Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Analysis of axonal growth and cell migration in 3D hydrogel cultures of embryonic mouse CNS tissue

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

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

Figure 1: Illustration of the collagen preparation described in this protocol.
Figure 2: Illustration of the main steps in the preparation of cell aggregates and explant cultures.
Figure 3: Examples of different explant culture and quantification methods.

References

  1. Banker, G. & Goslin, K. Culturing Nerve Cells (MIT Press, 1991).

  2. Brecknell, J.E. & Fawcett, J.W. Axonal regeneration. Biol. Rev. Camb. Philos. Soc. 71, 227–255 (1996).

    Article  CAS  Google Scholar 

  3. 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).

  4. Sotelo, C. The chemotactic hypothesis of Cajal: a century behind. Prog. Brain Res. 136, 11–20 (2002).

    Article  Google Scholar 

  5. Ramón y Cajal, S. Nuevo concepto de la histología de los centros nerviosos (Henrich, 1893).

  6. Marin, O. & Rubenstein, J.L. Cell migration in the forebrain. Annu. Rev. Neurosci. 26, 441–483 (2003).

    Article  CAS  Google Scholar 

  7. Marin, O., Valiente, M., Ge, X. & Tsai, L.H. Guiding neuronal cell migrations. Cold Spring Harb. Perspect Biol. 2, a001834 (2010).

    Article  Google Scholar 

  8. Tessier-Lavigne, M. & Goodman, C.S. The molecular biology of axon guidance. Science 274, 1123–1133 (1996).

    Article  CAS  Google Scholar 

  9. Dickson, B.J. Molecular mechanisms of axon guidance. Science 298, 1959–1964 (2002).

    Article  CAS  Google Scholar 

  10. 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).

  11. 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).

    Article  CAS  Google Scholar 

  12. Knoll, B., Weinl, C., Nordheim, A. & Bonhoeffer, F. Stripe assay to examine axonal guidance and cell migration. Nat. Protoc. 2, 1216–1224 (2007).

    Article  Google Scholar 

  13. von Philipsborn, A.C. et al. Growth cone navigation in substrate-bound ephrin gradients. Development 133, 2487–2495 (2006).

    Article  CAS  Google Scholar 

  14. Chen, H., He, Z. & Tessier-Lavigne, M. Axon guidance mechanisms: semaphorins as simultaneous repellents and anti-repellents. Nat. Neurosci. 1, 436–439 (1998).

    Article  CAS  Google Scholar 

  15. Jessell, T.M. & Sanes, J.R. Development. The decade of the developing brain. Curr. Opin. Neurobiol. 10, 599–611 (2000).

    Article  CAS  Google Scholar 

  16. 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).

    Article  CAS  Google Scholar 

  17. 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).

    Article  CAS  Google Scholar 

  18. 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).

    Article  CAS  Google Scholar 

  19. Dent, E.W. & Gertler, F.B. Cytoskeletal dynamics and transport in growth cone motility and axon guidance. Neuron 40, 209–227 (2003).

    Article  CAS  Google Scholar 

  20. 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).

    Article  CAS  Google Scholar 

  21. Castellani, V. & Bolz, J. in Protocols for Neuronal Cell Culture (eds. Fedoroff, S. & Richardson, A.) (Humana Press, 2001).

  22. Gahwiler, B.H. Organotypic monolayer cultures of nervous tissue. J. Neurosci. Methods 4, 329–342 (1981).

    Article  CAS  Google Scholar 

  23. 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).

    CAS  PubMed  Google Scholar 

  24. Billings-Gagliardi, S. & Wolf, M.K. A simple method for examining organotypic CNS cultures with Nomarski optics. In Vitro 13, 371–377 (1977).

    Article  CAS  Google Scholar 

  25. Chedotal, A. et al. Semaphorins III and IV repel hippocampal axons via two distinct receptors. Development 125, 4313–4323 (1998).

    CAS  PubMed  Google Scholar 

  26. 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).

    Article  CAS  Google Scholar 

  27. Klein, R. Eph/ephrin signaling in morphogenesis, neural development and plasticity. Curr. Opin. Cell Biol. 16, 580–589 (2004).

    Article  CAS  Google Scholar 

  28. 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).

    Article  CAS  Google Scholar 

  29. 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).

    Article  CAS  Google Scholar 

  30. 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).

    Article  CAS  Google Scholar 

  31. 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).

    Article  Google Scholar 

  32. 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).

    Article  CAS  Google Scholar 

  33. Dontchev, V.D. & Letourneau, P.C. Growth cones integrate signaling from multiple guidance cues. J. Histochem. Cytochem. 51, 435–444 (2003).

    Article  CAS  Google Scholar 

  34. Crank, J. The Mathematics of Diffusion 2nd edn (Clarendon Press, 1975).

  35. 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).

    Article  CAS  Google Scholar 

  36. 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).

    Article  CAS  Google Scholar 

  37. 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).

    Article  CAS  Google Scholar 

  38. 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).

    CAS  PubMed  Google Scholar 

  39. 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).

    Article  Google Scholar 

  40. Borrell, V. & Marin, O. Meninges control tangential migration of hem-derived Cajal-Retzius cells via CXCL12/CXCR4 signaling. Nat. Neurosci. 9, 1284–1293 (2006).

    Article  CAS  Google Scholar 

  41. Kothapalli, C.R. et al. A high-throughput microfluidic assay to study neurite response to growth factor gradients. Lab Chip 11, 497–507 (2011).

    Article  CAS  Google Scholar 

  42. 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).

    Article  CAS  Google Scholar 

  43. Goodhill, G.J. & Baier, H. Axon guidance: stretching gradients to the limit. Neural Comput. 10, 521–527 (1998).

    Article  CAS  Google Scholar 

  44. Cate, D.M., Sip, C.G. & Folch, A. A microfluidic platform for generation of sharp gradients in open-access culture. Biomicrofluidics 4, 44105 (2010).

    Article  CAS  Google Scholar 

  45. Keenan, T.M. & Folch, A. Biomolecular gradients in cell culture systems. Lab Chip 8, 34–57 (2008).

    Article  CAS  Google Scholar 

  46. 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).

    Article  CAS  Google Scholar 

  47. Bribian, A. et al. A novel role for anosmin-1 in the adhesion and migration of oligodendrocyte precursors. Dev. Neurobiol. 68, 1503–1516 (2008).

    Article  CAS  Google Scholar 

  48. Shahar, A., de Vellis, J., Vernadakis, A. & Haber, B. A Dissection and Tissue Culture Manual of the Nervous System (Liss, A. R., 1989).

  49. Courtes, S. et al. Reelin controls progenitor cell migration in the healthy and pathological adult mouse brain. PLoS ONE 6, e20430 (2011).

    Article  CAS  Google Scholar 

  50. Montolio, M. et al. A semaphorin 3A inhibitor blocks axonal chemorepulsion and enhances axon regeneration. Chem. Biol. 16, 691–701 (2009).

    Article  CAS  Google Scholar 

Download references

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

Authors

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

Correspondence to José Antonio del Río.

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

Reprints 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

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2011.445

This article is cited by

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.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing