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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.

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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.

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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.

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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.

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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)

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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

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