Successful Human Islet Isolation and Transplantation Indicating the Importance of Class 1 Collagenase and Collagen Degradation Activity Assay
Successful Human Islet Isolation and Transplantation Indicating the Importance of Class 1 Collagenase and Collagen Degradation Activity Assay
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DOI：
10.1097/tp.0b013e3181d21e9a
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发表时间：
2010-04-27
期刊：
影响因子：
6.2
通讯作者：
Hering, Bernhard J.
中科院分区：
文献类型：
--
作者：
Balamurugan, A. N.;Breite, Andrew G.;Hering, Bernhard J.
背景。纯化的组织解离酶（TDEs）对于临床移植所需的成功人胰岛分离至关重要，但对这些程序中使用的关键酶-溶组织梭菌I类（C1）和II类（C2）胶原酶的特性知之甚少。在这里，我们展示了三家供应商生产的纯化胶原酶产品中发现的C1胶原酶的差异，以及两家供应商之间C1的差异对人胰岛产量的影响。采用高效液相色谱法和胶原降解活性（CDA）对罗氏（Roche）、Serva/Nordmark （Uetersen, Germany）和VitaCyte （Indianapolis， IN）的胶原酶进行分析，CDA是一种优先检测完整C1胶原酶的检测方法。采用现行标准方法进行胰岛分离。这些研究表明，最高数量的完整C1与高特异性CDA（每毫克蛋白质的CDA单位）相关。特异性CDA最高的是VitaCyte产品，其次是罗氏和Serva/Nordmark产品。VitaCyte的产品成功地用于人胰岛分离（n = 14），获得的平均最终胰岛产量为419,100+/-150,900个胰岛当量（IEQ）（4147+/-1759个IEQ/g胰腺）。其中四种制剂已成功用于临床移植手术。与早期使用Serva NB1胶原酶和NB中性蛋白酶进行的27次分离相比，这些TDEs的结果明显更好，最终胰岛产量为217,500+/-152,400 IEQ（胰腺2134+/-1524 IEQ/g）。这些数据表明完整的C1和使用适当的分析方法将tde的生化特征与胰岛质量和产量联系起来的重要性。
Background. Purified tissue dissociation enzymes (TDEs) are critical to successful human islet isolation required for clinical transplantation, but little is known about the characteristics of the key enzymes-class I (C1) and class II (C2) collagenase from Clostridium histolyticum-used in these procedures. Here, we show the differences between the C1 collagenase found in purified collagenase products manufactured by three suppliers and the impact of differences in C1 between two suppliers on human islet yield.Methods. Collagenase from Roche, Serva/Nordmark (Uetersen, Germany), and VitaCyte (Indianapolis, IN) were analyzed by analytical high-performance liquid chromatography and collagen degradation activity (CDA), an assay that preferentially detects intact C1 collagenase. Human islet isolations were performed using current standard practices.Results. These studies showed that the highest amount of intact C1 that correlated with a high specific CDA (CDA unit per milligram of protein). The highest specific CDA was found in VitaCyte product followed by the Roche and Serva/Nordmark products. The products of VitaCyte were used successfully for human islet isolation (n = 14) with an average final islet yield obtained was 419,100+/-150,900 islet equivalent number (IEQ) (4147+/-1759 IEQ/g pancreas). Four of these preparations were used successfully in clinical transplantation procedures. These TDEs gave significantly better results when compared with earlier data where 27 isolations were performed using Serva NB1 collagenase and NB neutral protease where the final islet yield was 217,500+/-152,400 IEQ (2134+/-1524 IEQ/g pancreas).Conclusions. These data indicate the importance of intact C1 and the use of the appropriate analytical assays to correlate biochemical characteristics of TDEs to islet quality and yield.