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

Abstract

Islet culture aims to optimize islet survival and to reduce islet immunogenicity. To achieve these objectives, culture periods at 37 degrees C and 22-24 degrees C are mainly used. This study compares the influence of donor age (juvenile vs. adult), temperature (22 degrees C vs. 37 degrees 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). After 21 days at 22 degrees 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 degrees C, LTC at 22 degrees 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 degrees C was associated with a reduction of insulin content (85+/-9 vs. 152+/-10 microU/islet equivalents [IEQ], P<0.01), 24 hr-insulin secretion (82+/-7 vs. 552+/-91 microU/ day/IEQ, P<0.001), and integrated dynamic insulin response to glucose (1093+/-124 vs. 3074+/-708 microU/60 min/100 IEQ, P<0.05), compared with 37 degrees C LTC. Histologic analysis revealed disintegration of islet periphery after 22 degrees C, whereas smoothly shaped islets were present after 37 degrees C LTC. Integrity after 14 days at 37 degrees 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 degrees C (52+/-4.0% vs. 59+/-3.7%, not significant). This study demonstrates that survival of cultured porcine islets is increased at 22 degrees C, whereas in vitro function and viability are better preserved at 37 degrees C. Survival at 37 degrees C can be improved by adding homologous serum to the medium.