Lakey J R, Cavanagh T J, Zieger M A
Comprehensive Tissue Centre, Department of Surgery, University of Alberta, Edmonton, Canada.
Cell Transplant. 1998 Sep-Oct;7(5):479-87. doi: 10.1177/096368979800700507.
Density gradient separation of islets from exocrine tissue is usually performed with Ficoll. However, this reagent adds significantly to the cost of the isolation. The aim of this study was to evaluate the performance of Dextran as a potential low-cost substitute for Ficoll and to evaluate the effects of cold storage of the pancreatic digest prior to purification. Pancreases were procured from mongrel dogs, loaded with collagenase and mechanically dissociated. Washed pancreatic digest was collected and divided into two fractions that were purified using discontinuous gradients on the Cobe 2991 processor using identically prepared EuroFicoll (EF) or EuroDextran (ED) gradients. Alternate groups were suspended in EC and stored on ice, while the other fraction were resuspended in the 1.108-g/mL gradient layer (either EF or ED) and loaded into the COBE. This tissue layer was overlaid with layers of densities 1.096 and 1.037 g/mL and a HBSS cap, and centrifuged for 5 min at 800 x g. Purified islets were collected from the interface between the 1.037 and 1.096 layers and islet recovery, purity, and function were assessed. From a series of eight isolations, 72.9 +/- 8.2% (mean +/- SEM) of the islets were recovered from the EF purified gradients compared with 62.6 +/- 8.3% from ED gradients (p = NS, paired t-test). Gradients of ED that were run following hypothermic storage of the digest in cold EC solution (stored ED) had reduced islet recovery when compared with islet recovery from gradients prepared in EF(stored EF) (51.6 +/- 9.6% for ED stored vs. 72.9 +/- 11.9 for EF stored, p < 0.05). Islet recovery from EF gradients was equivalent between the stored and nonstored groups. The purity of preparations from the stored ED gradients was also reduced (71.3 +/- 4.3%) when compared with islets that were immediately purified after dissociation (82.5 +/- 4.8%, p < 0.05). Static glucose stimulation assay showed equivalence between the islets from ED and EF gradients. The stimulation index (SI) was 9.3 +/- 0.9 for EF islets compared with 7.9 +/- 1.4 for ED islets for digest purified immediately. However, if the digest was hypothermically stored in EC solution, a decrease in functional viability was observed in both the EF and the ED groups (7.7 +/- 1.4 and 5.9 +/- 0.8, respectively). Out of five alloxan-induced diabetic nude mice transplanted under the kidney capsule with 2000 islets isolated from the nonstored groups, three remained euglycemic >50 days posttransplant with either EF or ED islets. These experiments demonstrate effective recovery of equivalent numbers of canine islets using discontinuous gradients of ED or EF immediately following enzymatic digestion. However, following storage of the digest in cold EC solution results in a reduction in both islet recovery and function when gradients of ED are utilized.
通常使用菲可（Ficoll）从外分泌组织中进行胰岛的密度梯度分离。然而，这种试剂显著增加了分离成本。本研究的目的是评估葡聚糖作为菲可潜在低成本替代品的性能，并评估纯化前胰腺消化物冷藏的效果。从杂种狗获取胰腺，用胶原酶处理并进行机械解离。收集洗涤后的胰腺消化物并分为两部分，使用相同制备的欧洲菲可（EF）或欧洲葡聚糖（ED）梯度在Cobe 2991处理器上通过不连续梯度进行纯化。交替分组的样本悬浮于内皮细胞培养基（EC）中并保存在冰上，而另一部分重新悬浮于1.108 g/mL梯度层（EF或ED）中并加载到COBE中。该组织层用密度为1.096和1.037 g/mL的层以及汉克斯平衡盐溶液（HBSS）覆盖，并在800×g下离心5分钟。从1.037和1.096层之间的界面收集纯化的胰岛，并评估胰岛回收率、纯度和功能。在一系列八次分离中，从EF纯化梯度中回收了72.9±8.2%（平均值±标准误）的胰岛，而从ED梯度中回收了62.6±8.3%（p = 无显著性差异，配对t检验）。与从EF（冷藏EF）制备的梯度中回收的胰岛相比，消化物在冷EC溶液中低温储存后运行的ED梯度（冷藏ED）的胰岛回收率降低（冷藏ED为51.6±9.6%，冷藏EF为72.9±11.9%，p < 0.05）。冷藏组和未冷藏组从EF梯度中回收的胰岛数量相当。与解离后立即纯化的胰岛（82.5±4.8%，p < 0.05）相比，冷藏ED梯度制备物的纯度也降低了（71.3±4.3%）。静态葡萄糖刺激试验显示ED和EF梯度的胰岛之间相当。立即纯化的消化物中，EF胰岛的刺激指数（SI）为9.3±0.9，ED胰岛为7.9±1.4。然而，如果消化物在EC溶液中低温储存，EF和ED组的功能活力均降低（分别为7.7±1.4和5.9±0.8）。在五只经四氧嘧啶诱导的糖尿病裸鼠肾被膜下移植从非储存组分离的2000个胰岛后，三只移植EF或ED胰岛的小鼠在移植后>50天保持血糖正常。这些实验表明，酶消化后立即使用ED或EF的不连续梯度可有效回收等量的犬胰岛。然而，当使用ED梯度时，消化物在冷EC溶液中储存会导致胰岛回收率和功能降低。
