玻璃化法冷冻保存对人胚胰岛的影响

Cryopreservation of human fetal islets by vitrification

目的:观察玻璃化法对人胚胰岛冻存复苏后形态功能的影响.方法:取胎龄18-28wk中期水囊引产死胎,用胶原酶分离胰岛后,分别采用传统微机程控缓慢降温和玻璃化超快速降温方法冷冻保存胰岛,复苏后,从胰岛形态、超微结构、胰岛素分泌功能方面比较两种冻存方法的效果.结果:两种方法冷冻保存的胰岛,复苏后形态保持完整,电镜下胰岛B细胞分泌颗粒和线粒体丰富,线粒体嵴规整,胰岛素释放试验中,二者的胰岛素水平与冻存前无明显差异(P>0.05),胰岛素基因表达水平与冻存前相似.结论:玻璃化法冷冻保存胰岛能够维持细胞的正常结构和功能,其冷冻效果与微机程控缓慢降温相比无明显差别.

AIM： To evaluate the efficiency of cryopreservation of human fetal islets using vitrification. METHODS： Pancreatic islets of 18-28 weekfetus died from water bag were harvested by collagenase digestion. Islets were cultured and cryopreserved by computer-programmed slow freezing and vitrification, respectively. The morphology and ultrastructure of the thawed islets were studied in the two groups. The function of the thawed islets was assessed by glucose-stimulated release of insulin, which was measured by radioimmunoassay. The expression of insulin mRNA in the thawed islets was detected by reversed transcription polymerase chain reaction （RT-PCR）. RESULTS： Morphological studies of the thawed islets demonstrated that both cryopreservation methods retained the integrity of islets. Secretary granule and mitochondria in beta cells were abundant. Analysis of basal insulin secretion and the stimulation release in the two groups showed no significant difference from pro-cryopreservation （both P 〉 0.05）. CONCLUSION： The integrity and function of islets can be retained by vitrification, whose efficiency has no significant difference from that of computer-programmed slow freezing.