Evaluation of in vitro proliferative activity of human fetal neural stem/progenitor cells using indirect measurements of viable cells based on cellular metabolic activity
Evaluation of in vitro proliferative activity of human fetal neural stem/progenitor cells using indirect measurements of viable cells based on cellular metabolic activity
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DOI：
10.1002/jnr.10377
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发表时间：
2002-09-15
影响因子：
4.2
通讯作者：
Miyake, J
中科院分区：
文献类型：
--
作者：
Kanemura, Y;Mori, H;Miyake, J
为了扩大人类神经干/祖细胞（NSPC）培养物的临床应用，我们需要知道这些细胞可以活多久而不失去其增殖和分化的能力;因此，需要一种方便的方法来估计在神经球培养物中生长的人类NSPC的增殖活性，因为直接细胞计数是费力的并且可能不准确。在这里，我们从人胎儿前脑分离NSPCs并制备神经球培养物。我们确定了活细胞的数量，并估计其在长期培养中的增殖活性，使用两种方法，间接测量活细胞数，基于它们的代谢活性：WST-8测定，其中甲染料产生后，减少水溶性四唑盐WST-8脱氢酶活性，和ATP测定，测量总细胞血浆的ATP含量。我们将这些测定的结果与使用5-溴-2 '-脱氧尿苷掺入测定通过DNA合成估计的增殖活性进行了比较。我们发现活的人NSPCs的数量与WST-8和ATP测定中获得的代谢反应产物成正比。两种方法产生了相同的细胞生长曲线，显示出指数增殖期和长期培养中群体倍增时间的变化。他们还表明，人类NSPCs可以在体外扩增长达200天，而不会失去其增殖和分化的能力。我们的研究结果表明，基于代谢活性的活细胞的间接测量，特别是ATP测定，是确定完整神经球中活的人NSPCs数量的非常有效和可重复的方法。(C)2002 Wiley-Liss，Inc.
To scale up human neural stem/progenitor cell (NSPC) cultures for clinical use, we need to know how long these cells can live ex vivo without losing their ability to proliferate and differentiate; thus, a convenient method is needed to estimate the proliferative activity of human NSPCs grown in neurosphere cultures, as direct cell counting is laborious and potentially inaccurate. Here, we isolated NSPCs from human fetal forebrain and prepared neurosphere cultures. We determined the number of viable cells and estimated their proliferative activity in long-term culture using two methods that measure viable cell numbers indirectly, based on their metabolic activity: the WST-8 assay, in which a formazan dye is produced upon reduction of the water-soluble tetrazolium salt WST-8 by dehydrogenase activity, and the ATP assay, which measures the ATP content of the total cell plasma. We compared the results of these assays with the proliferative activity estimated by DNA synthesis using the 5-bromo-2'-deoxyuridine incorporation assay. We found the numbers of viable human NSPCs to be directly proportional to the metabolic reaction products obtained in the WST-8 and ATP assays. Both methods yielded identical cell growth curves, showing an exponentially proliferative phase and a change in the population doubling time in long-term culture. They also showed that human NSPCs could be expanded for up to 200 days ex vivo without losing their ability to proliferate and differentiate. Our findings indicated that indirect measurements of viable cells based on metabolic activity, especially the ATP assay, are very effective and reproducible ways to determine the numbers of viable human NSPCs in intact neurospheres. (C) 2002 Wiley-Liss, Inc.