Long-term survival, morphology and in vitro function of isolated pig islets under different culture conditions

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.