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Adult rat spinal cord culture on an organosilane surface in a novel serum-free medium

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  • Cell Growth/Differentiation/Apoptosis
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Summary

In this study, we have documented by morphological analysis, immunocytochemistry, and electrophysiology, the development of a culture system that promotes the growth and long-term survival of dissociated adult rat spinal cord neurons. This system comprises a patternable, nonbiological, cell growth-promoting organosilane substrate coated on a glass surface and an empirically derived novel serum-free medium, supplemented with specific growth factors (acidic fibroblast growth factor, heparin sulfate, neurotrophin-3, brain-derived neurotrophic factor, glial-derived neurotrophic factor, cardiotrophin-1, and vitronectin). Neurons were characterized by immunoreactivity for neurofilament 150, neuron-specific enolase, Islet-1 antibodies, electrophysiology, and the cultures were maintained for 4–6 wk. This culture system could be a useful tool for the study of adult mammalian spinal neurons in a functional in vitro system.

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References

  • Alexanian, A.; Nornes, H. Proliferation and regeneration of retrogradely labeled adult rat corticospinal neurons in culture. Exp. Neurol. 170:277–282; 2001.

    Article  PubMed  CAS  Google Scholar 

  • Anderson, K.; Potter, A.; Piccenna, L.; Quah, A.; Davies, K.; Cheema, S. Isolation and culture of motor neurons from the newborn mouse spinal cord. Brain Res. Brain Res. Protoc. 12:132–136; 2002.

    Article  Google Scholar 

  • Anderson, M. Differences in growth of neurons from normal and regenerated teleost spinal cord in vitro. In Vitro Cell. Dev. Biol. 29A:145–152; 1993.

    PubMed  CAS  Google Scholar 

  • Bamber, N.; Li, H.; Aebischer P.; Xu, X. Fetal spinal cord tissue in miniguidance channels promotes longitudinal axonal growth after grafting into hemisected adult rat spinal cords. Neural Plast. 6:103–121; 1999.

    PubMed  CAS  Google Scholar 

  • Brewer, G. Isolation and culture of adult rat hippocampal neurons. J. Neurosci. Methods 71:143–155; 1997.

    Article  PubMed  CAS  Google Scholar 

  • Campos, L.; Meng, Z.; Hu, G.; Chiu, D.; Ambron, R.; Martin, J. Engineering novel spinal circuits to promote recovery after spinal injury. J. Neurosci. Methods 24:2090–2101; 2004.

    CAS  Google Scholar 

  • Camu, W.; Henderson, C. Purification of embryonic rat motoneurons by panning on a monoclonal antibody to the low-affinity NGF receptor. J. Neurosci. Methods 44:59–70; 1992.

    Article  PubMed  CAS  Google Scholar 

  • Corcoran, J.; So, P.; Barber, R.; Vincent, K.; Mazarakis, N.; Mitrophanous, K. Kingsman, S.; Maden M. Retinoic acid receptor beta2 and neurite outgrowth in the adult mouse spinal cord in vitro. J. Cell Sci. 115:3779–3786; 2002a.

    Article  PubMed  CAS  Google Scholar 

  • Corcoran, J.; So, P.; Maden, M. Absence of retinoids can induce motoneuron disease in the adult rat and a retinoid defect is present in motoneuron disease patients. J. Cell Sci. 115:4735–4741; 2002b.

    Article  PubMed  CAS  Google Scholar 

  • Cuevas, P.; Carceller, F.; Gimenez-Gallego, G. Acidic fibroblast growth factor prevents post-axotomy neuronal death of the newborn rat facial nerve. Neurosci. Lett. 197:183–186; 1995.

    Article  PubMed  CAS  Google Scholar 

  • Das, M.; Molnar, P.; Devaraj, H.; Poeta, M.; Hickman J. Electrophysiological and morphological characterization of rat embryonic motoneurons in a defined system. Biotechnol. Prog. 19:1756–1761; 2003.

    Article  PubMed  CAS  Google Scholar 

  • Das, M.; Molnar, P.; Gregory, C.; Riedel, L.; Jamshidi, A.; Hickman, J. Long-term culture of embryonic rat cardiomyocytes on an organosilane surface in a serum-free medium. Biomaterials 25:5643–5647; 2004.

    Article  PubMed  CAS  Google Scholar 

  • Eckenstein, F.; Andersson, C.; Kuzis, K.; Woodward, W. Distribution of acidic and basic fibroblast growth factors in the mature, injured and developing rat nervous system. Prog. Brain Res. 103:55–64; 1994.

    PubMed  CAS  Google Scholar 

  • Ericson, J.; Thor, S.; Edlund, T.; Jessell, T.; Yamada, T. Early stages of motor neuron differentiation revealed by expression of homeobox gene Islet-1. Science 256:1555–1560; 1992.

    Article  PubMed  CAS  Google Scholar 

  • Estevez, A.; Crow, J.; Sampson, J., et al. Induction of nitric oxide-dependent apoptosis in motor neurons by zinc-deficient superoxide dismutase. Science 286:2498–2500; 1999.

    Article  PubMed  CAS  Google Scholar 

  • Frisen, J.; Haegerstrand, A.; Fried, K.; Piehl, F.; Cullheim S.; Risling, M. Adhesive/repulsive properties in the injured spinal cord: relation to myelin phagocytosis by invading macrophages. Exp. Neurol. 129:183–193; 1994.

    Article  PubMed  CAS  Google Scholar 

  • Fry, E. Central nervous system regeneration: mission impossible? Clin. Exp. Pharmacol. Physiol. 28:253–258; 2001.

    Article  PubMed  CAS  Google Scholar 

  • Geller, H.; Fawcett, J. Building a bridge: engineering spinal cord repair. Exp. Neurol. 174:125–136; 2002.

    Article  PubMed  Google Scholar 

  • Grimpe, B.; Silver, J. The extracellular matrix in axon regeneration. Prog. Brain Res. 137:333–349; 2002.

    PubMed  CAS  Google Scholar 

  • Haase, G.; Kennel, P.; Pettmann, B.; Vigne, E.; Akli, S.; Revah, F.; Schmalbruch, H.; Kahn, A. Gene therapy of murine motor neuron disease using adenoviral vectors for neurotrophic factors. Nat. Med. 3:429–436; 1997.

    Article  PubMed  CAS  Google Scholar 

  • Hanson, M. J.; Shen, S.; Wiemelt, A.; McMorris, F.; Barres, B. Cyclic AMP elevation is sufficient to promote the survival of spinal motor neurons in vitro. J. Neurosci. 18:7361–7371; 1998.

    PubMed  CAS  Google Scholar 

  • Henderson, C.; Camu, W.; Mettling, C. et al. Neurotrophins promote motor neuron survival and are present in embryonic limb bud. Nature 363:266–270; 1993.

    Article  PubMed  CAS  Google Scholar 

  • Henderson, C.; Phillips, H.; Pollock, R., et al. GDNF a potent survival factor for motoneurons present in peripheral nerve and muscle. Science 266:1062–1064; 1994.

    Article  PubMed  CAS  Google Scholar 

  • Henderson, C.; Yamamoto, Y.; Livet, J.; Arce, V.; Garces, A.; deLapeyriere, O. Role of neurotrophic factors in motoneuron development J. Physiol. Paris 92:279–281; 1998.

    Article  PubMed  CAS  Google Scholar 

  • Hughes, R.; Sendtner, M.; Thoenen, H. Members of several gene families influence survival of rat motoneurons in vitro and in vivo. J. Neurosci. Res. 36:663–671; 1993.

    Article  PubMed  CAS  Google Scholar 

  • Jacques, T.; Skepper, J.; Navaratnam, V. Fibroblast growth factor-1 improves the survival and regeneration of rat vagal preganglionic neurones following axon injury. Neurosci. Lett. 276:197–200; 1999.

    Article  PubMed  CAS  Google Scholar 

  • Kapfhammer, J. Axon sprouting in the spinal cord: growth promoting and growth inhibitory mechanisms. Anat. Embryol. (Berl) 196:417–426; 1997.

    Article  CAS  Google Scholar 

  • Kleinfeld D.; Kahler, K.; Hockberger, P. Controlled outgrowth of dissociated neurons on patterned substrates. J. Neurosci. 8:4098–4120; 1988.

    PubMed  CAS  Google Scholar 

  • Kuffler, D. Long-term survival and sprouting in culture by motoneurons isolated from the spinal cord of adult frogs J. Comp. Neurol. 302:729–738; 1990.

    Article  PubMed  CAS  Google Scholar 

  • Kuzis, K.; Coffin, J.; Eckenstein, F. Time course and age dependence of motor neuron death following facial nerve crush injury: role of fibroblast growth factor. Exp. Neurol. 157:77–87; 1999.

    Article  PubMed  CAS  Google Scholar 

  • Kwon, B.; Bordet, T.; Haase, G.; Castelnau-Ptakhine, L.; Rouhani, S.; Gilgenkrantz, H.; Kahn, A. In vivo electrotransfer of the cardiotrophin-1 gene into skeletal muscle slows down progression of motor neuron degeneration in pmn mice. Hum. Mol. Genet. 11:1615–1625; 2002.

    Article  Google Scholar 

  • Lindsay, R.; Neurotrophins and receptors. Prog. Brain Res. 103:3–14; 1994.

    Article  PubMed  CAS  Google Scholar 

  • Liu, R.; Morassutti, D.; Whittemore, S.; Sosnowski, J.; Magnuson, D. Electrophysiological properties of mitogen-expanded adult rat spinal cord and subventricular zone neural precursor cells. Exp. Neurol. 158:143–154; 1999.

    Article  PubMed  CAS  Google Scholar 

  • Maquet, V.; Martin, D.; Scholtes, F.; Franzen R.; Schoenen, J.; Moonen, G.; Jerme, R. Poly(D,L-lactide) foams modified by poly(ethylene oxide)-block-poly(D,L-lactide) copolymers and a-FGF: in vitro and in vivo evaluation for spinal cord regeneration. Biomaterials 22:1137–1146; 2001.

    Article  PubMed  CAS  Google Scholar 

  • Mey, J. Retinoic acid as a regulator of cytokine signaling after nerve injury. Z. Naturforsch. 56:163–176; 2001.

    CAS  Google Scholar 

  • Mrksich, M.; Whitesides, G. Using self-assembled monolayers to understand the interactions of man-made surfaces with proteins and cells. Annu. Rev. Biophys. Biomol. Struct. 25:55–78; 1996.

    Article  PubMed  CAS  Google Scholar 

  • Nacimiento, W.; Schmitt, A.; Brook, G. Nerve regeneration after spinal cord trauma. Neurobiological progress and clinical expectations. Nervenarzt 70:702–713; 1999.

    Article  PubMed  CAS  Google Scholar 

  • Nicholls, J.; Adams, W.; Eugenin, J.; Geiser, R.; Lepre, M.; Luque, J.; Wintzer, M. Why does the central nervous system not regenerate after injury? Surv. Ophthalmol. 43:S136-S141; 1999.

    Article  PubMed  Google Scholar 

  • Pennica, D.; Arce, V.; Swanson, T. et al. Cardiotrophin-1, a cytokine present in embryonic muscle, supports long-term survival of spinal motoneurons. Neuron 17:63–74; 1996.

    Article  PubMed  CAS  Google Scholar 

  • Price, P.; Brewer, G. Serum-free media for neural cell cultures. Adult and embryonic. In: Fedoroff, S.; Richardson, A., ed. Protocols for neural cell culture. Totowa, NJ: Humana Press; 2001:255–263.

    Chapter  Google Scholar 

  • Ravenscroft, M.; Bateman, K.; Shaffer, K., et al. Developmental neurobiology implications from fabrication and analysis of hippocampal neuronal networks on patterned silane-modified surfaces. J. Am. Chem. Soc. 120:12169–12177; 1998.

    Article  CAS  Google Scholar 

  • Sakamoto, T.; Kawazoe, Y.; Shen, J., et al. Adenoviral gene transfer of GDNF, BDNF and TGF beta 2, but not CNTF, cardiotrophin-1 or IGF1, protects injured adult motoneurons after facial nerve avulsion. J. Neurosci. Res. 72:54–64; 2003a.

    Article  PubMed  CAS  Google Scholar 

  • Sakamoto, T.; Kawazoe, Y.; Uchida, Y.; Hozumi, I.; Inuzuka, T.; Watabe, K. Growth inhibitory factor prevents degeneration of injured adult rat motoneurons. Neuroreport 14:2147–2151; 2003b.

    Article  PubMed  CAS  Google Scholar 

  • Schaffner, A.; Barker, J.; Stenger, D.; Hickman, J. Investigation of the factors necessary for growth of hippocampal neurons in a defined system. J. Neurosci. Methods 62:111–119; 1995.

    Article  PubMed  CAS  Google Scholar 

  • Schnaar, R.; Schaffner, A. Separation of cell types from embryonic chicken and rat spinal cord: characterization of motoneuron-enriched fractions. J. Neurosci. 1:204–217; 1981.

    PubMed  CAS  Google Scholar 

  • Seybold, V.; Abrahams, L. Primary cultures of neonatal rat spinal cord. Methods Mol. Med. 99:203–213; 2004.

    PubMed  Google Scholar 

  • Spargo, B.; Testoff, M.; Nielsen, T.; Stenger, D.; Hickman, J.; Rudolph, A. Spatially controlled adhesion, spreading, and differentiation of endothelial cells on self-assembled molecular monolayers. Proc. Natl. Acad. Sci. USA 91:11070–11074; 1994.

    Article  PubMed  CAS  Google Scholar 

  • Stenger, D.; Hickman, J.; Bateman, K. et al. Microlithographic determination of axonal/dendritic polarity in cultured hippocampal neurons. J. Neurosci. Methods 82:167–173; 1998.

    Article  PubMed  CAS  Google Scholar 

  • Stenger, D. A.; Georger, J. H.; Dulcey, C. S.; Hickman, J. J.; Rudolph, A. S.; Niel, T. B. Coplanar molecular assemblies of aminoalkylsilane and perfluorinated alkylsilane-characterization and geometric definition of mammalian-cell adhesion and growth. J. Am. Chem. Soc. 114:8435–8442; 1992.

    Article  CAS  Google Scholar 

  • Thoenen, H.; Sendtner, M. Neurotrophins: from enthusiastic expectations through sobering experiences to rational therapeutic approaches. Nat. Neurosci. 5:1046–1050; 2002.

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Nanoscience Technology Center, University of Central Florida, 32826, Orlando, Florida

    Mainak Das, Neelima Bhargava, Lisa Riedel, Peter Molnar & James J. Hickman

  2. Department of Bioengineering, Clemson University, 29634, Clemson, South Carolina

    Mainak Das, Cassie Gregory, Lisa Riedel, Peter Molnar & James J. Hickman

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  1. Mainak Das
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  2. Neelima Bhargava
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  3. Cassie Gregory
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  4. Lisa Riedel
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  5. Peter Molnar
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  6. James J. Hickman
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Correspondence to James J. Hickman.

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Das, M., Bhargava, N., Gregory, C. et al. Adult rat spinal cord culture on an organosilane surface in a novel serum-free medium. In Vitro Cell.Dev.Biol.-Animal 41, 343–348 (2005). https://doi.org/10.1007/s11626-005-0006-2

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  • DOI: https://doi.org/10.1007/s11626-005-0006-2

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