The authors thank Tom Yohannan for the editorial advice and M. Segura-Feliu for the technical assistance. This work was funded by the CERCA Programme and by the Commission for Universities and Research of the Department of Innovation, Universities, and Enterprise of the Generalitat de Catalunya (SGR2017-648). This work was funded by the Spanish Ministry of Research, Innovation and University (MEICO) through BFU2015-67777-R and and RTI2018-099773-B-100, the Spanish Prion Network (Prionet Spain AGL2017-90665-REDT), and the Institute Carlos III, CIBERNED (PRY-2018-2).

All animal experiments were performed under the guidelines and protocols of the Ethical Committee for Animal Experimentation (CEEA) of the University of Barcelona, and the protocol for the use of rodents in this study was reviewed and approved by the CEEA of the University of Barcelona (CEEA approval #276/16 and 141/15).&#160;
1. Purification of Rat Tail Collagen
<ol>
	<li>Collect adult Sprague-Dawley rat tails (8-9 weeks old) after sacrificing the animal following ethical guidelines and rin...

<ol>
	<li>Ram&#243;n y Cajal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Les nouvelles id&#233;es sur la structure du syst&#232;me nerveux chez l'homme et chez les vert&#233;br&#233;s. , (1894).</li><li>Ram&#243;n y Cajal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nuevo concepto de la histolog&#237;a de los centros nerviosos. , (1893).</li><li>Marin, O., Valiente, M., Ge, X., Tsai, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guiding+neuronal+cell+migrations.">Guiding neuronal cell migrations.</a> Cold Spring Harbor Perspectives in Biology. 2 (2), 001834 (2010).</li><li>Tessier-Lavigne, M., Goodman, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+biology+of+axon+guidance.">The molecular biology of axon guidance.</a> Science. 274 (5290), 1123-1133 (1996).</li><li>Dickson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+axon+guidance.">Molecular mechanisms of axon guidance.</a> Science. 298 (5600), 1959-1964 (2002).</li><li>Kolodkin, A. L., Tessier-Lavigne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+and+molecules+of+neuronal+wiring:+a+primer.">Mechanisms and molecules of neuronal wiring: a primer.</a> Cold Spring Harbor Perspectives in Biology. 3 (6), 001727 (2011).</li><li>Rosentreter, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+retinal+ganglion+cell+axons+to+striped+linear+gradients+of+repellent+guidance+molecules.">Response of retinal ganglion cell axons to striped linear gradients of repellent guidance molecules.</a> Journal of Neurobiology. 37 (4), 541-562 (1998).</li><li>Knoll, B., Weinl, C., Nordheim, A., Bonhoeffer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stripe+assay+to+examine+axonal+guidance+and+cell+migration.">Stripe assay to examine axonal guidance and cell migration.</a> Nature Protocols. 2 (5), 1216-1224 (2007).</li><li>von Philipsborn, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+cone+navigation+in+substrate-bound+ephrin+gradients.">Growth cone navigation in substrate-bound ephrin gradients.</a> Development. 133 (13), 2487-2495 (2006).</li><li>Chen, H., He, Z., Tessier-Lavigne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axon+guidance+mechanisms:+semaphorins+as+simultaneous+repellents+and+anti-repellents.">Axon guidance mechanisms: semaphorins as simultaneous repellents and anti-repellents.</a> Nature Neuroscience. 1 (6), 436-439 (1998).</li><li>Jessell, T. M., Sanes, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development.+The+decade+of+the+developing+brain.">Development. The decade of the developing brain.</a> Current Opinion in Neurobiology. 10 (5), 599-611 (2000).</li><li>Serafini, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+netrins+define+a+family+of+axon+outgrowth-promoting+proteins+homologous+to+C.+elegans+UNC-6.">The netrins define a family of axon outgrowth-promoting proteins homologous to C. elegans UNC-6.</a> Cell. 78 (3), 409-424 (1994).</li><li>Charron, F., Tessier-Lavigne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+brain+wiring+functions+for+classical+morphogens:+a+role+as+graded+positional+cues+in+axon+guidance.">Novel brain wiring functions for classical morphogens: a role as graded positional cues in axon guidance.</a> Development. 132 (10), 2251-2262 (2005).</li><li>Fournier, M. F., Sauser, R., Ambrosi, D., Meister, J. J., Verkhovsky, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+transmission+in+migrating+cells.">Force transmission in migrating cells.</a> Journal of Cell Biology. 188 (2), 287-297 (2010).</li><li>Dent, E. W., Gertler, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoskeletal+dynamics+and+transport+in+growth+cone+motility+and+axon+guidance.">Cytoskeletal dynamics and transport in growth cone motility and axon guidance.</a> Neuron. 40 (2), 209-227 (2003).</li><li>Lowery, L. A., Van Vactor, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+trip+of+the+tip:+understanding+the+growth+cone+machinery.">The trip of the tip: understanding the growth cone machinery.</a> Nature Reviews Molecular Cell Biology. 10 (5), 332-343 (2009).</li><li>Castellani, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Protocols for Neuronal Cell Culture. , (2001).</li><li>Gahwiler, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+monolayer+cultures+of+nervous+tissue.">Organotypic monolayer cultures of nervous tissue.</a> Journal of Neuroscience Methods. 4 (4), 329-342 (1981).</li><li>Bornstein, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstituted+rattail+collagen+used+as+substrate+for+tissue+cultures+on+coverslips+in+Maximow+slides+and+roller+tubes.">Reconstituted rattail collagen used as substrate for tissue cultures on coverslips in Maximow slides and roller tubes.</a> Laboratory Investigation. 7 (2), 134-137 (1958).</li><li>Billings-Gagliardi, S., Wolf, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+examining+organotypic+CNS+cultures+with+Nomarski+optics.">A simple method for examining organotypic CNS cultures with Nomarski optics.</a> In Vitro. 13 (6), 371-377 (1977).</li><li>Bell, E., Ivarsson, B., Merrill, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+a+tissue-like+structure+by+contraction+of+collagen+lattices+by+human+fibroblasts+of+different+proliferative+potential+in+vitro.">Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro.</a> Proceedings of the National Academy of Science. 76 (3), 1274-1278 (1979).</li><li>Lumsden, A. G., Davies, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Earliest+sensory+nerve+fibres+are+guided+to+peripheral+targets+by+attractants+other+than+nerve+growth+factor.">Earliest sensory nerve fibres are guided to peripheral targets by attractants other than nerve growth factor.</a> Nature. 306 (5945), 786-788 (1983).</li><li>Chedotal, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semaphorins+III+and+IV+repel+hippocampal+axons+via+two+distinct+receptors.">Semaphorins III and IV repel hippocampal axons via two distinct receptors.</a> Development. 125 (21), 4313-4323 (1998).</li><li>Kennedy, T. E., Serafini, T., de la Torre, J. R., Tessier-Lavigne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Netrins+are+diffusible+chemotropic+factors+for+commissural+axons+in+the+embryonic+spinal+cord.">Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal cord.</a> Cell. 78 (3), 425-435 (1994).</li><li>Klein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eph/ephrin+signaling+in+morphogenesis,+neural+development+and+plasticity.">Eph/ephrin signaling in morphogenesis, neural development and plasticity.</a> Current Opinion in Cell Biology. 16 (5), 580-589 (2004).</li><li>Wang, K. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+purification+of+a+mammalian+slit+protein+as+a+positive+regulator+of+sensory+axon+elongation+and+branching.">Biochemical purification of a mammalian slit protein as a positive regulator of sensory axon elongation and branching.</a> Cell. 96 (6), 771-784 (1999).</li><li>Emonard, H., Grimaud, J. A., Nusgens, B., Lapiere, C. M., Foidart, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstituted+basement-membrane+matrix+modulates+fibroblast+activities+in+vitro.">Reconstituted basement-membrane matrix modulates fibroblast activities in vitro.</a> Journal of Cell Physiology. 133 (1), 95-102 (1987).</li><li>Knapp, D. M., Helou, E. F., Tranquillo, R. 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The authors have nothing to disclose.

The growth of developing axons is mainly invasive and includes ECM degradation and remodeling. Using the procedure presented here, researchers can obtain a homogenous 3-D matrix formed by the natural type I collagen in which axons (or cells) can respond to a chemical gradient secreted by genetically-modified cells as they do in vivo. Different axonal responses to gradients of attractive or inhibitory cues (protein, lipids, etc.) can be easily compared to specific control (mock transfected cells). As advantages, we must m...

Neuronal axons, ending in axonal growth cones, migrate long distances through the extracellular matrix (ECM) of the embryo over specific pathways to reach their appropriate targets. The growth cone is the distal portion of the axon and it is specialized to sense the physical and molecular environment of the cell1,2. From a molecular point of view, growth cones are guided by at least four different molecular mechanisms: contact attraction, chemoattraction, contact repulsion, and chemorepulsion triggered by different axonal guidance cues3,4,5,6. Contact-mediated processes can be partially monitored in two-dimensional (2D) cultures on micro-patterned substrates (e.g., with stripes7,8 or spots9 containing the molecules). However, axons can navigate to their target in a non-diffusive manner by sensing several attractive and repulsive molecules from guidepost cells in the environment4,5,10. Here, we describe an easy method of 3-D culture to check whether a secreted molecule induces chemorepulsive or chemoattractive effects on developing axons.
The earliest studies aimed to determine the effects of axon guidance cues used explant cultures in three-dimensional (3-D) matrices to generate gradients simulating in vivo conditions11,12. This approach, together with in vivo experiments, allowed for the identification of four major families of guidance cues: Netrins, Slits, Semaphorins, and Ephrins4,5,6. These molecular cues and other factors13 are integrated by the growing axons, triggering the dynamics of adhesion complexes and transducing mechanical forces via the cytoskeleton14,15,16. To generate molecular gradients in 3-D cultures for axonal navigation, pioneering researchers used plasma clot substrates17, which was also used for organotypic slice preparations18. However, in 1958, a new protocol to generate 3-D collagen hydrogels was reported for studying with Maximow&#180;s devices19, a culture platform, used in several studies suitable for microscopic observations20. Another pioneer study reported collagen gel as a tool to embed human fibroblasts for studying the differentiation of fibroblasts into myofibroblasts in wound healing processes21. In parallel, Lumsden and Davies applied collagen from the bovine dermis to analyze the putative effect of nerve growth factor (NGF) on the guiding of sensory nerve fibers22. With the development of new culture platforms (e.g., multi-well plates) by different companies and laboratories, collagen cultures were adapted to these new devices6,23,24,25,26. In parallel, an extract of ECM material derived from the Engelbreth-Holm-Swarm tumor cell line was made commercially available to expand these studies27.
Recently, several protocols have been developed to generate molecular gradients with putative roles in axon guidance using 3-D hydrogels (e.g., collagen, fibrin, etc.)28. Alternatively, the candidate molecule can be immobilized at different concentration in a porous matrix (e.g., NGF29) or generated by culturing in a small region of the 3-D hydrogel cell aggregates secreting the molecule to generate a radial gradient4,23,24,25,26. The last possibility will be explained in this protocol.
The procedure presented here is an easy, fast and highly reproducible method based on the analysis of axonal growth in 3-D hydrogel cultures of the embryonic mouse brain. In comparison with other methods, the protocol is well suited for non-trained researchers and can be fully developed after a short training (1-2 weeks). In this protocol, we first isolate collagen from adult rat tails to further generate 3-D matrices in which genetically-modified cell aggregates are cultured in front of the embryonic neuronal tissue. These cell aggregates form radial chemical gradients of a candidate molecule which elicits a response for the growing axons. Finally, the evaluation of the effects of the molecule on growing axons can easily be performed using a phase contrast microscopy or, alternatively, immunocytochemical methods.

<table border="0" cellpadding="0" cellspacing="0" style="width:1176px;" width="1176">
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		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:455px;">Material</td>
			<td style="width:211px;"></td>
			<td style="width:344px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">3,3&#8242;-Diaminobenzidine tetrahydrochloride 10 mg tablets (DAB)</td>
			<td>Sigma</td>
			<td>D5905</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adult Sprague-Dawley rats (8 to 9 weeks old)&#160;</td>
			<td>Criffa-Credo, Lyon, France</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Avidin-biotin-peroxidase complex (ABC)</td>
			<td>Vector Labs</td>
			<td>PK-4000</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">B27 serum-free supplement 50x&#160;</td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bicinchoninic acid (BCA) protein assay kit</td>
			<td>Pierce</td>
			<td>23225</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cDNA plasmid vectors</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">COS1 cell lines</td>
			<td>ATTC</td>
			<td>CRL-1570</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">D-(+)-Glucose</td>
			<td>Sigma</td>
			<td>16325</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">D-(+)-glucose (45% solution in water) for complete Neurobasal medium</td>
			<td>Sigma</td>
			<td>G8769</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">D-MEM (Dulbecco&#39;s Modified Eagle Medium 1x ) for COS1 culture medium</td>
			<td>Invitrogen</td>
			<td>41966-029</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Dulbecco&#8217;s phosphate buffered saline 10x (without Ca2+ and Mg2+) (D-PBS) for cultures</td>
			<td>Invitrogen</td>
			<td>14200</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Ethanol&#160;</td>
			<td>merck</td>
			<td>108543</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Ethylenediaminetetraacetic acid dihydrate disodium salt (EDTA)</td>
			<td>Sigma</td>
			<td>E5134</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Fluorescence mounting media (e.g., Fluoromount-G or similar)</td>
			<td>Electron Microscopy Sciences (EMS)</td>
			<td>17984-25</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Gelatin powder&#160;</td>
			<td>Sigma</td>
			<td>G1890</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Glacial acetic acid (Panreac, cat. no. 211008)</td>
			<td>Panreac</td>
			<td>211008</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Hank&#8217;s balanced salt solution&#160;</td>
			<td>Invitrogen</td>
			<td>24020083</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Heat-inactivated foetal bovine serum&#160;</td>
			<td>Invitrogen</td>
			<td>10108-165</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Heat-inactivated horse serum&#160;</td>
			<td>Invitrogen</td>
			<td>26050-088</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Hydrogen peroxide (H2O2, 32 to 33% in water)</td>
			<td>Sigma</td>
			<td>316989</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamine 200 mM solution (100x) for complete Neurobasal and COS1 medium</td>
			<td>Invitrogen</td>
			<td>25030-024</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lipofectamine 2000 Reagent</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Mice pregnant female (embryonic day 12.5 to 16.5; E12.5-16.5)&#160;</td>
			<td>Criffa-Credo, Lyon, France</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Modified Minimum Essential Medium Eagle (MEM)</td>
			<td>Invitrogen</td>
			<td>11012-044</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Monoclonal antibody against class III &#946;-tubulin (clone TUJ-1)</td>
			<td>Biolegend</td>
			<td>801201</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-2 supplement 100x&#160;</td>
			<td>Invitrogen</td>
			<td>17502-048</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neurobasal medium&#160;</td>
			<td>Invitrogen</td>
			<td>21103049</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Merck</td>
			<td>1,040,051,000</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/streptomycin solution 100x&#160;</td>
			<td>Invitrogen</td>
			<td>15140-22</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate buffered saline 10x (PBS) for immunocytochemistry</td>
			<td>Invitrogen</td>
			<td>AM9624</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Secondary antibody: biotinylated horse anti-mouse&#160;</td>
			<td>Vector Labs</td>
			<td>BA-2000</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Serum-free medium (Opti-MEM)</td>
			<td>Invitrogen</td>
			<td>11058-021</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium azide&#160;</td>
			<td>Panreac</td>
			<td>162712</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium bicarbonate solution 7.5%&#160;</td>
			<td>Invitrogen</td>
			<td>25080-094</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile culture grade H2O&#160;</td>
			<td>Sigma</td>
			<td>W3500</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TritonTM X-100&#160;</td>
			<td>Sigma</td>
			<td>X100</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trizma base&#160;</td>
			<td>Sigma</td>
			<td>T1503</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin-EDTA (Trypsin (0.05% (wt/vol) with EDTA (1x)</td>
			<td>Invitrogen</td>
			<td>25300-054</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:455px;">Equipment</td>
			<td style="width:211px;"></td>
			<td style="width:344px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 large and 1 small curved scissors for dissection&#160;</td>
			<td>Fine tools Instruments or similar</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5-ml conical centrifuge tubes&#160;</td>
			<td>Eppendorf or similar</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15-ml conical centrifuge tubes&#160;</td>
			<td>Corning or similar</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2 haemostats&#160;&#160;</td>
			<td>Fine tools Instruments or similar</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2 small straight dissecting scissors</td>
			<td>Fine tools Instruments or similar</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">200-ml centrifuge tubes for centrifugation</td>
			<td>Nalgene or similar</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">200-ml sterile glass conical flasks</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-litre glass beaker</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4- and 6-well culture plates&#160;</td>
			<td>Nunc</td>
			<td>176740 and 140675</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Automatic pipette pumps and disposable 10 ml and 25 ml filter-containing sterile plastic pipettes.</td>
			<td colspan="2">Gilson, Brand, Eppendorf or similar&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Automatic pipettes, sterile filter tips and current sterile tips&#160;</td>
			<td>Gilson, Eppendorf or similar</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bench top microcentrifuge with angle fixed rotor&#160;</td>
			<td colspan="2">Eppendorf, Beckman Coulter or similar</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bench top refrigerated centrifuge with swing-bucket rotor (with 1.5, 15 and 50 ml tube adaptors)</td>
			<td colspan="2">Eppendorf, Beckman Coulter or similar</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell culture incubator at 37 &#186;C, 5% CO2 and 95% air</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dialysis tubing cellulose membrane</td>
			<td>Sigma</td>
			<td>D9402</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dialysis tubing closures&#160;</td>
			<td>Sigma</td>
			<td>Z37101-7</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable glass pipettes</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting microscope with dark field optics&#160;</td>
			<td>Olympus SZ51 or similar&#160;</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">High-speed refrigerated Beckman Coulter centrifuge or similar with angle fixed rotor</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laminar flow hood</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Large 100-mm, 60-mm and small 35-mm &#216; cell culture dishes&#160;</td>
			<td>Nunc</td>
			<td>&#160;150679 , 150288 and 150318, respectively</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnetic stirrer and magnetic spin bars</td>
			<td>IKA or similar</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">McIlwain tissue chopper</td>
			<td>Mickle Laboratory Engineering</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">One pair of fine straight forceps and one pair of curved forceps&#160;</td>
			<td>Fine tools Instruments or similar</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Razor blades for the tissue chopper</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpels (number 15 and 11)</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Two pairs of fine spatulas for transferring collagen and tissue pieces</td>
			<td>Fine tools Instruments or similar</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

generation of 3-d collagen-based hydrogels to analyze axonal growth and behavior during nervous system development

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is centered on culturing small pieces of embryonic or early postnatal rodent brains inside a 3-D hydrogel formed by the rat tail tendon-derived type I collagen with specific porosity. Tissue pieces are cultured inside the hydrogel and confronted to specific brain fragments or genetically-modified cell aggregates to produce and secrete molecules suitable for creating a gradient inside the porous matrix. The steps of this protocol are simple and reproducible but include critical steps to be considered carefully during its development. Moreover, the behavior of growing axons can be monitored and analyzed directly using a phase-contrast microscope or mono/multiphoton fluorescence microscope after fixation by immunocytochemical methods.

Here, we present a widely accessible methodology to study axonal growth in 3-D hydrogel collagen cultures of embryonic mouse nervous system. To this end, we isolated collagen from adult rat tails to generate 3-D matrices in which we cultured genetically-modified cell aggregates expressing Netrin-1 or Sema3E confronted with embryonic neuronal tissue (e.g., CA region of the hippocampus). These cell aggregates formed a radially distributed gradient of the candidate molecule inside the collagen matrix. Finally, to evaluate t...

Watch this Scientific Journal Video about Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development at JoVE.com

Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development

Here, we provide a method for analyzing the behavior of growing axons in 3D matrices, mimicking their natural development.

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is ...

This method is useful to order to analyze the role of some molecules in axonal guidance and neuronal migration during neuro system development. The main advantage of this technique is that the analysis of the results is quick and simple and doesn't require complex software which would imply a lot of training for researchers. Demonstrating the procedure will be Mirian Segura-Feliu, a technician from our laboratory and myself.To begin plate 200, 000 COS-1 cells into 35 millimeter Petri dishes and incubate with complete culture medium in a cell culture incubator in order to read 70 to 80%confluency overnight. On the following day, to transfect COS-1 cells with the DNA encoding the candidate molecule using liposome based transfection method. First mix 250 microliters of serum free medium and one to 2 micrograms DNA in a 1.5 milliliter centrifuge tube and incubate at room temperature for five minutes.To prepare a liposomal tube, add 240 microliters of the serum free medium and 10 microliters of liposomal transfection reagent, and incubate at room temperature for five minutes. After incubation, add the content of the DNA tube to the lipsomal tube. Gently mix and incubate at room temperature for 15 minutes.Replace the medium on the cultured cells with 1.5 milliliters of the serum free medium. Add the DNA liposomes mixture slowly and drop wise to the cells. Incubate in the carbon dioxide cell culture incubator for three hours.After completed incubation, replace the medium with the complete culture medium and incubate overnight in the incubator. On the following day, rinse the cells with 0.1 molar Dulbecco's PBS. Add 800 microliters trypsin EDTA per each dish and incubate for five to 15 minutes in the carbon dioxide incubator.Collect detached cells with two milliliters of complete culture medium into a 15 milliliter centrifuge tube and add 10 milliliters of the same medium. Centrifuge the cells at four degrees celsius at 130 times g for five minutes. Remove the supernatant and preserve the pellet containing COS-1 cells on ice.After preparing collagen working mixture, add 100 to 150 microliters of the collagen mixture to the pellet of the transfected cells and mix gently by pipetting up and down. Spread 45 to 50 microliters of collagen cell pellet mixture onto a 35 millimeter Petri dish to form a uniform band of collagen cells, approximately one to 1.5 centimeters long. Place the dish in the incubator at 37 degrees celsius and 5%carbon dioxide until the gelation is observed.Prepare a second strip containing control cells in a second culture dish and place it in the incubator. When gelation is completed, add three to four milliliters of 37 degrees celsius warmed COS-1 complete culture medium to each dish containing the gelled collagen cell strips and place them in the incubator. Use a fine scalpel or a tissue chopper to cut the collagen cell strips to generate small square pieces, 400 to 500 micrometers in length.Transfer all the sections from the same transfection condition to a Petri dish containing three to 3.5 milliliters of neuronal culture media. Check under a dissecting microscope the quality of the pieces and place the dishes in the incubator. Use scissors to cut embryo horns on embryonic day 16.5 from the abdominal cavity of a sacrificed pregnant female rat and place them into a large Petri dish containing cold HBSSg buffer.Place the dish in the laminar flow hood and use straight forceps to extract the embryos and place them into a new dish containing cold HBBSg. With small forceps, remove the skin, and then with curved and straight forceps, carefully dissect the brain and place into a dish containing cold HBSSg. Under a dissecting microscope, using a scalpel or fine scissors, cut the brain in half along the midline to separate both the hemispheres.Then, remove the diencephalon and with fine forceps, remove meninges and blood vessels from the brain pieces. Use 100%ethanol to clean all parts of the tissue chopper, especially PTFE cutting plate and the razor blade and keep the tissue chopper in the laminar flux hood under UV illumination for 15 minutes. Transfer each tissue piece to the cutting plate of the tissue chopper.After the tissue has been chopped, transfer the pieces from the 35 millimeter Petri dishes filled with three to four milliliters of complete NCM. Using fine tungsten needles, finish the tissue dissection in complete NCM. Check the quality of the obtained slices under the dissecting microscope, making sure that the layers are clearly identifiable in the dark field optics and dissect the region of interest with tungsten needles.Keep the good quality pieces in complete NCM medium in the carbon dioxide incubator. Visual demonstration is critical in the steps related to the preparation of the co-cultures which facilitates the words to understand the process. Place several sterile four well culture plates in the laminar flow hood and prepare a collagen working mixture as previously.Add 15 to 20 microliters of the hydrogel mixture into the bottom of each well to produce a circular collagen base. Place the dishes in the incubator until complete gelation is observed. And check the quality of the gelled collagen.With a pipette, transfer a small piece of COS-1 cell aggregate onto the hydrogel base, then use a pipette to place a tissue piece on the same base, close to the piece of cells aggregate at a distance of one explant size. After preparing a new working collagen mixture on ice, gently pipette 15 to 20 microliters of this new mixture to cover the explant and the cell aggregate, making a sandwich like hydrogel culture. Use a fine tungsten needle to reorientate the explant so it faces the cell aggregate at 500 to 600 micrometers.Return the plate to the incubator until the gelation is observed and then add 0.5 milliliters of complete NCM supplemented with 2%B27 supplement and keep cultures for 36 to 48 hours in the incubator. When hippocampal axons were confronted with Netrin-1, they grew preferentially towards the source of Netrin-1, indicating that Netrin-1 acts as a chemo attractive molecule for these axons. In the control condition, or mock transfection, all axons grew radially, without any directional preference.When hippocampal axons were confronted with Sema3E secreting cells, most of them grew opposite of the cell aggregate, indicating that Sema3E acts as a chemo repulsive molecule for these axons. This schematic representation of the axonal response and quantification method shows that in control conditions, the axons were equally distributed in both proximal and distal quadrants, shown with radial outgrowth. This indicated a proximal distal or PD ratio of one.When explants showed increased number of axons in the proximal quadrant in comparison to the distal, indicating chemo attraction, the PD ratio was greater than one. When the number of axons was higher in the distal quadrant than in the proximal one, indicating chemo repulsion, the PD ratio was lesser than one. Following this method, researchers can obtain essential information about the role of some molecules during neuronal development, which allows us to establish a starting point to further explore the functions.Although this technique is useful in neuronal development as this is, it could also be applied in pharmacological screening, angiogenesis, and tissue engineering as strategies.

generation-3-d-collagen-based-hydrogels-to-analyze-axonal-growth

Research

JoVE Journal

Neuroscience

5.6K visualizações.  Parc Científic de Barcelona. Este método é útil para analisar o papel de algumas moléculas na orientação axonal e migração neuronal durante o desenvolvimento do sistema neurológico. A principal vantagem dessa técnica é que a análise dos resultados é rápida e simples e não requer softwares complexos, o que implicaria muito treinamento para pesquisadores. Demonstrando o procedimento estará Mirian Segura-Feliu, técnica do nosso laboratório e eu.Para iniciar a placa 200.000 células COS-1 em placas d...