hPIs Encapsulation and Culturing

Cell cultures were prepared within 24/48 h after receiving islets. hPIs were incapsulated inside hydrogels or plated in suspension in 48-well with two different type of culture media: MIAMI medium #1A (Mediatech-Cellgro, VA, United States) (a CMRL-based culture medium commonly used in clinical trial for islet culture before transplantation) or GF medium (a serum-free medium with bFGF and EGF growth factors). 25 IEQ (low density) or 500 IEQ (high density) were incapsulated inside SAPs, previously dissolved in distilled water and diluted with 584 mM sucrose solution (ratio 1:1). A droplet of 25 µL was placed onto glass coverslip in 48-well, medium was added to start SAP gelation and to obtain free-floating samples. Same concentration of islets was used for samples in suspension in 48-well. In these conditions, the samples were maintained in culture up to 14-days (T14) and 28-days (T28) at 24°C, 20% O₂, 5% CO₂ in a humidified atmosphere. As positive control, suspensions of islets in MIAMI medium or GF medium were maintained in culture for 1 day (T1). hPIs incapsulated insides hydrogel were monitored individually during culture time and brightfield images from day 1 to day 28 were acquired weekly via Zeiss light microscope at ×5 magnification.