The Importance of Tryptic-like Activity in Purified Enzyme Blends for Efficient Islet Isolation
The Importance of Tryptic-like Activity in Purified Enzyme Blends for Efficient Islet Isolation
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DOI：
10.1097/tp.0b013e31819499f0
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发表时间：
2009-02-15
期刊：
影响因子：
6.2
通讯作者：
Brandhorst, Daniel
中科院分区：
文献类型：
--
作者：
Brandhorst, Heide;Friberg, Andrew;Brandhorst, Daniel
背景从人胰腺中分离胰岛关键地依赖于一种有效的酶混合物。以前的研究仅关注胶原酶和中性蛋白酶/嗜热菌蛋白酶的存在。尽管这些组分的表征得到了改进，但与批次相关的功效变异性仍然存在，这表明有效的胰岛裂解需要另外的迄今为止被忽视的酶。在胶原酶NB I批次中鉴定了胰蛋白酶样酶的不同活性，根据胰蛋白酶样酶和胶原酶活性之间的匹配比率（TLA比率）进行选择。大鼠和人胰腺用目前的标准程序处理。将TLA比率从1.3%增加到10%可使大鼠胰腺分离时间减少50%，而不影响糖尿病裸鼠的胰岛产量、活力或移植后功能。将用于人胰腺处理的TLA比率从1.3%提高到12.6%导致再循环时间的显著减少和人胰岛产量的递增增加，而不影响纯度、体外功能或培养后的恢复。优化的胰腺消化与更高百分比的胰岛制剂符合临床移植的质量标准相关。我们的结论是，TLA是所有的有效成分，应包括在适量的人胰岛分离的酶混合物，以优化效率和最小化批次相关的变异性。
Background. The isolation of islets from the human pancreas critically depends oil an efficient enzyme blend. Previous studies have solely focused oil the presence of collagenase and neutral protease/thermolysin. Despite improved characterization of these components, the lot-related variability in efficacy still persists suggesting that additional so far disregarded enzymes are required for efficient islet cleavage.Methods. Varying activities of a tryptic-like enzyme were identified within collagenase NB I lots, which were selected according to a matched ratio between tryptic-like and collagenase activity (TLA-ratio). Rat and human pancreata were processed with current standard procedures.Results. Increasing the TLA-ratio from 1.3% to 10% reduced pancreas dissociation time in rats by 50% without affecting islet yield, viability, or posttransplant function in diabetic nude mice. Enhancing the TLA-ratio from 1.3% to 12.6% for human pancreas processing resulted in a significant reduction of recirculation time and increased incrementally human islet yield without affecting purity, in vitro function or recovery after culture. Optimized pancreas digestion correlated with a higher percentage of islet preparations fulfilling quality criteria for clinical transplantation.Conclusions. We conclude that TLA is all effective component that should be included in moderate amounts ill enzyme blends for human islet isolation to optimize the efficiency and minimize the lot-related variability.