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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Abstract
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.