Kenmochi T, Miyamoto M, Sasaki H, Une S, Nakagawa Y, Moldovan S, Benhamou P Y, Brunicardi F C, Tanaka H, Mullen Y
Department of Surgery, UCLA School of Medicine, USA.
Pancreas. 1998 Nov;17(4):367-77. doi: 10.1097/00006676-199811000-00007.
The most critical factors that affect the outcome of clinical pancreatic islet transplantation are the number and quality of donor islets available for transplantation. Toward this goal, we attempted to obtain islets that are both of better quality and higher number than are obtainable by the islet-isolation process that is now widely used. We paid special attention to two critical components of the isolation procedure: minimizing the exposure of pancreatic tissue and freed islets to warm enzyme solution, and development of a preservation solution suitable for islets during cold storage of digested pancreatic tissue-free islets. For this purpose, we developed both a two-step procedure for pancreas digestion and a new cold preservation solution, the LAP-1 solution (Los Angeles preservation solution 1). In this study, we evaluated the effect of four preservation solutions by storing digested pancreatic tissues on ice for 90 min. After the cold storage, islets were purified on three layers of Euro-Ficoll solutions in a 50-ml tube, and the islet yield, viability, and function were determined. These experiments were performed by using samples from 10 consecutive islet isolations. Results with LAP-1, original University of Wisconsin solution (oUW), and modified UW solution (mUW;UW without hydroxyethyl starch) were compared with those obtained with Hank's balanced salt solution (HBSS). The islet yield was significantly higher in the LAP-1 and mUW groups as compared with the HBSS group (p < 0.01). The islet purity was significantly better in the LAP-1, oUW, and mUW groups than the HBSS (p < 0.001). The islet viability was lowest in the HBSS group immediately after purification (vs. LAP-1, oUW, and mUW, p < 0.05) and further decreased during culture (p < 0.01). Both the number and viability of cultured islets were the highest with LAP-1 solution but without statistical significance between mUW and oUW. Electron microscopic examination showed only slight damage to cell membranes immediately after purification of islets stored in LAP-1 solution and their complete recovery within 1-2 days of culture. These islets also exhibited normal insulin responses to high glucose by static incubation and perifusion assays.
影响临床胰岛移植结果的最关键因素是可用于移植的供体胰岛的数量和质量。为了实现这一目标，我们试图获取质量更好、数量更多的胰岛，这比目前广泛使用的胰岛分离方法所能获得的胰岛要好。我们特别关注分离过程中的两个关键环节：尽量减少胰腺组织和游离胰岛与温热酶溶液的接触，以及开发一种适合在消化后的无胰腺组织胰岛冷藏期间使用的保存溶液。为此，我们开发了一种两步胰腺消化程序和一种新的冷藏保存溶液，即LAP-1溶液（洛杉矶保存溶液1）。在本研究中，我们通过将消化后的胰腺组织在冰上储存90分钟来评估四种保存溶液的效果。冷藏后，在一个50毫升的试管中，在三层欧洲菲可溶液上纯化胰岛，并测定胰岛产量、活力和功能。这些实验使用了连续10次胰岛分离的样本。将LAP-1、原始威斯康星大学溶液（oUW）和改良威斯康星大学溶液（mUW；不含羟乙基淀粉的UW）的结果与用汉克平衡盐溶液（HBSS）获得的结果进行比较。与HBSS组相比，LAP-1组和mUW组的胰岛产量显著更高（p < 0.01）。LAP-1组、oUW组和mUW组的胰岛纯度明显优于HBSS组（p < 0.001）。纯化后，HBSS组的胰岛活力立即最低（与LAP-1组、oUW组和mUW组相比，p < 0.05），并且在培养过程中进一步下降（p < 0.01）。用LAP-1溶液培养的胰岛数量和活力最高，但mUW组和oUW组之间无统计学意义。电子显微镜检查显示，储存在LAP-1溶液中的胰岛纯化后，细胞膜仅受到轻微损伤，并且在培养1-2天内完全恢复。这些胰岛在静态孵育和灌流试验中对高葡萄糖也表现出正常的胰岛素反应。
