Effects of different culture media on optimization of primary neuronal cell culture for in vitro models assay

Document Type : Physiology

Authors

1 Department of Basic Sciences, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran

2 Department of Microbiology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran

3 Department of Surgery and Radiology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran

Abstract

Background: In vitro model studies are becoming increasingly popular for experimental research designs. They include isolation and expansion of cells of a particular tissue, such as the nervous tissue which contributes to understanding the underlying mechanisms in many pathologies. It enables  the scrutinization of intracellular signaling pathways responsible for cell death. OBJECTIVES: In the literature, there are different methods for the isolation and culture of rat embryonic cortical neurons. However, this study developed a feasible, rapid and easily performable method. METHODS: Isolation of neurons was performed without using enzymatic digestion. Primary cortical cultures neurite outgrowth and neuron numbers per field of common mediums were compared for neuronal cells isolation and expansion. In this study, three different culture mediums were considered: Medium I: Neurobasal medium, B-27 and L-glutamine; Medium II: DMEM, FBS and L-glutamine; and Medium III: DMEM/F-12, FBS and L-glutamine. RESULTS: High survival rate and number of neurons was obtained with the current method. The best neuronal growth was achieved by Medium I, while Medium II and III had moderate effect on the neurite outgrowth. CONCLUSIONS: Enzyme-free treatment was introduced and Medium I was used as an alternative method for optimal neuron isolation and expansion. The neuronal cultures are similar to nervous tissue in physiological aspects. Hence, Medium I is more similar to the in vivo condition compared to Mediums II and III.

Keywords

Article Title [Persian]

تأثیر محیط کشت های مختلف بر روی بهینه سازی کشت اولیه سلول های عصبی برای ارزیابی مدل های برون تنی

Authors [Persian]

  • محمد حسین گرانمایه 1
  • علی باغبان زاده 1
  • عباس برین 2
  • جمیله سالار آملی 1
  • محمد مهدی دهقان 3
1 گروه علوم پایه، دانشکده دامپزشکی دانشگاه تهران، تهران، ایران
2 گروه میکروبیولوژی، دانشکده دامپزشکی دانشگاه تهران، تهران، ایران
3 گروه جراحی و رادیولوژی، دانشکده دامپزشکی دانشگاه تهران، تهران، ایران
Abstract [Persian]

زمینه مطالعه:  مطالعه مدل‌های برون‌تنی برای طراحی تحقیقات تجربی رو به افزایش است. از جمله این نوع مطالعات جداسازی و کشت سلول‌ها از بافت‌های مختلف مانند بافت‌های عصبی می‌باشد، که در شناخت مکانیسم‌های اساسی آسیب‌ها می‌تواند بسیار کمک کننده باشد و ما را قادر می‌سازد تا مسیرهای سیگنالینگ درون سلولی مسئول مرگ سلولی را به دقت مورد بررسی قرار دهیم. هدف: اگر چه تا کنون، روش‌های مختلفی برای کشت نورون‌های قشری جنین رت توصیف شده است. با این حال، مطالعه موجود روش عملی و سریعی را معرفی می‌کند که می‌تواند در این نوع مطالعات مورد استفاده قرار گیرد. روش کار: در این مطالعه، از سه محیط کشت مختلف، به ترتیبی که توضیح داده می‌شود برای جداسازی و کشت نورون‌ها در کشت اولیه قشری بدون استفاده از هضم آنزیمی استفاده گردید: محیط‌های مورد استفاده عبارت بودند از: محیط I: محیط نوروبازال، B-27 و ال-گلوتامین؛ محیط II: DMEM، FBS و ال-گلوتامین؛ و محیط III:رFBS،ز12-DMEM/F، و ال-گلوتامین. نتایج: میزان زنده مانی نورونی در این مطالعه بسیار قابل توجه بود و بهترین رشد نورونی در محیط I مشاهده گردید، در حالیکه محیط‌های II و III اثر متوسطی بر روی رشد نوریت داشتند. نتیجه‌گیری نهایی: نتایج این مطالعه نشان می‌دهد محیط I بیشترین تشابه با شرایط برون تنی را در مقایسه با محیط‌های II و III ایجاد نموده و سلول‌های نورونی بیشتر قادر به حفظ خصوصیات فیزیولوژیک خود در این محیط می‌باشند.

Keywords [Persian]

  • نورونهای قشری
  • جداسازی
  • کشت اولیه سلول
  • رت

References

Barclay, D.C., Hallbergson, A.F., Montague, J.R., Mudd, L.M. (2005) Reversal of ethanol toxicity in embryonic neurons with growth factors and estrogen. Brain Res Bull. 67: 459-465.
Brewer, G.J., Torricelli, J.R. (2007) Isolation and culture of adult neurons and neurospheres. Nat Protoc. 2: 1490-1498.
Briz, V., Molina-Molina, J.M., Sánchez-Redondo, S., Fernández, M.F., Grimalt, J.O., Olea, N., Rodríguez-Farré, E., Suñol, C. (2011) Differential estrogenic effects of the persistent organochlorine pesticides dieldrin, endosulfan, and lindane in primary neuronal cultures. Toxicol Sci. 120: 413-427.
Chen, T., Fei, F., Jiang, X.F., Zhang, L., Qu, Y., Huo, K., Fei, Z. (2012) Down-regulation of Homer1b/c attenuates glutamate-mediated excitotoxicity through endoplasmic reticulum and mitochondria pathways in rat cortical neurons. Free Radic Biol Med. 52: 208-217.
Choi, D.W., Maulucci-Gedde, M., Kriegstein, A.R. (1987) Glutamate neurotoxicity in cortical cell culture. J Neurosci. 7: 357-368.
Dhandapani, K.M., Wade, F.M., Wakade, C., Mahesh, V.B., Brann, D.W. (2005) Neuroprotection by stem cell factor in rat cortical neurons involves AKT and NFκB. J Neurochem. 95: 9-19.
Harrill, J.A., Freudenrich, T.M., Machacek, D.W., Stice, S.L., Mundy, W.R. (2010) Quantitative assessment of neurite outgrowth in human embryonic stem cell-derived hN2™ cells using automated high-content image analysis. Neurotoxicology. 31: 277-290.
Harrill, J.A., Freudenrich, T.M., Robinette, B.L., Mundy, W.R. (2011) Comparative sensitivity of human and rat neural cultures to chemical-induced inhibition of neurite outgrowth. Toxicol Appl Pharmacol. 256: 268-280.
Harrill, J.A., Mundy, W.R. (2011) Quantitative assessment of neurite outgrowth in PC12 cells. Methods Mol Biol. 758: 331-348.
Harrill, J.A., Robinette, B.L., Freudenrich, T., Mundy, W.R. (2013) Use of high content image analyses to detect chemical-mediated effects on neurite sub-populations in primary rat cortical neurons. Neurotoxicology. 34: 61-73.
Hasan, M.R., Kim, J.H., Kim, Y.J., Kwon, K.J., Shin, C.Y., Kim, H.Y., Han, S.H., Choi, D.H., Lee, J. (2013) Effect of HDAC inhibitors on neuroprotection and neurite outgrowth in primary rat cortical neurons following ischemic insult. Neurochem Res. 38: 1921-1934.
Heber, S., Herms, J., Gajic, V., Hainfellner, J., Aguzzi, A., Rulicke, T., Kretzschmar, H., Von Koch, C., Sisodia, S., Tremml, P., Lipp, H.P., Wolfer, D.P., Muller, U. (2000) Mice with combined gene knock-outs reveal essential and partially redundant functions of amyloid precursor protein family members. J Neurosci. 20: 7951-7963.
Jeerage, K.M., Oreskovic, T.L., Hume, S.L. (2012) Neurite outgrowth and differentiation of rat cortex progenitor cells are sensitive to lithium chloride at non-cytotoxic exposures. Neurotoxicology. 33: 1170-1179.
Kim, H.J., Magrane, J. (2011) Isolation and culture of neurons and astrocytes from the mouse brain cortex. Methods Mol Biol. 793: 63-75.
Kim, J.Y., Jeong, H.Y., Lee, H.K., Kim, S., Hwang, B.Y., Bae, K., Seong, Y.H. (2012) Neuroprotection of the leaf and stem of Vitis amurensis and their active compounds against ischemic brain damage in rats and excitotoxicity in cultured neurons. Phytomedicine. 19: 150-159.
Koh -y J., Choi, D.W. (1988) Vulnerability of cultured cortical neurons to damage by excitotoxins: Differential susceptibility of neurons containing NADPH-diaphorase. J Neurosci. 8: 2153-2163.
Koo, K.A., Kim, S.H., Oh, T.H., Kim, Y.C. (2006) Acteoside and its aglycones protect primary cultures of rat cortical cells from glutamate-induced excitotoxicity. Life Sci. 79: 709-716.
Kuwagata, M. (2012) Current problems of in vivo developmental neurotoxicity tests and a new in vivo approach focusing on each step of the developing central nervous system. Congenit Anom. 52: 129-139.
Li, D., Gu, X., Lu, L., Liang, L. (2010) Effects of phenylalanine on the survival and neurite outgrowth of rat cortical neurons in primary cultures: Possible involvement of brain-derived neurotrophic factor. Mol Cell Biochem. 339: 1-7.
Li, L., Liu, Q.R., Xiong, X.X., Liu, J.M., Lai, X.J., Cheng, C., Pan, F., Chen, Y., Yu, S.B., Yu, A.C.H., Chen, X.Q. (2014) Neuroglobin promotes neurite outgrowth via differential binding to PTEN and Akt. Mol Neurobiol. 49: 149-162.
Liu, D., Bi, Y., Liu, Z., Liu, H., Li, Z. (2014) The expression of vesicular glutamate transporter 3 and vesicular monoamine transporter 2 induced by brain-derived neurotrophic factor in dorsal root ganglion neurons in vitro. Brain Res Bull. 100: 93-106.
Maekawa, F., Tsuboi, T., Oya, M., Aung, K.H., Tsukahara, S., Pellerin, L., Nohara, K. (2013) Effects of sodium arsenite on neurite outgrowth and glutamate AMPA receptor expression in mouse cortical neurons. Neurotoxicology. 37: 197-206.
Nabiuni, M., Rasouli, J., Parivar, K., Kochesfehani, H.M., Irian, S., Miyan, J.A. (2012) In vitro effects of fetal rat cerebrospinal fluid on viability and neuronal differentiation of PC12 cells. Fluids Barriers CNS. 9: 8.
Noshita, T., Murayama, N., Oka, T., Ogino, R., Nakamura, S., Inoue, T. (2012) Effect of bFGF on neuronal damage induced by sequential treatment of amyloid β and excitatory amino acid in vitro and in vivo. Eur J Pharmacol. 695: 76-82.
Pacifici, M., Peruzzi, F. (2012) Isolation and culture of rat embryonic neural cells: a quick protocol. J Vis Exp. JoVE. 63: e3965, doi:10.3791/3965.
Pinton, S., Souza, A. C., Sari, M.H.M., Ramalho, R.M., Rodriguesb, C.M.P., Nogueiraa, C.W. (2013) P,p’-Methoxyl-diphenyl diselenide protects against amyloid-β induced cytotoxicity in vitro and improves memory deficits in vivo. Behav Brain Res. 247: 241-247.
Radio, N.M., Freudenrich, T.M., Robinette, B.L., Crofton, K.M., Mundy, W.R. (2010) Comparison of PC12 and cerebellar granule cell cultures for evaluating neurite outgrowth using high content analysis. Neurotoxicol Teratol. 32: 25-35.
Wu, H., Mahmood, A., Qu, C., Xiong, Y., Chopp, M. (2012) Simvastatin attenuates axonal injury after experimental traumatic brain injury and promotes neurite outgrowth of primary cortical neurons. Brain Res. 1486: 121-130.
Xu, J., Xilouri, M., Bruban, J., Shioi, J., Shao, Z., Papazoglou, I., Vekrellis, K., Robakis, N.K. (2011) Extracellular progranulin protects cortical neurons from toxic insults by activating survival signaling. Neurobiol Aging. 32: 2326.e2325-2326.e2316.
Yan, T., Chopp, M., Ye, X., Liu, Z., Zacharek, A., Cui, Y., Roberts, C., Buller, B., Chen, J. (2012) Niaspan increases axonal remodeling after stroke in type 1 diabetes rats. Neurobiol Dis. 46: 157-164.
Yu, X., An, L. (2002) A serum- and antioxidant-free primary culture model of mouse cortical neurons for pharmacological screen and studies of neurotrophic and neuroprotective agents. Cell Mol Neurobiol. 22: 197-206.
Zeron, M.M., Hansson, O., Chen, N., Wellington, C.L., Leavitt, B.R., Brundin, P., Hayden, M.R., Raymond, L.A. (2002) Increased sensitivity to N-methyl-D-aspartate receptor-mediated excitotoxicity in a mouse model of Huntington’s disease. Neuron. 33: 849-860.
Zhang, J., Li, L., Chen, X., Zhang, B., Wang, Y., Yamamoto, K. (2000) Effects of a traditional Chinese medicine, Qing Nao Yi Zhi Fang, on glutamate excitotoxicity in rat fetal cerebral neuronal cells in primary culture. Neurosci Lett. 290: 21-24.
Iranian Journal of Veterinary Medicine
Volume 9, Issue 3 - Serial Number 3
October 2015
Pages 163-170

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