Abstract
Metabolism and differentiation of cultured cells are influenced by changes in cellular morphology. In this study, we investigated the differences in cell proliferation and neuronal differentiation of NT2 cells in monolayer (2D) and spheroid (3D) cultures. In the monolayer culture, the cells adhered and extended on a tissue culture plate. For the spheroid culture, we fabricated a microwell chip comprising 195 circular microwells (600 ìm in diameter) on a cutture plate, and the surface was modified with polyethylene glycol to promote spheroid formation. The cells were aggregated in each microwell and formed spheroids within 24 hours of culture, and the spheroid morphology was maintained thoughout the culture period. Although the cell proliferation ability in monolayer culture was higher than that in spheroid culture, the neuronal differentiation ability of NT2 spheroid culture was higher than that in monolayer culture. Furthermore, the neuronal differentiation of NT2 spheroids was dramatically enhanced by retinoic acid treatment. These results indicate that NT2 cell properties are influenced by differences in cell morphologies, and that spheroid culture is a promising technique to induce neuronal differentiation.
Highlights
The central nervous system manages neural communication and plays essential roles in maintaining normal physiology
Among various stem cells including embryonic stem (ES) cells, induced pluripotent stem cells, mesenchymal stem cells (MSCs), and so on, human embryonal carcinoma cell line NTERA-2 clone D1 (NT2) cells are known as a typical cell model for neuronal differentiation in vitro (Serra et al, 2007; Abolpour Mofrad et al, 2016), and have been utilized for developmental neurotoxicity testing (Hill et al, 2008) and neuronal transplantation studies (Kondziolka and Wechsler, 2008)
In the microwell chip culture, the cells were aggregated in each microwell and formed spheroids within 24 hours of culture, and the spheroid morphology was maintained throughout the culture period
Summary
The central nervous system manages neural communication and plays essential roles in maintaining normal physiology. Nerve cells have been used for various applications such as neural tissue engineering and pharmacological, toxicological, and fundamental cell biology studies. As one of the strategies for the success of such applications, it is important to control the neural differentiation of stem cells. NT2 cells can differentiate into neurons by treatment with retinoic acid (RA), an inducer of mammalian neural development (Coyle et al, 2011). Some researchers have reported that the neural differentiation of NT2 cells is promoted by the formation of spheroids (spherical multi-cellular aggregates), which are formed by the rearrangement and compaction of cell aggregates (Serra et al, 2009). The control of spheroid size is an important issue, because it affects the differentiation fate of stem cells
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