Chadwick D R, Robertson G S, Toomey P, Contractor H, Rose S, James R F, Bell P R, London N J
Department of Surgery, Leicester Royal Infirmary, UK.
Cell Transplant. 1993 Jul-Aug;2(4):355-61. doi: 10.1177/096368979300200419.
Euro-Ficoll and bovine serum albumin (BSA) are two of the most commonly used density gradient media for the purification of pancreatic islets. Euro-Ficoll is based upon Euro-Collins, a cold storage medium, and must, therefore, be used at 4 degrees C. The ionic composition of BSA, however, is likely to contribute to hypothermic cellular swelling, and this may influence the efficiency of islet purification using this medium at 4 degrees C. Experience in this laboratory also suggested that batch-to-batch variation in islet purity using BSA was related to differences in BSA osmolality. The aim of this study was to assess the effect of gradient medium temperature and osmolality on the purification of human and porcine islets using BSA. Pancreata were collagenase-digested, and islets were purified on continuous linear density gradients of BSA. The distribution of insulin and amylase in each gradient was assayed, and used to calculate the median density of islets and exocrine tissue, and the efficiency of islet purification (% amylase contamination at a fixed insulin yield), using: 1) gradient osmolalities of 300, 400, and 500 mOsm/kg H2O (seven porcine pancreata), and 2) gradients at 4 degrees C and at 22 degrees C (eight human and seven porcine pancreata). Increase in density gradient osmolality produced increases in porcine exocrine tissue density which exceeded changes in islet density, resulting in improved islet purity, maximal at a BSA osmolality of 400 mOsm/kg H2O. For human pancreata there was no significant change in pancreatic tissue densities nor islet purity with temperature.(ABSTRACT TRUNCATED AT 400 WORDS)
欧洲菲可（Euro - Ficoll）和牛血清白蛋白（BSA）是用于纯化胰岛的两种最常用的密度梯度介质。欧洲菲可基于欧洲柯林斯（Euro - Collins）这种冷藏介质，因此必须在4摄氏度下使用。然而，BSA的离子组成可能会导致低温下细胞肿胀，这可能会影响在4摄氏度下使用这种介质进行胰岛纯化的效率。本实验室的经验还表明，使用BSA时胰岛纯度的批次间差异与BSA渗透压的差异有关。本研究的目的是评估梯度介质温度和渗透压对使用BSA纯化人胰岛和猪胰岛的影响。胰腺用胶原酶消化，胰岛在BSA的连续线性密度梯度上进行纯化。测定每个梯度中胰岛素和淀粉酶的分布，并用于计算胰岛和外分泌组织的中位密度以及胰岛纯化效率（在固定胰岛素产量下的淀粉酶污染百分比），使用：1）300、400和500 mOsm/kg H₂O的梯度渗透压（七个猪胰腺），以及2）4摄氏度和22摄氏度的梯度（八个人类胰腺和七个猪胰腺）。密度梯度渗透压的增加导致猪外分泌组织密度的增加超过了胰岛密度的变化，从而提高了胰岛纯度，在BSA渗透压为400 mOsm/kg H₂O时达到最大值。对于人胰腺，胰腺组织密度和胰岛纯度随温度没有显著变化。（摘要截断于400字）
