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

Document Type : Research Articles

Authors

• Seyed Majid Jalalian Hosseini ¹
• Javad Baharara ²
• Shahrbanoo Oryan ³
• Mohammad Amin Kerachian ^{4, 5}

¹ Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran

² Department of Biology and Research Center for Animal Development Applied Biology, Mashhad Branch, Islamic Azad University, Mashhad, Iran

³ Department of Zoology, Faculty of Biological Sciences, Kharazmi University, Tehran, Iran

⁴ Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran

⁵ Department of Medical Genetics, Faculty of Medicine, University of Medical Sciences, Mashhad, Iran

https://doi.org/10.22067/jcmr.2022.78495.1047

Abstract

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.

Keywords

• Neural differentiation
• Glutamine
• PC-12 cells
• Alginate

20.1001.1.20089147.2022.14.1.7.7

References

Aamodt J. M. aznd Grainger D. W. (2016) Extracellular matrix-based biomaterial scaffolds and the host response. Biomaterials 86:68-82.

Abe H., Ino K., Li C.-Z., Kanno Y., Inoue K. Y., Suda A., Kunikata R., Matsudaira M., Takahashi Y. and Shiku H. (2015) Electrochemical imaging of dopamine release from three-dimensional-cultured PC12 cells using large-scale integration-based amperometric sensors. Analytical chemistry 87:6364-6370.

Abnosi M., Najafabadi M., Soleimani Mehranjani M., Momeni H. and Safarabadi F. (2010) Preparation of Natural Scaffold from Sciatic Nerve and Viability Evaluation of Seeded Rat Bone Marrow Mesenchymal Stem Cells. Journal of Cell & Tissue 1.43-52

Agarwal G., Roy A., Kumar H. and Srivastava A. (2022) Graphene-collagen cryogel controls neuroinflammation and fosters accelerated axonal regeneration in spinal cord injury. Biomaterials Advances:212971.1-12.

Akter F. 2016. Principles of tissue engineering. In Tissue engineering made easy. Elsevier. 3-16.

Andersen T., Auk-Emblem P. and Dornish M. (2015) 3D cell culture in alginate hydrogels. Microarrays 4:133-161.

Baiguera S., Del Gaudio C., Lucatelli E., Kuevda E., Boieri M., Mazzanti B., Bianco A. and Macchiarini P. (2014) Electrospun gelatin scaffolds incorporating rat decellularized brain extracellular matrix for neural tissue engineering. Biomaterials 35:1205-1214.

Ban K. and Kozar R. A. (2010) Glutamine protects against apoptosis via downregulation of Sp3 in intestinal epithelial cells. American Journal of Physiology-Gastrointestinal and Liver Physiology 299:G1344-G1353

Bernal A. and Arranz L. (2018) Nestin-expressing progenitor cells: function, identity and therapeutic implications. Cellular and Molecular Life Sciences 75:2177-2195.

Brose S. A., Marquardt A. L. and Golovko M. Y. (2014) Fatty acid biosynthesis from glutamate and glutamine is specifically induced in neuronal cells under hypoxia. Journal of neurochemistry 129:400-412.

Chen S., Xia Y., Zhu G., Yan J., Tan C., Deng B., Deng J., Yin Y. and Ren W. (2018) Glutamine supplementation improves intestinal cell proliferation and stem cell differentiation in weanling mice. Food & nutrition research 62.1-9.

Cheravi M., Baharara J., Yaghmaei P. and Roudbari N. H. (2022) The effect of cerebrospinal fluid-derived exosomes on neural differentiation of adipose mesenchymal stem cells in alginate hydrogel scaffold. Nova Biologica Reperta 8:265-278.

Darbinian N. 2013. Cultured Cell Line Models of Neuronal Differentiation: NT2, PC12. In Neuronal Cell Culture. Springer. 23-33

Geranmayeh M., Baghbanzadeh A., Barin A., Salar-Amoli J. and Dehghan M. (2015) Effects of different culture media on optimization of primary neuronal cell culture for in vitro models assay. Iranian Journal of Veterinary Medicine 9:163-170.

Gordon J., Amini S. and White M. K. 2013. General overview of neuronal cell culture. In Neuronal Cell Culture. Springer. 1-8.

Katebi S., Esmaeili A., Ghaedi K. and Zarrabi A. (2019) Superparamagnetic iron oxide nanoparticles combined with NGF and quercetin promote neuronal branching morphogenesis of PC12 cells. International journal of nanomedicine 14:2157.-2169

Kim Y., Kang K., Jeong J., Paik S. S., Kim J. S., Park S. A., Kim W. D., Park J. and Choi D. (2017) Three-dimensional (3D) printing of mouse primary hepatocytes to generate 3D hepatic structure. Annals of surgical treatment and research 92:67-72.

Li J., Ghazwani M., Liu K., Huang Y., Chang N., Fan J., He F., Li L., Bu S. and Xie W. (2017) Regulation of hepatic stellate cell proliferation and activation by glutamine metabolism. PLoS One 12:e0182679.1-17.

Martins J. P., Ferreira M. P., Ezazi N. Z., Hirvonen J. T., Santos H. A., Thrivikraman G., França C. M., Athirasala A., Tahayeri A. and Bertassoni L. E. (2018) 3D printing: Prospects and challenges. Nanotechnologies in Preventive and Regenerative Medicine:299-379.

Mobini S., Song Y. H., McCrary M. W. and Schmidt C. E. (2019) Advances in ex vivo models and lab-on-a-chip devices for neural tissue engineering. Biomaterials 198:146-166.

Raizel R. and Tirapegui J. (2018) Role of glutamine, as free or dipeptide form, on muscle recovery from resistance training: A review study. Nutrire 43:1-10.

Razavi S., Khosravizadeh Z., Bahramian H. and Kazemi M. (2015) Time-Dependent Effect of Encapsulating Alginate Hydrogel on Neurogenic Potential. Cell Journal (Yakhteh) 17:304-311.

Rubin H. (2019) Deprivation of glutamine in cell culture reveals its potential for treating cancer. Proceedings of the National Academy of Sciences 116:6964-6968.

Sarkadi B., Meszaros K., Krencz I., Canu L., Krokker L., Zakarias S., Barna G., Sebestyen A., Papay J. and Hujber Z. (2020) Glutaminases as a novel target for SDHB-associated pheochromocytomas /paragangliomas. Cancers 12:599.1-25.

Suh J.-K., Nejad J. G., Lee Y.-S., Kong H.-S., Lee J.-S. and Lee H.-G. (2022) Effects of L-glutamine supplementation on degradation rate and rumen fermentation characteristics in vitro. Animal bioscience 35:422-433.

Wang H., Tang C., Jiang Z., Zhou X., Chen J., Na M., Shen H. and Lin Z.(2017 (Glutamine promotes Hsp70 and inhibits α-Synuclein accumulation in pheochromocytoma PC12 cells. Experimental and Therapeutic Medicine 14:1253-1259.

Wang Q., Zhang C., Zhang L., Guo W., Feng G., Zhou S., Zhang Y., Tian T., Li Z. and Huang F. (2014) The preparation and comparison of decellularized nerve scaffold of tissue engineering. J Biomed Mater Res A 102:4301-4308.

Wiatrak B., Kubis-Kubiak A., Piwowar A. and Barg E. (2020) PC12 cell line: cell types, coating of culture vessels, differentiation and other culture conditions. Cells 9:9581-15..

Yi S., Xu L. and Gu X. (2019) Scaffolds for peripheral nerve repair and reconstruction. Experimental neurology 319:112761.1-11.

Yousefi Taemeh S., Mehrzad J. and Dehghani H. (2021) Effect of glutamine stability on the long-term culture and line establishment of chicken primordial germ cells. Journal of Cell and Molecular Research 13:44-53.

Zhao Y., Wang Q., Wang Y., Li J., Lu G. and Liu Z. (2019) Glutamine protects against oxidative stress injury through inhibiting the activation of PI3K/Akt signaling pathway in parkinsonian cell model. Environmental health and preventive medicine 24:1-7.