Kenmochi T, Miyamoto M, Une S, Nakagawa Y, Moldovan S, Navarro R A, Benhamou P Y, Brunicardi F C, Mullen Y
Department of Surgery, UCLA School of Medicine and Veterans Administration Medical Center, West Los Angeles, California 90073, USA.
Pancreas. 2000 Mar;20(2):184-90. doi: 10.1097/00006676-200003000-00012.
A new approach, involving a two-step digestion process and Los Angeles preservation solution #1 (LAP-1), a cold storage solution, was developed for isolation of high-quality islets from human pancreata for transplantation. This approach markedly improves the islet yield, purity and viability, and the isolation success rate. In this method, the pancreas was digested first in warm collagenase solution for up to 20 minutes. After decanting the enzyme solution, partially digested tissue was dissociated by gentle agitation in cold LAP-1 solution without additional collagenase. The digested tissues were stored in cold LAP-1 solution until islet purification on Euro-Ficoll. Forty-six islet isolations were performed consecutively by the new method (group 1). These results were compared to those obtained earlier with 46 consecutive isolations, using our previous method that had been used before development of the new method (group 2). Our old method was a modification of Ricordi's method involving only warm collagenase digestion and the storage of digested tissues in cold Hanks balanced salt solution. All pancreata were partial, containing the body and tail. There were no significant differences in both groups with regard to the donor age, cold ischemic time, harvesting conditions, and pancreatic weight. Pancreas digestion was completed in approximately 1 hour in both groups. The isolation success rate as determined by viable islets after 2 days in culture was 93.5% (43 of 46 cases) in group 1, and 56.5% (26 of the 46) in group 2. Immediately after isolation, the new method yielded a total of 335,739 +/- 36,244 islets equivalent to 150 microm (IEQ) and 6,233 +/- 681 IEQ/g of pancreas with 83 +/- 2.5% purity, whereas the old method yielded a total of 195,587 +/- 25,242 IEQ and 3,763 +/- 5,509 IEQ/g with 69.2 +/- 4.7% purity. Isolated islets in group 1 maintained a good three-dimensional structure, displayed normal insulin release to high glucose stimulation in vitro, and restored euglycemia after transplantation into streptozotocin-diabetic athymic mice. The two-step digestion method provides a sufficient number of islets for transplantation from a single pancreas.
一种新方法被开发出来用于从人类胰腺中分离高质量胰岛以进行移植，该方法包括两步消化过程以及使用洛杉矶保存液#1（LAP - 1），一种冷藏溶液。这种方法显著提高了胰岛产量、纯度和活力，以及分离成功率。在该方法中，胰腺首先在温热的胶原酶溶液中消化长达20分钟。倒出酶溶液后，部分消化的组织在冷的LAP - 1溶液中通过轻柔搅拌解离，无需额外的胶原酶。消化后的组织保存在冷的LAP - 1溶液中，直到在欧洲菲可（Euro - Ficoll）上进行胰岛纯化。通过新方法连续进行了46次胰岛分离（第1组）。将这些结果与之前使用我们在新方法开发之前使用的旧方法连续进行46次分离所获得的结果进行比较（第2组）。我们的旧方法是对里科尔迪（Ricordi）方法的改进，仅涉及温热胶原酶消化以及将消化后的组织保存在冷的汉克斯平衡盐溶液中。所有胰腺均为部分胰腺，包含胰体和胰尾。两组在供体年龄、冷缺血时间、采集条件和胰腺重量方面均无显著差异。两组的胰腺消化均在约1小时内完成。培养2天后通过有活力的胰岛确定的分离成功率在第1组为93.5%（46例中的43例），在第2组为56.5%（46例中的26例）。分离后立即测定，新方法产生的胰岛总数为335,739±36,244个等效于150微米的胰岛（IEQ），每克胰腺6,233±681个IEQ，纯度为83±2.5%，而旧方法产生的胰岛总数为共195,587±25,242个IEQ，每克3,763±5,509个IEQ，纯度为69.2±4.7%。第1组分离出的胰岛保持良好的三维结构，在体外对高葡萄糖刺激显示出正常的胰岛素释放，并在移植到链脲佐菌素诱导的糖尿病无胸腺小鼠后恢复了正常血糖水平。两步消化法能从单个胰腺中提供足够数量的胰岛用于移植。
