van der Burg M P, Guicherit O R, Frölich M, Prins F A, Bruijn J A, Gooszen H G
Department of Surgery, University Hospital, Leiden, The Netherlands.
Cell Transplant. 1994 Jul-Aug;3(4):315-24. doi: 10.1177/096368979400300408.
Allogeneic islet transplantation in Type I diabetic patients is considerably hampered by the variable outcome of islet isolation and purification. After collagenase digestion of the pancreas, islet isolation is traditionally performed under hypothermic conditions in physiological solutions such as Hanks and RPMI. The University of Wisconsin solution (UWS) has been shown superior for hypothermic preservation of the pancreas. We, therefore, compared the UWS and RPMI for canine islet isolation and subsequent purification in either a conventional hyperosmotic density gradient of dextran in Hanks, or a novel normosmotic density gradient of Percoll in UWS. The isolation solution did not affect islet yield before purification (51% of the native islet mass). Loss of amylase (30%) and swelling of the acinar cells were observed in RPMI. In contrast, no loss of amylase and slight shrinkage of the acinar cells were observed in the UWS. Cell swelling affected the density separation and viability of the cells. Dextran density separation resulted in a 15% purity and 41% recovery of the islets isolated in RPMI, as compared to a 93% purity and 52% recovery of islets isolated in UWS. Percoll density separation improved the purity (99%) and recovery (74%) of islets isolated in UWS. Islets isolated in UWS demonstrated a superior basal and glucose stimulated insulin release during perifusion. Electron microscopy demonstrated a well-preserved islet ultrastructure after isolation in both solutions--except for slightly swollen mitochondria after isolation in RPMI. Autotransplantation of islets in pancreatectomised dogs was successful both after isolation in UWS and RPMI. We conclude that prevention of cell swelling during isolation and purification in the UWS resulted in an improved yield of viable and consistent virtually pure islets. Prevention of cell swelling during islet isolation should facilitate the analysis and control of other factors affecting outcome in man.
I型糖尿病患者的同种异体胰岛移植受到胰岛分离和纯化结果多变的严重阻碍。胰腺经胶原酶消化后，传统上胰岛分离是在低温条件下于生理溶液（如汉克斯溶液和RPMI培养基）中进行。威斯康星大学溶液（UWS）已被证明在胰腺低温保存方面更具优势。因此，我们比较了UWS和RPMI用于犬胰岛分离及随后在汉克斯溶液中传统的高渗葡聚糖密度梯度或UWS中新型等渗 Percoll密度梯度下的纯化效果。分离溶液在纯化前不影响胰岛产量（占天然胰岛质量的51%）。在RPMI中观察到淀粉酶损失（30%）和腺泡细胞肿胀。相比之下，在UWS中未观察到淀粉酶损失，腺泡细胞仅有轻微收缩。细胞肿胀影响细胞的密度分离和活力。葡聚糖密度分离使在RPMI中分离的胰岛纯度达到15%，回收率为41%，而在UWS中分离的胰岛纯度为93%，回收率为52%。Percoll密度分离提高了在UWS中分离的胰岛的纯度（99%）和回收率（74%）。在UWS中分离的胰岛在灌注期间表现出更好的基础胰岛素释放和葡萄糖刺激的胰岛素释放。电子显微镜显示，两种溶液分离后胰岛超微结构保存良好——除了在RPMI中分离后线粒体略有肿胀。在胰腺切除的犬中，无论是在UWS还是RPMI中分离后进行胰岛自体移植均获成功。我们得出结论，在UWS中进行分离和纯化过程中防止细胞肿胀可提高有活力且一致的几乎纯的胰岛产量。在胰岛分离过程中防止细胞肿胀应有助于分析和控制影响人类移植结果的其他因素。
