González-Clemente J M, Goyti C, Mendola J, Conget J I, Casamitjana R, Gomis R, Vilardell E
Endocrinology and Nutrition Unit, Hospital Clínic, School of Medicine, University of Barcelona, Spain.
Metabolism. 1997 Jun;46(6):644-9. doi: 10.1016/s0026-0495(97)90007-x.
Cryopreservation is an effective method of islet storage and may facilitate clinical trials of islet transplantation. It was the aim of the present study to evaluate the in vitro viability of cryopreserved rat islets, including the response to nonglucose secretagogues and glucose oxidation. After pancreatic digestion via intraductal injection of collagenase, 75- to 200-micron Wistar rat islets were handpicked and cultured in RPMI 1640 (glucose 11.1 mmol/L) and randomized into two groups: control (cultured 20 to 24 hours at 37 degrees C) and cryopreserved (after 20 to 24 hours of culture at 37 degrees C, islets were cryopreserved according to Rajotte's protocol: freezing velocity, -0.25 degree C/min; thawing velocity, 200 degrees C/min). In the two groups, we evaluated recovery, insulin content per islet, staining viability (ethidium bromide/orange acridine; semiquantitative scoring, measuring the viable area of the islet from 0 = less viable to 3 = more viable), insulin secretion after glucose and nonglucose secretagogues, and oxidation of D-[U-14C]glucose. The results for the control group were always higher for the following: recovery (95.4% +/- 1.2% v 83.0% +/- 2.1%, P = .00), insulin content (2,203.9 +/- 335.2 v 1,443.3 +/- 171.8 microU/islet, P = .03), insulin secretion after 5.5 mmol/L glucose (61.3 +/- 8.0 v 28.3 +/- 3.4 microU/islet/90 min, p = .00), 16.7 mmol/L glucose (151.4 +/- 16.1 v 98.7 +/- 14.1 microU/islet/90 min, p = .03), 10 mmol/L L-leucine +10 mmol/L L-glutamine (125.6 +/- 27.9 v 56.8 +/- 6.4 microU/islet/90 min, P = .05), and 10 mmol/L L-arginine (202.5 +/- 27.5 v 128.8 +/- 14.2 microU/islet/90 min, P = .01), and glucose oxidation at 5.5 mmol/L (12.5 +/- 1.1 v 7.9 +/- 0.6 pmol/islet/120 min, P = .00) and at 16.7 mmol/L (26.1 +/- 2.6 v 14.3 +/- 1.6 pmol/islet/120 min, P = .00). No significant differences in staining viability were found between groups (2.35 and 2.48, respectively, P = .55). However, cryopreserved and control islets showed a significant increase in insulin secretion and glucose oxidation after increasing the glucose concentration from 5.5 to 16.7 mmol/L. We conclude that when glucose is increased, cryopreserved islets keep the capacity to increase insulin secretion, but cryopreservation produces a significant decrease in several islet viability characteristics. This decrease may be due to a decline of beta-cell number per islet and/or a decrease in the content of insulin per beta cell.
冷冻保存是胰岛储存的一种有效方法，可能会促进胰岛移植的临床试验。本研究的目的是评估冷冻保存的大鼠胰岛的体外活力，包括对非葡萄糖促分泌剂的反应和葡萄糖氧化。通过导管内注射胶原酶消化胰腺后，挑选出75至200微米的Wistar大鼠胰岛，在RPMI 1640（葡萄糖11.1 mmol/L）中培养，并随机分为两组：对照组（在37℃培养20至24小时）和冷冻保存组（在37℃培养20至24小时后，根据拉乔特方案对胰岛进行冷冻保存：冷冻速度，-0.25℃/分钟；解冻速度，200℃/分钟）。在两组中，我们评估了回收率、每个胰岛的胰岛素含量、染色活力（溴化乙锭/吖啶橙；半定量评分，测量胰岛的存活面积，从0 = 存活能力较低到3 = 存活能力较高）、葡萄糖和非葡萄糖促分泌剂刺激后的胰岛素分泌以及D-[U-14C]葡萄糖的氧化。对照组在以下方面的结果总是更高：回收率（95.4%±1.2%对83.0%±2.1%，P = .00）、胰岛素含量（2,203.9±335.2对1,443.3±171.8微单位/胰岛，P = .03）、5.5 mmol/L葡萄糖刺激后的胰岛素分泌（61.3±8.0对28.3±3.4微单位/胰岛/90分钟，p = .00）、16.7 mmol/L葡萄糖刺激后的胰岛素分泌（151.4±16.1对98.7±14.1微单位/胰岛/90分钟，p = .03）、10 mmol/L L-亮氨酸 + 10 mmol/L L-谷氨酰胺刺激后的胰岛素分泌（125.6±27.9对56.8±6.4微单位/胰岛/90分钟，P = .05）、10 mmol/L L-精氨酸刺激后的胰岛素分泌（202.5±27.5对1,28.8±14.2微单位/胰岛/90分钟，P = .01），以及5.5 mmol/L时的葡萄糖氧化（12.5±1.1对7.9±0.6皮摩尔/胰岛/120分钟，P = .00）和16.7 mmol/L时的葡萄糖氧化（26.1±2.6对十四点三±1.6皮摩尔/胰岛/120分钟，P = .00）。两组之间在染色活力方面未发现显著差异（分别为2.35和2.48，P = .55）。然而，当葡萄糖浓度从5.5 mmol/L增加到16.7 mmol/L后，冷冻保存的胰岛和对照胰岛的胰岛素分泌和葡萄糖氧化均显著增加。我们得出结论，当葡萄糖浓度升高时，冷冻保存的胰岛保持增加胰岛素分泌的能力，但冷冻保存会导致胰岛的几个活力特征显著下降。这种下降可能是由于每个胰岛中β细胞数量的减少和/或每个β细胞中胰岛素含量的降低。
