Bulk cryopreservation of isolated islets of Langerhans
Bulk cryopreservation of isolated islets of Langerhans
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DOI：
10.1016/0963-6897(95)02025-x
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发表时间：
1996-05-01
影响因子：
3.3
通讯作者：
Rajotte, RV
中科院分区：
文献类型：
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作者：
Lakey, JRT;Warnock, GL;Rajotte, RV
目前分离人类朗格汉斯胰岛的方法有限，成功逆转长期存在的1型糖尿病需要多个捐赠者。分离胰岛的冷冻保存是一种有效的存储和汇集胰岛的方法，由于目前的做法是在每个单独的冷冻管中放置少量的胰岛，所以当前的冷冻保存方案很繁琐。在本研究中，我们研究了血液冷冻袋在缓慢降温和快速解冻两种方法冷冻保存分离胰岛中的应用。使用放置在500毫升Crycell(Baxter)血液冷冻袋中的热电偶产生的冷冻和解冻曲线显示，为了持续实现与在玻璃管中观察到的相似的核形成和冷却曲线，在-7.4℃下需要更长的平衡期，当已知数量的大鼠胰岛被放置在冷冻袋中并且以蔗糖稀释法逐步添加和移除冷冻保护剂二甲基亚砜(DMSO)时，胰岛回收率与玻璃管相比为92+/-4.8比90+/-2.3%(n=4，p=ns，Mann-Whitney U检验)。将纯化的犬胰岛冷冻保存在单个冷冻袋和多个玻璃管中，其回收率相似(冷冻袋78.8+/-12.5%，玻璃管82.3+/-5.3%；n=6，p=ns)。两组体外功能相当。冷冻袋和玻璃管冷冻保存的犬胰岛在葡萄糖灌流(超过基础胰岛素分泌刺激)时的胰岛素释放刺激指数分别为3.2±1.0和2.3±1.3(n=6，p=ns)。这些值显著低于未冷冻的对照胰岛(6.9+/-2.4，p<；0.05)。当2000只冷冻保存在冷冻袋或玻璃管中的犬类胰岛被移植到糖尿病裸鼠体内时，移植后的动物血糖正常。我们的结论是，新鲜分离的犬胰岛冷冻保存在单个冷冻袋中的存活率与玻璃管法相当，将胰岛批量冷冻保存在单个冷冻袋中将有助于有效的低温组织库，以支持正在进行的胰岛移植临床试验。
Current methods to isolate human islets of Langerhans are limited and multiple donors are required for successful reversal of longstanding Type 1 diabetes mellitus. Cryopreservation of isolated islets is an effective method of storing and pooling islets, Current cryopreservation protocols are cumbersome due to current practices of placing small aliquots of islets per individual freezer tube. In the present study, we examined the application of a blood freezer bag for the cryopreservation of isolated islets by slow cooling and rapid thawing. Freezing and thawing profiles generated using thermocouples placed inside a 500 mL Cryocyte (Baxter) blood freezer bag showed that a longer equilibration period at -7.4 degrees C was necessary to consistently achieve nucleation and cooling profiles similar to those observed in glass tubes, When known numbers of rat islets were placed in the freezer bag and the cryoprotectant dimethyl sulfoxide (DMSO) was added in a stepwise fashion and removed using a sucrose dilution, the islet recovery compared with glass tubes was 92 +/- 4.8 vs. 90 +/- 2.3% (n = 4, p = ns, Mann-Whitney U-test). When purified canine islets were cryopreserved in a single freezer bag or in multiple glass tubes, the recovery was similar (78.8 +/- 12.5% recovery for freezer bag vs, 82.3 +/- 5.3% for glass tubes; n = 6, p = ns). In vitro function was equivalent for both groups. The stimulation index of insulin release during glucose perifusion (stimulated over basal insulin secretion) for canine islets cryopreserved in a freezer bag vs, glass tubes was 3.2 +/- 1.0 and 2.3 +/- 1.3, respectively (n = 6, p = ns). These values were significantly lower than the nonfrozen control islets (6.9 +/- 2.4, p < 0.05). When 2000 canine islets cryopreserved in either a freezer bag, or glass tubes were transplanted into diabetic nude mice, the animals became and remained normoglycemic posttransplant. We conclude that the survival of freshly isolated canine islets cryopreserved in a single freezer bag is equivalent to the glass tube method, Bulk cryopreservation of islets in a single freezer bag will facilitate effective low temperature tissue banking to support ongoing clinical trials of islet transplantation.