Introduction: Neonatal porcine islets (NPIs) represent a promising alternative source for curing type 1 diabetes. The neonatal pancreas is still immature, and NPIs isolated from it need to be differentiated in vitro before transplantation. However, there is no consensus on the optimal age of donors and the duration and conditions of in vitro culture. Knowledge of the molecular and functional properties of NPIs from commonly used protocols is a prerequisite for further targeted improvements.

Methods: Two NPI culture media were selected for in vitro maturation of NPIs isolated from 3-5-day old piglets. Culture condition A is based on Ham's F10 adjusted to 10 mM glucose, and supplemented with BSA, nicotinamide, glutamine, CaCl2, and IBMX (Ellis et al.). Condition B is for the first 3 days Ham's F12/M199 supplemented with nicotinamide, glutamine, zinc sulfate, and vitamins (imitating CMRL1066 media), and further BSA, GLP1 analogs, insulin, and protease inhibitors (Kemter et al.), followed by Ham's F10-based medium from day 3 on. Each NPI isolate was divided and examined under both culture conditions. Characterization of marker expression by flow cytometry was performed on day 1, 3, and 9. Dynamic glucose-stimulated insulin secretion (GSIS) was performed on day 10. Further parameters such as islet mass and morphology were also monitored.

Results: Flow cytometric analysis revealed an increase in viability and in the ratio of insulin-, glucagon-, somatostatin-, PDX1- and Nkx6-1-positive cells. While the ratio of insulin-positive cells with high granularity increased, the expression levels of hormones and beta cell identity markers initially dropped from day 1 to day 3, but increased to culture day 9. Islet mass continuously decreased, particularly during the first 3 days of culture. Mass loss within the first 3 days after isolation was distinctly increased in media condition A but associated with higher viability and a lower proportion of insulin-positive cells within the dead cell population at culture day 1. NPIs cultured in media condition A exhibited better biphasic insulin secretion upon high glucose concentrations. Insulin response to high glucose with forskolin was distinctly stronger, without difference between media conditions due to high isolate-to-isolate variability.

Conclusion: Media conditions affect each parameter of NPI-characterization over time, with distinct isolate-to-isolate variability. Over culture time, enrichment of endocrine cells and recovery of marker expression levels support the maintenance of islet cell identity. For more detailed molecular characterization of NPIs during in vitro maturation, scRNAseq analysis is ongoing.