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A coculture assay to visualize and monitor interactions between migrating glioma cells and nerve fibers

Nature Protocols volume 4, pages 923–927 (2009)Cite this article

Abstract

Glioma-cell migration is usually assessed in dissociated cell cultures, spheroid cultures, acute brain slices and intracranial implantation models. However, the interactions between migrating glioma cells and neuronal tracts remain poorly understood. We describe here a protocol for the coculture of glioma cells with myelinated axons in vitro. Unlike other methods, this protocol allows the creation of in vitro conditions that largely mimic the complex in vivo environment. First, long retinal axons from embryonic chicken are formed in an organotypic culture. Glioma cells are then positioned in the vicinity of the explants to allow them to contact the axons, interact with them and eventually migrate along them. High-resolution video microscopy and confocal microscopy can be used to monitor the migratory behavior. This protocol, which takes about 5 days to complete, could be applied to different types of tumor cells that interact with neurites, and is suitable for pharmacological and genetic approaches aimed at elucidating mechanisms underlying tumor migration.

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Figure 1: Diagrams showing the removal of eyes from chicken embryos at various stages, isolation and culture of retinas and coculture with glioma cells.
Figure 2: Interactions between C6 cells and chicken axons in culture.
Figure 3: Chicken axons in culture are associated with myelin.
Figure 4: Classification of phenotypes of glioma-cell–axon interactions.

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Acknowledgements

We thank Mechthild Langkamp-Flock and Mechthild Wissing for technical assistance, and English Science Editing for editing the manuscript. The work was supported by the DFG (Grant Th 386/14-1 to S.T.), by Innovative Medical Research (IMF) Münster (Grant OE 62 08 02 to P.O.) and Wilhelm-Sander-Foundation (Grant 2005.058.2 to W.P. and V.S.).

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Authors and Affiliations

  1. Department of Experimental Ophthalmology, School of Medicine, University Eye Hospital Münster, Domagkstrasse, Münster, Germany

    Patrick Oellers, Tobias Stupp, Petar Charalambous & Solon Thanos

  2. Department of Ophthalmology, University Hospital Essen, Hufelandstr., Essen, Germany

    Maurice Schallenberg

  3. Interdisciplinary Center for Clinical Research (IZKF), Münster, Germany

    Petar Charalambous & Solon Thanos

  4. Institute of Neuropathology, School of Medicine, University Hospital Münster, Domagkstrasse, Münster, Germany

    Volker Senner & Werner Paulus

Authors
  1. Patrick Oellers
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  2. Maurice Schallenberg
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  3. Tobias Stupp
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  4. Petar Charalambous
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  5. Volker Senner
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  6. Werner Paulus
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  7. Solon Thanos
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Corresponding author

Correspondence to Solon Thanos.

Supplementary information

Supplementary Video 1

Migration of C6 cells on chick retinal axons in culture. After axons have grown in vitro, C6 cells are added to the vicinity of axons and contact the axons. It is evident that several cells either move in different directions along the axons or cross the axons. The experiments have been approved by the local authorities and the University of Münster (file reference 9.93.2.10.36.07.095) (MOV 483 kb)

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Oellers, P., Schallenberg, M., Stupp, T. et al. A coculture assay to visualize and monitor interactions between migrating glioma cells and nerve fibers. Nat Protoc 4, 923–927 (2009). https://doi.org/10.1038/nprot.2009.62

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