Background. Islet culture aims to optimize islet survival and to reduce islet immunogenicity. To achieve these objectives, culture periods at 37°C and 22-24°C are mainly used. Methods. This study compares the influence of donor age (juvenile vs. adult), temperature (22°C vs. 37°C), and serum supplementation (10% newborn calf serum [NCS] with 10% pig serum) on morphological integrity and in vitro function of porcine islets during long- term culture (LTC). Results. After 21 days at 22°C, the survival rate of cultured islets isolated from juvenile donors was lower than of adult islets (23±0.9% vs. 88±2.8%, P<0.001). Compared with 37°C, LTC at 22°C increased survival of adult islets and DNA recovery (92±2.5% vs. 45±4.8%, P<0.001; 72±4.1% vs. 30±5.1%, P<0.001) and reduced viability (62±8% vs. 89±5%, P<0.05). LTC at 22°C was associated with a reduction of insulin content (85±9 vs. 152±10 μU/islet equivalents [IEQ], P<0.01), 24 hr-insulin secretion (82±7 vs. 552±91 μU/day/IEQ, P<0.001), and integrated dynamic insulin response to glucose (1093±124 vs. 3074±708 μU/60 min/100 IEQ, P<0.05), compared with 37°C LTC. Histologic analysis revealed disintegration of islet periphery after 22°C, whereas smoothly shaped islets were present after 37°C LTC. Integrity after 14 days at 37°C was significantly better preserved when medium CMRL 1066 was supplemented with 10% porcine serum, compared with 10% NCS (40±2.3% vs. 21±6.7%, P<0.05), contrasting with 22°C (52±4.0% vs. 59±3.7%, not significant). Conclusions. This study demonstrates that survival of cultured porcine islets is increased at 22°C, whereas in vitro function and viability are better preserved at 37°C. Survival at 37°C can be improved by adding homologous serum to the medium.