Abstract
BackgroundNeural stem cells (NSCs) play an important role in developing potential cell-based therapeutics for neurodegenerative disease. Microfluidics has proven a powerful tool in mechanistic studies of NSC differentiation. However, NSCs are prone to differentiate when the nutrients are limited, which occurs unfavorable by fast medium consumption in miniaturized culture environment. For mechanistic studies of NSCs in microfluidics, it is vital that neuronal cell differentiation is triggered by controlled factors only. Thus, we studied the correlation between available cell medium and spontaneous neuronal cell differentiation of C17.2 NSCs in standard culture medium, and proposed the necessary microfluidic design criteria to prevent undesirable cell phenotype changes.Methodology/Principal FindingsA series of microchannels with specific geometric parameters were designed to provide different amount of medium to the cells over time. A medium factor (MF, defined as the volume of stem cell culture medium divided by total number of cells at seeding and number of hours between medium replacement) successfully correlated the amount of medium available to each cell averaged over time to neuronal cell differentiation. MF smaller than 8.3×104 µm3/cell⋅hour produced significant neuronal cell differentiation marked by cell morphological change and significantly more cells with positive β-tubulin-III and MAP2 staining than the control. When MF was equal or greater than 8.3×104 µm3/cell⋅hour, minimal spontaneous neuronal cell differentiation happened relative to the control. MF had minimal relation with the average neurite length.Significance MFs can be controlled easily to maintain the stem cell status of C17.2 NSCs or to induce spontaneous neuronal cell differentiation in standard stem cell culture medium. This finding is useful in designing microfluidic culture platforms for controllable NSC maintenance and differentiation. This study also offers insight about consumption rate of serum molecules involved in maintaining the stemness of NSCs.
Highlights
Neural stem cells (NSCs) have recently attracted significant interest for their promise in treating neurodegenerative disorders, such as Alzheimer’s disease, ischemia and Parkinson’s disease. [1– 12] Despite progress in neuronal cell differentiation and transplantation of NSCs, future success will require further understanding of the neuronal cell differentiation mechanisms. [2,4,5,7,9–11,13–20] Microfluidics has recently been shown to be a powerful tool in stem cell research, due to the advantage of precise control of individual environmental cues, single cell analysis, real-time measurement and easy integration with electrical stimulation. [21–46]
In vivo neuronal cell differentiation of NSCs occurs when there is a shortage of blood and oxygen supply, as studied in disease models like ischemia. [4,15,16,53–58] For in vitro cultures, serum withdrawal is often used to induce neuronal differentiation of NSC. [59–61] Based on the available knowledge up to date, we hypothesize that NSCs could undergo neuronal cell differentiation even in the regular NSC culture media if the volume of media available is limited, which after cell metabolism quickly becomes nutrient depleted
The medium factor (MF) successfully predicts the outcome of C17.2 NSCs in standard culture medium
Summary
Neural stem cells (NSCs) have recently attracted significant interest for their promise in treating neurodegenerative disorders, such as Alzheimer’s disease, ischemia and Parkinson’s disease. [1– 12] Despite progress in neuronal cell differentiation and transplantation of NSCs, future success will require further understanding of the neuronal cell differentiation mechanisms. [2,4,5,7,9–11,13–20] Microfluidics has recently been shown to be a powerful tool in stem cell research, due to the advantage of precise control of individual environmental cues, single cell analysis, real-time measurement and easy integration with electrical stimulation. [21–46].Concentration gradients of cytokine or growth hormone have been created in microfluidic devices to quantitatively study chemical and biological cues that initiate or facilitate neuronal cell differentiation. [22,47–50] Microfluidics have been used to introduce mechanical or topographical stimulation for the analysis of non-chemical cues on neuronal cell differentiation. [51,52] The use of microfluidics in NSC research, presents an issue with regard to dynamic nutrient concentration. As the culture volume is miniaturized, nutrient consumption from cell metabolism is much more pronounced than conventional bulk culture, while it is well established that NSCs are extremely sensitive to serum depletion. The MF was defined as the volume of culture medium normalized to the total number of cells at seeding and the feeding period It was controlled using microchannels of various heights, since it is otherwise difficult to reduce the height of culture media to below one millimeter in conventional bulk culture, considering the meniscus. We studied the correlation between available cell medium and spontaneous neuronal cell differentiation of C17.2 NSCs in standard culture medium, and proposed the necessary microfluidic design criteria to prevent undesirable cell phenotype changes
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