Abstract
This protocol uses rat tail–derived type I collagen hydrogels to analyze key processes in developmental neurobiology, such as chemorepulsion and chemoattraction. The method is based on culturing small pieces of brain tissue from embryonic or early perinatal mice inside a 3D hydrogel formed by rat tail–derived type I collagen or, alternatively, by commercial Matrigel. The neural tissue is placed in the hydrogel with other brain tissue pieces or cell aggregates genetically modified to secrete a particular molecule that can generate a gradient inside the hydrogel. The present method is uncomplicated and generally reproducible, and only a few specific details need to be considered during its preparation. Moreover, the degree and behavior of axonal growth or neural migration can be observed directly using phase-contrast, fluorescence microscopy or immunocytochemical methods. This protocol can be carried out in 4 weeks.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
References
Banker, G. & Goslin, K. Culturing Nerve Cells (MIT Press, 1991).
Brecknell, J.E. & Fawcett, J.W. Axonal regeneration. Biol. Rev. Camb. Philos. Soc. 71, 227–255 (1996).
Ramón y Cajal, S. Les nouvelles idées sur la structure du système nerveux chez l'homme et chez les vertébrés. Ed. française rev. et augm. par l'auteur, tr. de l'espagnol par L. Azoulay. edn, (Reinwald,, 1894).
Sotelo, C. The chemotactic hypothesis of Cajal: a century behind. Prog. Brain Res. 136, 11–20 (2002).
Ramón y Cajal, S. Nuevo concepto de la histología de los centros nerviosos (Henrich, 1893).
Marin, O. & Rubenstein, J.L. Cell migration in the forebrain. Annu. Rev. Neurosci. 26, 441–483 (2003).
Marin, O., Valiente, M., Ge, X. & Tsai, L.H. Guiding neuronal cell migrations. Cold Spring Harb. Perspect Biol. 2, a001834 (2010).
Tessier-Lavigne, M. & Goodman, C.S. The molecular biology of axon guidance. Science 274, 1123–1133 (1996).
Dickson, B.J. Molecular mechanisms of axon guidance. Science 298, 1959–1964 (2002).
Kolodkin, A.L. & Tessier-Lavigne, M. Mechanisms and molecules of neuronal wiring: a primer. Cold Spring Harb. Perspect. Biol. 3, doi: 10.1101/cshperspect.a001727 (2011).
Rosentreter, S.M. et al. Response of retinal ganglion cell axons to striped linear gradients of repellent guidance molecules. J. Neurobiol. 37, 541–562 (1998).
Knoll, B., Weinl, C., Nordheim, A. & Bonhoeffer, F. Stripe assay to examine axonal guidance and cell migration. Nat. Protoc. 2, 1216–1224 (2007).
von Philipsborn, A.C. et al. Growth cone navigation in substrate-bound ephrin gradients. Development 133, 2487–2495 (2006).
Chen, H., He, Z. & Tessier-Lavigne, M. Axon guidance mechanisms: semaphorins as simultaneous repellents and anti-repellents. Nat. Neurosci. 1, 436–439 (1998).
Jessell, T.M. & Sanes, J.R. Development. The decade of the developing brain. Curr. Opin. Neurobiol. 10, 599–611 (2000).
Serafini, T. et al. The netrins define a family of axon outgrowth-promoting proteins homologous to C. elegans UNC-6. Cell 78, 409–424 (1994).
Charron, F. & Tessier-Lavigne, M. Novel brain wiring functions for classical morphogens: a role as graded positional cues in axon guidance. Development 132, 2251–2262 (2005).
Fournier, M.F., Sauser, R., Ambrosi, D., Meister, J.J. & Verkhovsky, A.B. Force transmission in migrating cells. J. Cell Biol. 188, 287–297 (2010).
Dent, E.W. & Gertler, F.B. Cytoskeletal dynamics and transport in growth cone motility and axon guidance. Neuron 40, 209–227 (2003).
Lowery, L.A. & Van Vactor, D. The trip of the tip: understanding the growth cone machinery. Nat. Rev. Mol. Cell Biol. 10, 332–343 (2009).
Castellani, V. & Bolz, J. in Protocols for Neuronal Cell Culture (eds. Fedoroff, S. & Richardson, A.) (Humana Press, 2001).
Gahwiler, B.H. Organotypic monolayer cultures of nervous tissue. J. Neurosci. Methods 4, 329–342 (1981).
Bornstein, M.B. Reconstituted rattail collagen used as substrate for tissue cultures on coverslips in Maximow slides and roller tubes. Lab. Invest. 7, 134–137 (1958).
Billings-Gagliardi, S. & Wolf, M.K. A simple method for examining organotypic CNS cultures with Nomarski optics. In Vitro 13, 371–377 (1977).
Chedotal, A. et al. Semaphorins III and IV repel hippocampal axons via two distinct receptors. Development 125, 4313–4323 (1998).
Kennedy, T.E., Serafini, T., de la Torre, J.R. & Tessier-Lavigne, M. Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal cord. Cell 78, 425–435 (1994).
Klein, R. Eph/ephrin signaling in morphogenesis, neural development and plasticity. Curr. Opin. Cell Biol. 16, 580–589 (2004).
Wang, K.H. et al. Biochemical purification of a mammalian slit protein as a positive regulator of sensory axon elongation and branching. Cell 96, 771–784 (1999).
Emonard, H., Grimaud, J.A., Nusgens, B., Lapiere, C.M. & Foidart, J.M. Reconstituted basement-membrane matrix modulates fibroblast activities in vitro. J. Cell Physiol. 133, 95–102 (1987).
Knapp, D.M., Helou, E.F. & Tranquillo, R.T. A fibrin or collagen gel assay for tissue cell chemotaxis: assessment of fibroblast chemotaxis to GRGDSP. Exp. Cell Res. 247, 543–553 (1999).
Kapur, T.A. & Shoichet, M.S. Immobilized concentration gradients of nerve growth factor guide neurite outgrowth. J. Biomed. Mater. Res. A 68, 235–243 (2004).
Ciofani, G., Raffa, V., Menciassi, A., Micera, S. & Dario, P. A drug delivery system based on alginate microspheres: mass-transport test and in vitro validation. Biomed. Microdevices 9, 395–403 (2007).
Dontchev, V.D. & Letourneau, P.C. Growth cones integrate signaling from multiple guidance cues. J. Histochem. Cytochem. 51, 435–444 (2003).
Crank, J. The Mathematics of Diffusion 2nd edn (Clarendon Press, 1975).
Rosoff, W.J., McAllister, R., Esrick, M.A., Goodhill, G.J. & Urbach, J.S. Generating controlled molecular gradients in 3D gels. Biotechnol. Bioeng. 91, 754–759 (2005).
Del Rio, J.A. et al. MAP1B is required for Netrin 1 signaling in neuronal migration and axonal guidance. Curr. Biol. 14, 840–850 (2004).
Nobrega-Pereira, S. et al. Postmitotic Nkx2-1 controls the migration of telencephalic interneurons by direct repression of guidance receptors. Neuron 59, 733–745 (2008).
Alcantara, S., Ruiz, M., De Castro, F., Soriano, E. & Sotelo, C. Netrin 1 acts as an attractive or as a repulsive cue for distinct migrating neurons during the development of the cerebellar system. Development 127, 1359–1372 (2000).
Simo, S. et al. Reelin induces the detachment of postnatal subventricular zone cells and the expression of the Egr-1 through Erk1/2 activation. Cereb. Cortex 17, 294–303 (2007).
Borrell, V. & Marin, O. Meninges control tangential migration of hem-derived Cajal-Retzius cells via CXCL12/CXCR4 signaling. Nat. Neurosci. 9, 1284–1293 (2006).
Kothapalli, C.R. et al. A high-throughput microfluidic assay to study neurite response to growth factor gradients. Lab Chip 11, 497–507 (2011).
Sundararaghavan, H.G., Monteiro, G.A., Firestein, B.L. & Shreiber, D.I. Neurite growth in 3D collagen gels with gradients of mechanical properties. Biotechnol. Bioeng. 102, 632–643 (2009).
Goodhill, G.J. & Baier, H. Axon guidance: stretching gradients to the limit. Neural Comput. 10, 521–527 (1998).
Cate, D.M., Sip, C.G. & Folch, A. A microfluidic platform for generation of sharp gradients in open-access culture. Biomicrofluidics 4, 44105 (2010).
Keenan, T.M. & Folch, A. Biomolecular gradients in cell culture systems. Lab Chip 8, 34–57 (2008).
Rochat, A., Omlin, F.X. & Droz, B. Substrate-dependent migration of myelin-associated glycoprotein immunoreactive cells in cultured explants of dorsal root ganglia from chick embryos. Dev. Neurosci. 10, 236–244 (1988).
Bribian, A. et al. A novel role for anosmin-1 in the adhesion and migration of oligodendrocyte precursors. Dev. Neurobiol. 68, 1503–1516 (2008).
Shahar, A., de Vellis, J., Vernadakis, A. & Haber, B. A Dissection and Tissue Culture Manual of the Nervous System (Liss, A. R., 1989).
Courtes, S. et al. Reelin controls progenitor cell migration in the healthy and pathological adult mouse brain. PLoS ONE 6, e20430 (2011).
Montolio, M. et al. A semaphorin 3A inhibitor blocks axonal chemorepulsion and enhances axon regeneration. Chem. Biol. 16, 691–701 (2009).
Acknowledgements
This study was supported by FP7-PRIORITY, Ministerio de Ciencia e Innovación (MICINN) (BFU2009-10848), SGR2009-366 (Generalitat de Catalunya) and Instituto Carlos Tercero (CIBERNED and Biomarkers of Early Stages of Alzheimer's Disease–Prevention (BESAD-P)) grants to J.A.d.R. We thank all the members from the laboratories of E. Soriano (Institute for Research in Biomedicine, Barcelona), A. Chedotal (UMR S968, Paris), C. Sotelo (Instituto de Neurociencias, Alicante), M. Tessier-Lavigne (Genentech) and A.L. Kolodkin (The Johns Hopkins University School of Medicine) for their contributions and collaborations during all these years in improving the techniques explained in this paper. We also thank members of the A. Raya laboratory (IBEC, Barcelona) for advice in dark-field photodocumentation and F. Llorens and S. Nocentini (IBEC, Barcelona) for reading and commenting on the manuscript. We also thank the Language Advisory Service at the University of Barcelona for their editorial help.
Author information
Authors and Affiliations
Contributions
J.A.d.R. performed the experiments illustrated in the manuscript. J.A.d.R. and V.G. collaborated to write the manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Supplementary information
Supplementary Fig 1
Photomicrographs illustrating current problems in explant cultures in hydrogels. (a) In this culture, the collagen solution was contaminated with yeast (arrows). Although cortical axons could still grow and growth cones (arrows in b) were observed, puncta-like DAB deposits corresponding to microbial contamination (arrowheads in b) were also seen. Derived results should be considered carefully and further experiments with uncontaminated collagen should be performed. (c) Low power photomicrograph in dark field optics of a non-homogeneous collagen due to extensive manipulation with the tungsten needle. A detail is shown in the insert box (d) Photomicrograph of a very bad explant co-culture with several experimental mistakes, some of them showed in (a-c). First, the distance between the two pieces is too small and the explant is broken (1). Second, a general contamination can be seen in the collagen containing cells (2). In addition, extensive manipulation of the explant has been performed since the collagen is not homogeneous and some holes are present (3). Lastly, high contamination increases the background in the collagen most probably through cell debris and contaminating microorganisms (4). Scale bars: a and d = 500 µm; b = 50 µm. (TIFF 1566 kb)
Supplementary Fig 2
Photographs illustrating some control experiments of transfection efficiency. (a) Fluorescence photomicrographs illustrating examples of transfected cells with SEMA 3 cDNA (red). HEK-293 cells were immunostained using α-SEMA antibody (Santa cruz Biotechnologies, cat. no. SC-1148) and Alexa Fluor-568 tagged secondary antibody. Cells were counterstained with DAPI. (b) Example of Western blot detection of production of Netrin-1 in transfected cells 48 h after transfection. Actin was detected as control protein. (c-e) Example of the "hanging drop" procedure (see Box1, Optional procedure 1 for details). In this example, SEMA 3A-AP transfected cells as pre-clotted aggregates (PCCC) (c-d) or non-clotted (HDCC) (c,e) were cultured in 20 µl- drops. Phase contrast photomicrographs in (d) and (e) are examples of PCCC and HDCC drops respectively. (f) NBT/BCIP development of the AP activity in the media of HDCC and PCCC drops at different concentrations. Notice that AP activity was similar between both culture types. A lane containing media from Mock transfected PCCC cultures is also shown. Scale bars: a = 50 µm; d and e = 500 µm. (TIFF 6999 kb)
Rights and permissions
About this article
Cite this article
Gil, V., del Río, J. Analysis of axonal growth and cell migration in 3D hydrogel cultures of embryonic mouse CNS tissue. Nat Protoc 7, 268–280 (2012). https://doi.org/10.1038/nprot.2011.445
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2011.445
This article is cited by
-
Growth cone repulsion to Netrin-1 depends on lipid raft microdomains enriched in UNC5 receptors
Cellular and Molecular Life Sciences (2021)
-
New functions of Semaphorin 3E and its receptor PlexinD1 during developing and adult hippocampal formation
Scientific Reports (2018)
-
Domain-Specific Activation of Death-Associated Intracellular Signalling Cascades by the Cellular Prion Protein in Neuroblastoma Cells
Molecular Neurobiology (2016)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.