Kühtreiber W M, Ho L T, Kamireddy A, Yacoub J A W, Scharp D W
Prodo Laboratories Inc, Irvine, California 92618, USA.
Transplant Proc. 2010 Jul-Aug;42(6):2027-31. doi: 10.1016/j.transproceed.2010.05.099.
The general consensus among transplant centers is that a cold ischemia time (CIT) beyond 8 hours results in reduced yields and quality of human islets. We sought to optimize the isolation process and enzymes for pancreata with extended CIT. We processed 16 extended CIT pancreata (13.2 +/- 0.7 hours). Donors averaged 50.8 +/- 2.6 (standard error of the mean) years old with a body mass index of 28.6 +/- 1.5. Glands were shipped in cold organ preservation solution without oxygenated perfluorocarbon. Isolations were performed under a protocol optimized for digestion with the new cGMP collagenase from Roche. Purification used continuous Euroficoll/University of Wisconsin gradients. Islets were cultured in two types of Prodo cGMP islet culture media and/or in Miami 1A media. Glucose-stimulated insulin secretion assays were performed after 8 to 16 days of culture. Prepurification yield averaged 415 +/- 41 KIEQ postpurification, 359 +/- 29 KIEQ (purification loss 13.5%); and postculture 317 +/- 27 KIEQ (culture loss 11.7%). Our process liberated an average of 4278 IEQ/g of pancreas (97 +/- 5 g). Most islets were recovered in the purest fraction (purity 79.7% +/- 1.9%). Culture loss in our enhanced culture media was 11.7%. After 2 to 3 days in culture, viability was 92% +/- 1%. Islets exhibited compactness and dithizone staining. Glucose-stimulated insulin secretion assays performed after 3 to 23 days in our PIM(R) media resulted in a stimulation index of 6.8 +/- 1.7 (G50 to G350). We concluded that our human islet isolation process permitted the recovery of large numbers of high-quality human islets from extended CIT pancreata and that our cGMP islet culture media was superior to the current standard CMRL-based media.
移植中心的普遍共识是，冷缺血时间（CIT）超过8小时会导致人胰岛的产量和质量下降。我们试图优化针对CIT延长的胰腺的分离过程和酶。我们处理了16个CIT延长的胰腺（13.2±0.7小时）。供体平均年龄为50.8±2.6（平均标准误差）岁，体重指数为28.6±1.5。腺体在无氧全氟碳的冷器官保存溶液中运输。分离按照针对罗氏公司新的cGMP胶原酶消化优化的方案进行。纯化使用连续的欧洲菲可/威斯康星大学梯度。胰岛在两种Prodo cGMP胰岛培养基和/或迈阿密1A培养基中培养。培养8至16天后进行葡萄糖刺激的胰岛素分泌测定。纯化前平均产量为415±41 KIEQ，纯化后为359±29 KIEQ（纯化损失13.5%）；培养后为317±27 KIEQ（培养损失11.7%）。我们的过程平均每克胰腺释放出4278 IEQ（97±5克）。大多数胰岛在最纯的部分中回收（纯度79.7%±1.9%）。我们增强型培养基中的培养损失为11.7%。培养2至3天后，活力为92%±1%。胰岛表现出紧密性和双硫腙染色。在我们的PIM(R)培养基中培养3至23天后进行的葡萄糖刺激的胰岛素分泌测定得出刺激指数为6.8±1.7（G50至G350）。我们得出结论，我们的人胰岛分离过程能够从CIT延长的胰腺中回收大量高质量的人胰岛，并且我们的cGMP胰岛培养基优于当前基于CMRL的标准培养基。
