We established a 3D model of human NT2/D1-derived early neural progenitor cells immobilised in alginate microfibres as a system for testing the neurotoxicity of energy drinks and their components, either alone, together, or in combination with alcohol. The in vitro system supports the retinoic acid-induced neurogenesis of NT2/D1 cells, and the proliferative capacity of the NT2/D1-derived early neural progenitor cells was maintained in the 3D environment. Cell cycle distribution and the expression of pluripotency markers (SOX2, OCT4 and NANOG), early neural markers (SOX3, PAX6 and miR-219), and Cyclin D1 (a marker of neural commitment), showed profiles characteristic of early neural progenitors. Treatments with an energy drink and its major components (caffeine and taurine) — either alone, together, or in combination with alcohol — had different effects on the proliferative capacity of the NT2/D1-derived early neural progenitor cells in the 2D and 3D models. In the 2D-cultured cells, all treatments except for caffeine led to a significant decrease, while cells within the 3D model exhibited a significant increase after treatment with caffeine, or after combined treatment with energy drink and alcohol. Preliminary findings suggesting that there were no treatment effects on OCT4 and PAX6 protein expression in either model, should be further confirmed. This human cell-based 3D model could potentially represent a rapid and cost-effective system for assessing the acute and long-term neurotoxicity of various compounds.
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