Investigating the Effect of Stable Glutamine on the Neuronal Differentiation of PC-12 Cells in 2D- and 3D-Culture Media

Glutamine (Gln) is an essential amino acid with a wide range of cellular functions and is necessary for cell proliferation. It is usually added to the culture media in the form of L-glutamine, which is highly unstable and degrades in a temperature-dependent manner during the culture period. Although, Gln is beneficial for the cells, its degradation produces ammonia which is toxic and negatively affect cell culture. Pheochromocytoma cells (PC-12), originating from cancerous cells of the rat adrenal gland, are considered as a suitable model to study the differentiating effects of different factors. Previous studies showed the importance of Gln in the normal growth and differentiation of the cells. Alginate, is one of the biomaterials currently used as a natural scaffold for the induction of neuronal differentiation. In the present experimental research, the effect of stable and elevated levels of Gln on the growth and neuronal differentiation of PC-12 cells was compared under 2D- and 3D- (sodium alginate hydrogel beads) culture conditions. The cells’ viabilities were determined and compared between experimental groups using live/dead cell staining by Acridine orange/Propidium iodide (AO/PI), and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test. Furthermore, cells were stained using cresyl violet to detect neuronal Nissl bodies. The induction of differentiation was confirmed using immunocytochemical analysis of Nestin and β-tubulin III proteins and Cells’ nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Results showed that high concentration of Gln can induce neuronal differentiation in PC-12 cells under both 2D- and 3D- culture conditions and increases the expression of progenitor and mature neuronal markers Nestin and β-tubulin III, respectively.