluate 3D cell culture models for type 1 diabetes research. Insulin secretion, metabolic activity, live cell fluorescence staining, and gene expression assays were used to compare the viability and functionality of spheroids composed of 100 beta-cells, 1:1 beta-cell/endothelial, and 1:3 beta-cell/endothelial. Monoculture and βH5/HUVEC cocultures formed compact spheroids within 7 days, with average diameter 140μm. This pilot study indicated that stimulated insulin release from 0 to 20mM glucose increased from 8-fold for monoculture and 1:1 coculture spheroids to over 20-fold for 1:3 EndoC-βH5/HUVEC spheroids. Metabolic activity was also 12 higher in the 1:3 EndoC-βH5/HUVEC group compared to other groups. Stimulating monoculture beta-cell spheroids with 20mM glucose +1μg/mL glycine-modified INGAP-P increased the insulin stimulation index 2-fold compared to glucose alone. Considering their availability and consistent phenotype, EndoC-βH5-based spheroids present a useful 3D cell model for in vitro testing and drug screening applications.

Islet transplantation offers a long-term cure for Type 1 Diabetes (T1D), freeing patients from daily insulin injections. Therapeutic peptides have shown potential to increase the insulin output of pancreatic islets, maximizing the impact of grafted cells. The islet neogenesis-associated protein (INGAP), and its bioactive core (INGAP-P), stimulate beta-cell function and viability, offering the possibility for islet treatment prior to implant. However, dosing efficacy is limited by low circulation time and enzyme degradation. This proof-of-concept study presents the investigation of novel molecular variants of INGAP-P to find a more bioactive form. Custom-designed peptide variants of INGAP-P were synthesized and tested for their effect on the insulin secretion and gene expression of live human islets. We exposed the live islets of five donors to varying glucose concentrations with INGAP-P variants in solution. We identified four peptide variants (I9, I15Tyr, I19 and I15Cys) which displayed statistically significant enhancements over negative controls (representing a 1.6–2.8-fold increase in stimulation index). This is the first study that has assessed these INGAP-P variants in human islets. It highlights the potential for customized peptides for type 1 diabetes therapy and provides a foundation for future peptide-screening experiments.