Abstract
In order to develop a simplified method for long-term primary culture of highly-pure rat embryonic hippocampal neurons of low-density (103 cells/cm2), we optimized and modified conventional culturing methods. The modifications of our simplified method include: (1) combinational application of two growth substrates, tail collagen and poly-L-lysine, to coat plastic culture dishes and coverslips for a better neuronal attachment; (2) dissociation of hippocampal tissues with combinational use of two milder enzymes (collagenase and dispase) and trypsin of a lower concentration to minimize enzymatic damages to cultured neurons; (3) a cell pre-plating step to preliminarily eliminate the contaminating non-neuronal cells; (4) a modified culture medium as a critical step to promote highly pure neurons of low-density for a long term; and (5) appropriately reduced frequency and volume of refreshment of the culture medium. Using our modified method, the β-tubulin III-immunostained and Hoechst 33342 counterstained neurons harvested a steady and healthy growth with a longer culture time of over 35 days, and a clear distinction between TAU-1- and MAP2-immunoreactive neurites was apparent at the early culturing period. In addition, the purity of neurons was over 95% at the different time points in comparison with the control culture using conventional serum-free method in which most neurons degenerated and died within 5 days. Thus, our modified method proved to be a simple, feasible as well as time- and resource-saving approach for a long-term survival of pure rat embryonic hippocampal neurons of low-density.
References
Sasaki Y, Fukushima N, Yoshida A, Ueda H (1998) Low-density induced apoptosis of cortical neurons is inhibited by serum factors. Cell Mol Neurobiol 18:487–496
Fujita R, Yoshida A, Mizuno K, Ueda H (2001) Cell density-dependent death mode switch of cultured cortical neurons under serum-free starvation stress. Cell Mol Neurobiol 21:317–324
Martin DL (1992) Synthesis and release of neuroactive substances by glial cells. Glia 5:81–94
Pellitteri R, Zicca A, Mancardi GL, Savio T, Cadoni A (2001) Schwann cell-derived factors support serotoninergic neuron survival and promote neurite outgrowth. Eur J Histochem 45:367–376
Walsh E, Ueda Y, Nakanishi H, Yoshida K (1992) Neuronal survival and neurite extension supported by astrocytes co-cultured in transwells. Neurosci Lett 138:103–106
Price PJ (1975) Preparation and use of rat-tail collagen. Methods in Cell Sci 1:43–44
Phifer CB, Terry LM (1986) Use of hypothermia for general anesthesia in preweanling rodents. Physiol Behav 38:887–890
Goslin K, Asmussen H, Banker G (1998) Rat hippocampal neurons in low density culture. Culturing nerve cells. MIT Press, Cambridge, pp 339–370
James CD, Davis R, Meyer M, Turner A, Turner S, Withers G, Kam L, Banker G, Craighead H, Isaacson M, Turner J, Shain W (2000) Aligned microcontact printing of micrometer-scale poly-L-lysine structures for controlled growth of cultured neurons on planar microelectrode arrays. IEEE Trans Biomed Eng 47:17–21
Woerly S, Maghami G, Duncan R, Subr V, Ulbrich K (1993) Synthetic polymer derivatives as substrata for neuronal adhesion and growth. Brain Res Bull 30:423–432
Ruegg UT, Hefti F (1984) Growth of dissociated neurons in culture dishes coated with synthetic polymeric amines. Neurosci Lett 49:319–324
Maxwell GD (1976) Substrate dependence of cell migration from explanted neural tubes in vitro. Cell Tiss Res 172:325–330
Bunge RP, Bunge MB (1978) Evidence that contact with connective tissue matrix is required for normal interaction between Schwann cells and nerve fibers. J Cell Biol 78:943–950
Elsdall T, Bard J (1972) Collagen substrate for studies on cell behavior. J Cell Biol 54:626–637
Lee HY, Greene LA, Mason CA, Manzini MC (2009) Isolation and culture of post-natal mouse cerebellar granule neuron progenitor cells and neurons. J Vis Exp 16:990
Tafti M, Ghyselinck NB (2007) Functional implication of the vitamin A signaling pathway in the brain. Arch Neurol 64:1706–1711
Dringen R (2000) Metabolism and functions of glutathione in brain. Prog Neurobio 62:649–671
Butterfield DA, Koppal T, Subramaniam R, Yatin S (1999) Vitamin E as an antioxidant/free radical scavenger against amyloid beta-peptide-induced oxidative stress in neocortical synaptosomal membranes and hippocampal neurons in culture: insights into Alzheimer’s disease. Rev Neurosci 10:141–149
Ono K, Yoshiike Y, Takashima A, Hasegawa K, Naiki H, Yamada M (2004) Vitamin A exhibits potent antiamyloidogenic and fibril-destabilizing effects in vitro. Exp Neurol 189:380–392
Diaz-Hernandez JI, Almeida A, Delgado-Esteban M, Fernandez E, Bolanos JP (2005) Knockdown of glutamate-cysteine ligase by small hairpin RNA reveals that both catalytic and modulatory subunits are essential for the survival of primary neurons. J Biol Chem 280:38992–39001
Mizui T, Kinouchi H, Chan PH (1992) Depletion of brain glutathione by buthionine sulfoximine enhances cerebral ischemic injury in rats. Am J Physiol 262:H313–H317
Sagara JI, Fujiwara K, Sakakura Y, Sato H, Bannai S, Makino N (2007) Beneficial effect of antioxidants in purified neurons derived from rat cortical culture. Brain Res 1131:11–16
Sagara JI, Miura K, Bannai S (1993) Maintenance of neuronal glutathione by glial cells. J Neurochem 61:1672–1676
Slivka A, Spina MB, Cohen G (1987) Reduced and oxidized glutathione in human and monkey brain. Neurosci Lett 74:112–118
Dringen R, Hirrlinger J (2003) Glutathione pathways in the brain. Bio Chem 384:505–516
Martinez-Cruz F, Pozo D, Osuna C, Espinar A, Marchante C, Guerrero JM (2002) Oxidative stress induced by phenylketonuria in the rat: prevention by melatonin, vitamin E and vitamin C. J Neurosci Res 69:550–558
Ioudina M, Uemura E, Greenlee HW (2004) Glucose insufficiency alters neuronal viability and increases susceptibility to glutamate toxicity. Brain Res 1004(1–2):188–192
Liu D, Chan SL, de Souza-Pinto NC, Slevin JR, Wersto RP, Zhan M, Mustafa K, de Cabo R, Mattson MP (2006) Mitochondrial UCP4 mediates an adaptive shift in energy metabolism and increases the resistance of neurons to metabolic and oxidative stress. Neuromol Med 8:389–414
Albrecht J, Sonnewald U, Waagepetersen HS, Schousboe A (2007) Glutamine in the central nervous system: function and dysfunction. Front Biosci 12:332–343
Yang H, Liang Z, Li J, Cheng X, Luo N, Ju G (2006) Optimized and efficient preparation of astrocyte cultures from rat spinal cord. Cytotechnology 52:87–97
Geshi M, Takenouchi N, Yamauchi N, Nagai T (2000) Effects of sodium pyruvate in nonserum maturation medium on maturation, fertilization, and subsequent development of bovine oocytes with or without cumulus cells. Biol Reprod 63:1730–1734
Suh SW, Aoyama K, Matsumori Y, Liu J, Swanson RA (2005) Pyruvate administered after severe hypoglycemia reduces neuronal death and cognitive impairment. Diabetes 54:1452–1458
Andrae U, Singh J, Ziegler-Skylakakis K (1985) Pyruvate and related alphaketoacids protect mammalian cells in culture against hydrogen peroxideinduced cytotoxicity. Toxicol Lett 28:93–98
Needels DL, Nieto-Sampedro M, Cotman CW (1986) Induction of a neurite-promoting factor in rat brain following injury or deafferentation. Neurosci 18:517–526
Acknowledgments
This work was supported by Natural Science Foundation of China (Nos. 30973088, 30872829 and 30571998).
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Hao Yang and Rui Cong contributed equally to this work.
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Yang, H., Cong, R., Na, L. et al. Long-Term Primary Culture of Highly-Pure Rat Embryonic Hippocampal Neurons of Low-Density. Neurochem Res 35, 1333–1342 (2010). https://doi.org/10.1007/s11064-010-0189-0
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DOI: https://doi.org/10.1007/s11064-010-0189-0