Variation in human islet viability based on different membrane integrity stains
Variation in human islet viability based on different membrane integrity stains
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DOI：
10.3727/000000004783983701
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发表时间：
2004-01-01
影响因子：
3.3
通讯作者：
Lakey, J. R. T.
中科院分区：
文献类型：
--
作者：
Barnett, M. J.;McGhee-Wilson, D.;Lakey, J. R. T.
膜完整性荧光染色常规用于评价胰岛活力。结果被用作胰岛产品放行标准的决定因素之一，并用于评估不同培养条件的有效性。最近，已经观察到，基于所使用的活力测定，新鲜分离的胰岛的活力染色存在变化。本研究比较了三种膜完整性染色法对分离的人胰岛活力的评估。目前用于评估胰岛活力的标准方法二乙酸异黄绿素/碘化丙啶（FDA/PI）显示出强烈的细胞外荧光，减少了完整胰岛的差异染色。我们进一步评估了SYTO-13/溴化乙锭（SYTO/EB）和钙黄绿素AM/乙锭同二聚体（C/EthD）作为替代活力测定，并发现FDA/PI与SYTO/EB或C/EthD染色之间存在相当大的差异。从尸体胰腺经胶原酶消化、机械分离和连续Ficoll梯度离心纯化后获得人胰岛制剂。对于每种制备物，对每种染色剂的50个胰岛的两个重复样品进行计数，并计算活力百分比。SYTO/EB和C/EthD的结果几乎相同[分别为57.6 +/- 7.3%和57.9 +/-7.2%（平均值+/- SEM），N = 11]。然而，与SYTO/EB相比，FDA/PI染色的胰岛显示出持续升高的值。胰岛活性的准确评估仍然是胰岛产品释放的关键决定因素。FDA/PI评分和视觉质量之间的差异，与替代染色相比，表明FDA/PI染色可能不是评估胰岛活力的最佳方法。
Membrane integrity fluorescent staining is used routinely to evaluate islet viability. Results are used as one of the determining factors in islet product release criteria, and are used to assess the efficacy of different culture conditions. Recently, it has been observed that there is variation in the viability staining of freshly isolated islets based on which viability assay is used. This investigation compares three membrane integrity stains for the viability assessment of isolated human islets. Fluorescein diacetate/propidium iodide (FDA/PI), the current standard method for assessing islet viability, demonstrates intense extracellular fluorescence, reducing the differential staining of intact islets. We further evaluated SYTO-13/ethidium bromide (SYTO/EB) and calcein AM/ethidium homodimer (C/EthD) as alternative viability assays, and found considerable variation between FDA/PI and either SYTO/EB or C/EthD staining. Preparations of human islets were obtained from cadaveric pancreata after collagenase digestion, mechanical separation, and purification by continuous Ficoll gradient centrifugation. For each preparation, two replicate samples of 50 islets were counted for each stain, and the percent viability calculated. The results for SYTO/EB and C/EthD were nearly identical [57.6 +/- 7.3% and 57.9 +/- 7.2%, respectively (mean +/- SEM), N = 11]. FDA/PI-stained islets, however, showed consistently elevated values when compared to SYTO/EB. Accurate assessment of islet viability remains a critical determinant of islet product release. The discrepancies found between FDA/PI scoring and visual quality, compared with alternative stains, suggests that the FDA/PI stain may not be the optimal approach to assess islet viability.