Name: a co-culture method to study neurite outgrowth in response to dental pulp paracrine signals
Uploaded: 2020-02-14T14:00:00
Description: Watch this Scientific Journal Video about A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals at JoVE.com

This work was supported by a) the National Institutes of Health/NIAMS (grant numbers R01 AR062507 and R01 AR053860 to RS), b) the University of Alabama at Birmingham Dental Academic Research Training (DART) grant (number T90DE022736 (PI MacDougall)) to SBP from the National Institute of Dental and Craniofacial Research/National Institutes of Health, c) a UAB Global Center for Craniofacial, Oral and Dental Disorders (GC-CODED) Pilot and Feasibility grant to SBP and d) the National Institute of Dental and Craniofacial Research/National Institutes of Health K99 DE024406 grant to SBP.

All experiments with mice were approved by the UAB Institutional Animal Care and Use Committee (IACUC).
1. Plate Preparation
NOTE: Coverslips can be used to image DP cells at the end of the assay. Be sure the plate lid is on during all incubation and rinsing steps outside of the sterile tissue culture hood to prevent contamination during sample processing.
<ol>
	<li>Coverslip preparation
	<ol>
		<li>Autoclave circular coverslips.</li>
		<li>Filter...

<ol>
	<li>Moe, K., Sijaona, A., Shrestha, A., Kettunen, P., Taniguchi, M., Luukko, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semaphorin+3A+controls+timing+and+patterning+of+the+dental+pulp+innervation.">Semaphorin 3A controls timing and patterning of the dental pulp innervation.</a> Differentiation. 84 (5), 371-379 (2012).</li><li>Kollar, E. J., Lumsden, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tooth+morphogenesis:+the+role+of+the+innervation+during+induction+and+pattern+formation.">Tooth morphogenesis: the role of the innervation during induction and pattern formation.</a> Journal de biologie buccale. 7 (1), 49-60 (1979).</li><li>Lillesaar, C., Fried, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurites+from+trigeminal+ganglion+explants+grown+in+vitro+are+repelled+or+attracted+by+tooth-related+tissues+depending+on+developmental+stage.">Neurites from trigeminal ganglion explants grown in vitro are repelled or attracted by tooth-related tissues depending on developmental stage.</a> Neuroscience. 125 (1), 149-161 (2004).</li><li>Fried, K., Lillesaar, C., Sime, W., Kaukua, N., Patarroyo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target+finding+of+pain+nerve+fibers:+Neural+growth+mechanisms+in+the+tooth+pulp.">Target finding of pain nerve fibers: Neural growth mechanisms in the tooth pulp.</a> Physiology &amp; Behavior. 92 (1-2), 40-45 (2007).</li><li>Pagella, P., Jim&#233;nez-Rojo, L., Mitsiadis, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+innervation+in+developing+and+regenerating+orofacial+tissues.">Roles of innervation in developing and regenerating orofacial tissues.</a> Cellular and Molecular Life Sciences. 71 (12), 2241-2251 (2014).</li><li>Luukko, K., Kettunen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+tooth+morphogenesis+and+innervation+by+local+tissue+interactions,+signaling+networks,+and+semaphorin+3A.">Integration of tooth morphogenesis and innervation by local tissue interactions, signaling networks, and semaphorin 3A.</a> Cell Adhesion &amp; Migration. , 1-9 (2016).</li><li>Smit, M., Leng, J., Klemke, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+for+neurite+outgrowth+quantification.">Assay for neurite outgrowth quantification.</a> BioTechniques. 35 (2), 254-256 (2003).</li><li>de Almeida, J. F. A., Chen, P., Henry, M. A., Diogenes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+of+the+apical+papilla+regulate+trigeminal+neurite+outgrowth+and+targeting+through+a+BDNF-dependent+mechanism.">Stem cells of the apical papilla regulate trigeminal neurite outgrowth and targeting through a BDNF-dependent mechanism.</a> Tissue engineering. Part A. 20 (23-24), 3089-3100 (2014).</li><li>Pagella, P., Miran, S., Mitsiadis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Developing+Tooth+Germ+Innervation+Using+Microfluidic+Co-culture+Devices.">Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices.</a> Journal of Visualized Experiments. (102), e53114 (2015).</li><li>Coelen, R. J., Jose, D. G., May, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+hexadimethrine+bromide+(polybrene)+on+the+infection+of+the+primate+retroviruses+SSV+1/SSAV+1+and+BaEV.">The effect of hexadimethrine bromide (polybrene) on the infection of the primate retroviruses SSV 1/SSAV 1 and BaEV.</a> Archives of Virology. 75 (4), 307-311 (1983).</li><li>Caroni, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+growth-associated+proteins+in+the+neurons+of+adult+transgenic+mice.">Overexpression of growth-associated proteins in the neurons of adult transgenic mice.</a> Journal of neuroscience methods. 71 (1), 3-9 (1997).</li><li>Ali&#263;, I., 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=Neural+stem+cells+from+mouse+strain+Thy1+YFP-16+are+a+valuable+tool+to+monitor+and+evaluate+neuronal+differentiation+and+morphology.">Neural stem cells from mouse strain Thy1 YFP-16 are a valuable tool to monitor and evaluate neuronal differentiation and morphology.</a> Neuroscience Letters. 634, 32-41 (2016).</li><li>Howroyd, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+the+Trigeminal+Ganglion+of+Nonrodent+Species+Used+in+Toxicology+Studies.">Dissection of the Trigeminal Ganglion of Nonrodent Species Used in Toxicology Studies.</a> Toxicologic Pathology. , (2019).</li><li>Schwieger, J., Esser, K. H., Lenarz, T., Scheper, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+long-term+spiral+ganglion+neuron+culture+with+reduced+glial+cell+number:+Effects+of+AraC+on+cell+composition+and+neurons.">Establishment of a long-term spiral ganglion neuron culture with reduced glial cell number: Effects of AraC on cell composition and neurons.</a> Journal of Neuroscience Methods. 268, 106-116 (2016).</li><li>Liu, R., Lin, G., Xu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Efficient+Method+for+Dorsal+Root+Ganglia+Neurons+Purification+with+a+One-Time+Anti-Mitotic+Reagent+Treatment.">An Efficient Method for Dorsal Root Ganglia Neurons Purification with a One-Time Anti-Mitotic Reagent Treatment.</a> PLoS ONE. 8 (4), 60558 (2013).</li><li>Burry, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimitotic+drugs+that+enhance+neuronal+survival+in+olfactory+bulb+cell+cultures.">Antimitotic drugs that enhance neuronal survival in olfactory bulb cell cultures.</a> Brain Research. 261 (2), 261-275 (1983).</li><li>Katzenell, S., Cabrera, J. R., North, B. J., Leib, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+Purification,+and+Culture+of+Primary+Murine+Sensory+Neurons.">Isolation, Purification, and Culture of Primary Murine Sensory Neurons.</a> Methods in molecular biology. 1656, 229-251 (2017).</li><li>Dussor, G. O., Price, T. J., Flores, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activating+transcription+factor+3+mRNA+is+upregulated+in+primary+cultures+of+trigeminal+ganglion+neurons.">Activating transcription factor 3 mRNA is upregulated in primary cultures of trigeminal ganglion neurons.</a> Molecular Brain Research. 118 (1-2), 156-159 (2003).</li><li>Lillesaar, C., Arenas, E., Hildebrand, C., Fried, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+of+rat+trigeminal+neurones+to+dental+pulp+cells+or+fibroblasts+overexpressing+neurotrophic+factors+in+vitro.">Responses of rat trigeminal neurones to dental pulp cells or fibroblasts overexpressing neurotrophic factors in vitro.</a> Neuroscience. 119 (2), 443-451 (2003).</li><li>Lillesaar, C., Eriksson, C., Fried, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+tooth+pulp+cells+elicit+neurite+growth+from+trigeminal+neurones+and+express+mRNAs+for+neurotrophic+factors+in+vitro.">Rat tooth pulp cells elicit neurite growth from trigeminal neurones and express mRNAs for neurotrophic factors in vitro.</a> Neuroscience Letters. 308 (3), (2001).</li><li>Lillesaar, C., Eriksson, C., Johansson, C. S., Fried, K., Hildebrand, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tooth+pulp+tissue+promotes+neurite+outgrowth+from+rat+trigeminal+ganglia+in+vitro.">Tooth pulp tissue promotes neurite outgrowth from rat trigeminal ganglia in vitro.</a> Journal of neurocytology. 28 (8), 663-670 (1999).</li><li>Chmilewsky, F., Ayaz, W., Appiah, J., About, I., Chung, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+Growth+Factor+Secretion+From+Pulp+Fibroblasts+is+Modulated+by+Complement+C5a+Receptor+and+Implied+in+Neurite+Outgrowth.">Nerve Growth Factor Secretion From Pulp Fibroblasts is Modulated by Complement C5a Receptor and Implied in Neurite Outgrowth.</a> Scientific reports. 6, 31799 (2016).</li><li>Pagella, P., Neto, E., Jim&#195;&#169;nez-Rojo, L., Lamghari, M., Mitsiadis, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics+co-culture+systems+for+studying+tooth+innervation.">Microfluidics co-culture systems for studying tooth innervation.</a> Frontiers in Physiology. 5, 326 (2014).</li><li>Miura, T., Yokokawa, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+culture+on+a+chip:+Developmental+biology+applications+of+self-organized+capillary+networks+in+microfluidic+devices.">Tissue culture on a chip: Developmental biology applications of self-organized capillary networks in microfluidic devices.</a> Development, Growth &amp; Differentiation. 58 (6), 505-515 (2016).</li></ol>

The daily activities of the oral cavity require that teeth sense external stimuli and internal inflammation in order to permit proper usage and maintenance. However, only limited information is available regarding the signals that drive the developmental processes of tooth innervation. This protocol provides a method to isolate and co-culture primary DP cells and TG neurons in order to study the cross-communication between the two populations. Several variables were optimized and leave open further avenues of research, a...

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. Failure to sense tooth pain associated with dental caries and trauma leads to disease progression. Thus, proper innervation is a requirement for normal tooth growth, function and care.
While most organs are fully functional and innervated by the time of birth, tooth development extends into adult life, with tooth innervation and mineralization occurring in concert during postnatal stages1,2. Interestingly, the dental pulp (DP) mesenchyme initially secretes repellant signals during embryogenesis to prevent axon entry into the developing tooth organ, which later shifts to the secretion of attractant factors as the tooth nears eruption3,4. During postnatal stages, afferent axons from the trigeminal (TG) nerve penetrate into and throughout the tooth around the time dentin deposition begins (reviewed in Pagella, P. et al.5). Several in vivo studies have demonstrated that neuronal-mesenchymal interactions guide tooth innervation in mice (reviewed in Luukko, K. et al.6), but few details of the molecular mechanisms are available.
Cell co-cultures provide controlled environments in which investigators can manipulate interactions between neuronal and mesenchymal populations. Co-culture experiments make it possible to delve deeper into the signaling pathways guiding tooth innervation and development. However, several of the conventional methods used to study cells in co-culture present technical challenges. For instance, crystal violet staining of neurite outgrowth can non-specifically stain Schwann cells included in TG bundle dispersions, and there may be peaks in color intensity with relatively small responses7. Microfluidic chambers offer an attractive option, but are considerably more expensive than transwell filters8,9 and only permit the investigation of neuronal responses to DP secretions. To address these issues, we have developed a protocol that allows for: a) precise staining and imaging of TG neurite outgrowth in response to DP secretions, b) genetic modification of DP cells and/or TG neurons to investigate specific signaling pathways, and c) investigation of DP cell responses to factors secreted by TG neurons. This protocol provides the ability to precisely investigate several features of tooth innervation in the controlled environment of an in vitro co-culture assay.

&#160; &#160;

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5-Fluoro-2&#39;-deoxyuridine</td>
			<td>Sigma-Aldrich</td>
			<td>F0503</td>
			<td>Used as a mitotic Inhibitor at 15 &#956;M concentration in co-culture media, Day 2</td>
		</tr>
		<tr>
			<td>24 Well Cell Culture Plate</td>
			<td>Corning</td>
			<td>3524</td>
			<td>Co-culture plate</td>
		</tr>
		<tr>
			<td>Alexa-546 anti-chicken</td>
			<td>Invitrogen</td>
			<td>A-11040</td>
			<td>Secondary to stain neurite outgrowth labeled by anti-GFP antibody, 1:500 dilution</td>
		</tr>
		<tr>
			<td>Anti-GFP Antibody</td>
			<td>Aves Lab, Inc</td>
			<td>GFP-1010</td>
			<td>Primary antibody to label Thy1-YFP neurons, 1:200 dilution</td>
		</tr>
		<tr>
			<td>Anti-Neurofilament 200 antibody</td>
			<td>Sigma-Aldrich</td>
			<td>NO142</td>
			<td>Monoclonal primary antibody to label neurons, 1:1000 dilution, alternative if YFP mice are not available</td>
		</tr>
		<tr>
			<td>B6;129- Tgfbr2tm1Karl/J</td>
			<td>The Jackson Laboratory</td>
			<td>12603</td>
			<td>Tgfbr2f/f mouse model used for dental pulp cells in optimized protocol</td>
		</tr>
		<tr>
			<td>B6.Cg-Tg(Thy1-YFP)16Jrs/J</td>
			<td>The Jackson Laboratory</td>
			<td>3709</td>
			<td>Thy1-YFP mouse model genotype used for trigeminal neurons</td>
		</tr>
		<tr>
			<td>Collagenase Type II</td>
			<td>Millipore</td>
			<td>234155-100MG</td>
			<td>Used to disperse trigeminal neurons</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10437</td>
			<td>Additive to co-culture media</td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Fine Science Tools</td>
			<td>11413-11</td>
			<td>Fine forceps for TG dissection</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma-Aldrich</td>
			<td>L2020</td>
			<td>Coats the transwell inserts at final concentration of 10 &#956;g/ml, stock solution is assumed at 1.5 mg/ml</td>
		</tr>
		<tr>
			<td>Lysis Buffer (Buffer RLT)</td>
			<td>Qiagen</td>
			<td>79216</td>
			<td>Extracts RNA from dental pulp cells post co-culture</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Gibco</td>
			<td>25030081</td>
			<td>Additive to co-culture media</td>
		</tr>
		<tr>
			<td>Micro-dissecting scissors</td>
			<td>Sigma-Aldrich</td>
			<td>S3146-1EA</td>
			<td>Dissection scissors to open skull</td>
		</tr>
		<tr>
			<td>Microscope Cover Glass</td>
			<td>Fisherbrand</td>
			<td>12-545-81</td>
			<td>Circlular coverslip for optional cell culturing and immunofluorescence processing</td>
		</tr>
		<tr>
			<td>Minimal Essential Medium a</td>
			<td>Gibco</td>
			<td>12571063</td>
			<td>Co-culture media base</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15070063</td>
			<td>Antibiotic additive to co-culture media</td>
		</tr>
		<tr>
			<td>Phosphatase Inhibitor</td>
			<td>Sigma-Aldrich</td>
			<td>04 906 837 001</td>
			<td>Additive to RIPA Buffer for extracting protein from dental pulp cells post co-culture</td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>Millipore</td>
			<td>TR-1003-G</td>
			<td>Used to aid in dental pulp cell transfection</td>
		</tr>
		<tr>
			<td>Poly-D-Lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P7280</td>
			<td>Coverslip coating to aid dental pulp cellular adhesion</td>
		</tr>
		<tr>
			<td>Protease Inhibitors</td>
			<td>Millipore</td>
			<td>05 892 791 001</td>
			<td>Additive to RIPA Buffer for extracting protein from dental pulp cells post co-culture</td>
		</tr>
		<tr>
			<td>RNAse/DNAse free eppendorf tubes</td>
			<td>Denville</td>
			<td>C-2172</td>
			<td>Presterilized 1.7 ml tubes for RNA, DNA or protein collection at the end of assay</td>
		</tr>
		<tr>
			<td>ThinCert Cell Culture Insert</td>
			<td>Greiner Bio-One</td>
			<td>662631</td>
			<td>Transwell inserts for trigeminal neurons in co-culture assays</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%)</td>
			<td>Gibco</td>
			<td>25200056</td>
			<td>Used fto disperse dental pulp cells</td>
		</tr>
		<tr>
			<td>Trypsin Type II</td>
			<td>Sigma-Aldrich</td>
			<td>T-7409</td>
			<td>Used to disperse trigeminal neurons</td>
		</tr>
		<tr>
			<td>Ultra Fine Forceps</td>
			<td>Fine Science Tools</td>
			<td>11370-40</td>
			<td>Ultra fine forceps for dissection</td>
		</tr>
		<tr>
			<td>Uridine</td>
			<td>Sigma-Aldrich</td>
			<td>U3750</td>
			<td>Used as a mitotic Inhibitor at 1 &#956;M concentration in co-culture media, Day 2</td>
		</tr>
		<tr>
			<td>Vacuum Filtration System</td>
			<td>Millipore</td>
			<td>SCNY00060</td>
			<td>Steriflip disposable filter, 50 &#956;m nylon net filter</td>
		</tr>
		<tr>
			<td>Vial forceps</td>
			<td>Fine Science Tools</td>
			<td>110006-15</td>
			<td>Long forceps for tissue transfer to conicals</td>
		</tr>
	</tbody>
</table>

a co-culture method to study neurite outgrowth in response to dental pulp paracrine signals

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. Without sensory innervation, daily oral activities would cause irreparable damage. Despite its importance, the roles of innervation in tooth development and maintenance have been largely overlooked. Several studies have demonstrated that DP cells secrete extracellular matrix proteins and paracrine signals to attract and guide TG axons into and throughout the tooth. However, few studies have provided detailed insight into the crosstalk between the DP mesenchyme and neuronal afferents. To address this gap in knowledge, researchers have begun to utilize co-cultures and a variety of techniques to investigate these interactions. Here, we demonstrate the multiple steps involved in co-culturing primary DP cells with TG neurons dispersed on an overlying transwell filter with large diameter pores to allow axonal growth through the pores. Primary DP cells with the gene of interest flanked by loxP sites were utilized to facilitate gene deletion using an Adenovirus-Cre-GFP recombinase system. Using TG neurons from the Thy1-YFP mouse allowed for precise afferent imaging, with expression well above background levels by confocal microscopy. The DP responses can be investigated via protein or RNA collection and analysis, or alternatively, through immunofluorescent staining of DP cells plated on removable glass coverslips. Media can be analyzed using techniques such as proteomic analyses, although this will require albumin depletion due to the presence of fetal bovine serum in the media. This protocol provides a simple method that can be manipulated to study the morphological, genetic, and cytoskeletal responses of TG neurons and DP cells in response to the controlled environment of a co-culture assay.

These results show that TG neurite outgrowth was increased in the presence of primary DP cells in the underlying well compared to the control of TG neurite monoculture (Figure 2A,C). There is some assay-to-assay variability in neurite outgrowth. Thus, a TG neuron monoculture should be included in all assays as a control to detect the basal levels of neurite outgrowth. Primary cells from the Tgfbr2f/f mouse were used in this protocol after infection of Ad-Cre-GFP a...

Watch this Scientific Journal Video about A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals at JoVE.com

A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals

We describe the isolation, dispersion and plating of dental pulp (DP) primary cells with trigeminal (TG) neurons cultured atop overlying transwell filters. Cellular responses of DP cells can be analyzed with immunofluorescence or RNA/protein analysis. Immunofluorescence of neuronal markers with confocal microscopy permits the analysis of neurite outgrowth responses.

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. ...

This protocol provides detailed outline of how to co-culture dental pulp stem cells with trigeminal neurons. With it, we can investigate multiple responses driven by their crosstalk. The main advantage of this technique is the ability to study and manipulate either or both cell populations and precisely measure the outcomes.This method has direct relevance to neural research and could provide insight into research on how dental pulp stem cells repair neural tissue damaged by traumatic events or neurodegenerative diseases. There's several stages of this technique to learn and it can be hard to keep tract of them all. This protocol details each stage to help with that.The dental pulp and ganglia are difficult to disperse and require various vortexing and pipetting. Even then you'll not get full dispersion so optimization and replicates will be required. After euthanization of the mouse, place the head on the disposable underpad so that the mouth is towards the ceiling and the base of the neck is flat on the work surface.Use a razor blade and a sawing motion to separate the mandible from the maxilla. Remove the tongue with scissors or forceps to allow easier access to the molars. Place the open head in the dish atop a sterile gauze pad and place the specimen under the dissecting microscope.Remove the alveolar bone tissue surrounding the first molars. Insert forceps into the alveolar opening and tease the tissue away from the tooth toward the buccle or lingual side of the mouth. Collect all maxillary first molars and gently transfer the submandibular and maxillary first molars to a separate cell culture dish with 1X PBS on ice.Remove the enamel outer organ surrounding the outside of each maxillary first molar. With a set of forceps rotate the molar so that cusps are down and the open root is exposed. There's an oval opening on the bottom of the tooth and opaque dental pulp tissue encapsulated by a thin layer of dentin and enamel.Using the tip of the forceps, gently loosen the dental pulp by running one arm of the forceps around the internal circumference of the mineralized tissue. Remove the dental pulp tissue out of the mineralized structure and transfer to a third dish containing 1X PBS. Remove the enamel outer organ if it was not already separated.Transfer all dental pulp tissue to 0.25%trypsin EDTA in a 50 milliliter conical tube. Vortex the mixture and place it in a 37 degree Celsius warm water bath for 10 minutes. Under a sterile hood, add warmed co-culture media to a final ratio of at least one-to-one media to trypsin to inactivate the enzyme.Pipet the media up and down multiple times with a 10 milliliter pipet to further disperse the dental pulp in the media, avoid large bubbles. Transfer one milliliter of the dispersed dental pulp to each well of 24-well tissue culture plate. Place the plate in an incubator at 37 degree Celsius.And allow the cells to attach and migrate out from the undispersed tissue for 48 hours before changing media. After euthanization and removal of skin, insert the tip of a pair of micro dissecting scissors into the base of the skull. Cut along the sagittal suture of the skull.Make four small horizontal cuts to along the coronal sutures by the ears until along the lambdoid sutures of the base of the skull. This creates two flaps of bone. Use the forceps to peel back the two flaps of bone to reveal the brain.Locate the trigeminal ganglia housed in the dura mater between the brain and bone of maxillary process. Cut the three branches that travel to the eyes, maxillae, and mandible. And use straight edge fine forceps to transfer the ganglia to cold 1X PBS in a dish on ice.Once all trigeminal bundles are harvested use file forceps to transfer ganglia to 50 milliliter conical tube containing five milligrams per milliliter of sterile filtered collagenase type II.Vortex the mixture and place the tube in the 37 degree Celsius water bath for 25 to 30 minutes. Every five to ten minutes, take the tube out of the water bath, vortex, and return to the bath. Centrifuge the collagenase trigeminal neuron solution for two minutes at 643xg.Under a tissue culture hood, gently aspirate the collagenase with a micropipet and add five milliliters of 1%sterile filtered trypsin type II.Then place the tube into a 37 degree Celsius water bath for 25 to 30 minutes and within this time frame vortex the tube briefly every five minutes. Add media at a one-to-one ratio of trypsin to media to deactivate the remaining trypsin. Count the number of cells and dilute the mixture to 200, 000 cells per milliliter in media.Place coated transwell filters into wells with dental pulp. Pipet 250 microliters onto the transwell filter and culture the cells at 37 degree Celsius overnight. The next day, replace the media with one milliliter of co-culture media supplemented with one micromolar uridine and 15 micromolar 5-Fluoro-2'deoxyuridine to stop the over proliferation of mesenchymal cells that may prevent neurite outgrowth.You must add mitotic inhibitors on day two or neurite growth will not occur. In this study, trigeminal neurite outgrowth was increased in the presence of primary dental pulp cells in the underlying well compared to the control of trigeminal neurite monoculture. Primary cells isolated from the TGF-beta receptor 2 flox/flox mouse were equivalent in number after injections of either ade-Cre-GFP or ade-eGFP.Semi-quantitative PCR demonstrates that the adenovirus-Cre-GFP deleted the flanked gene transforming growth factor beta receptor 2 with adenovirus-eGFP serving as a control viral vector. In the cultures with transforming growth factor beta receptor 2 deletion, neurite outgrowth was decreased. Bright-field imaging of transwell filters after crystal violet staining of cell populations shows that large pores are prevalent.The large arrow points out a cell with mesenchymal morphology whereas the small arrow points to a cell of neuronal morphology. Crystal violet stained both cells without bias. In addition, immunofluorescence staining of beta-III tubulin with an alexa 488 secondary antibody shows non-specific staining of multiple cells making imaging of afferent structures difficult.Because neither dental pulp cells nor trigeminal neurons disperse easily, each researcher will need to optimize their plating procedures. If you culture dental pulp cells alone in separate wells you can use immunofluorescence or you can quantify RNA or protein levels to determine how the dental pulp cells are responding to the neurons. Remember to bleach any containers or tips that come in contact with adenovirus.Be sure to discard of razors properly in a sharpy bin.

a-co-culture-method-to-study-neurite-outgrowth-response-to-dental

Research

JoVE Journal

Developmental Biology

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often located distally from the regulated gene in intergenic regions or even within the body of another gene. The position independent nature of enhancer activity makes it difficult to match enhancers with the genes they regulate. Deletion of an enhancer region provides direct evidence for enhancer activity and is the gold standard to reveal an enhancer's role in endogenous gene transcription. Conventional homologous recombination based deletion methods have been surpassed by recent advances in genome editing technology which enable rapid and precisely located changes to the genomes of numerous model organisms. CRISPR/Cas9 mediated genome editing can be used to manipulate the genome in many cell types and organisms rapidly and cost effectively, due to the ease with which Cas9 can be targeted to the genome by a guide RNA from a bespoke expression plasmid. Homozygous deletion of essential gene regulatory elements might lead to lethality or alter cellular phenotype whereas monoallelic deletion of transcriptional enhancers allows for the study of cis-regulation of gene expression without this confounding issue. Presented here is a protocol for CRISPR/Cas9 mediated deletion in F1 mouse embryonic stem (ES) cells (Mus musculus129 x Mus castaneus). Monoallelic deletion, screening and expression analysis is facilitated by single nucleotide polymorphisms (SNP) between the two alleles which occur on average every 125 bp in these cells.

Experimental validation of enhancer activity is best approached by loss-of-function analysis. Presented here is an efficient protocol that uses CRISPR/Cas9 mediated deletion to study allele-specific regulation of gene transcription in F1 ES cells which contain a hybrid genome (Mus musculus129 x Mus castaneus).

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often ...

Using Touch-evoked Response and Locomotion Assays to Assess Muscle Performance and Function in Zebrafish

Zebrafish muscle development is highly conserved with mammalian systems making them an excellent model to study muscle function and disease. Many myopathies affecting skeletal muscle function can be quickly and easily assessed in zebrafish over the first few days of embryogenesis. By 24 hr post-fertilization (hpf), wildtype zebrafish spontaneously contract their tail muscles and by 48 hpf, zebrafish exhibit controlled swimming behaviors. Reduction in the frequency of, or other alterations in, these movements may indicate a skeletal muscle dysfunction. To analyze swimming behavior and assess muscle performance in early zebrafish development, we utilize both touch-evoked escape response and locomotion assays.
Touch-evoked escape response assays can be used to assess muscle performance during short burst movements resulting from contraction of fast-twitch muscle fibers. In response to an external stimulus, which in this case is a tap on the head, wildtype zebrafish at 2 days post-fertilization (dpf) typically exhibit a powerful burst swim, accompanied by sharp turns. Our method quantifies skeletal muscle function by measuring the maximum acceleration during a burst swimming motion, the acceleration being directly proportional to the force produced by muscle contraction.
In contrast, locomotion assays during early zebrafish larval development are used to assess muscle performance during sustained periods of muscle activity. Using a tracking system to monitor swimming behavior, we obtain an automated calculation of the frequency of activity and distance in 6-day old zebrafish, reflective of their skeletal muscle function. Measurements of swimming performance are valuable for phenotypic assessment of disease models and high-throughput screening of mutations or chemical treatments affecting skeletal muscle function.

Zebrafish are an excellent model to study muscle function and disease. During early embryogenesis zebrafish begin regular muscle contractions producing rhythmic swimming behavior, which is altered when the muscle is disrupted. Here we describe a touch-evoked response and locomotion assay to examine swimming performance as a measure of muscle function.

Zebrafish muscle development is highly conserved with mammalian systems making them an excellent model to study muscle function and disease. Many ...

Application of Impermeable Barriers Combined with Candidate Factor Soaked Beads to Study Inductive Signals in the Chick

The chick embryo provides a superb vertebrate model that can be used to dissect developmental questions in a direct way. Its accessibility and robustness following surgical intervention are key experimental strengths. Mica plates were the first barriers used to prevent chick limb bud initiation1. Protocols that use aluminum foil as an impermeable barrier to wing bud or leg bud induction and or initiation are described. We combine this technique with bead placement lateral to the barrier to exogenously supply candidate endogenous factors that have been blocked by the barrier. The results are analyzed using in situ hybridization of subsequent gene expression. Our main focus is on the role of retinoic acid signaling in the induction and later initiation of the chick embryo fore and hindlimb. We use BMS 493 (an inverse agonist of retinoic acid receptors (RAR)) soaked beads implanted in the lateral plate mesoderm (LPM) to mimic the effect of a barrier placed between the somites (a source of retinoic acid (RA)) and the LPM from which limb buds grow. Modified versions of these protocols could also be used to address other questions on the origin and timing of inductive cues. Provided the region of the chick embryo is accessible at the relevant developmental stage, a barrier could be placed between the two tissues and consequent changes in development studied. Examples may be found in the developing brain, axis extension and in organ development, such as liver or kidney induction.

Protocols using impermeable barriers to block induction events between tissues of the main body axis and flank in the chick embryo required for limb formation are described. Beads soaked in candidate inductive signals are used to overcome the effect of barrier placement and analysis of gene expression confirms this.

The chick embryo provides a superb vertebrate model that can be used to dissect developmental questions in a direct way. Its accessibility and robustness ...

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature maintenance and pathological malformations can result in severe ischemic diseases, while overly abundant vascular development is associated with cancer and inflammatory disorders. A promising form of pro-angiogenic therapy is the use of angiogenic cell sources, which can provide regulatory factors as well as physical support for newly developing vasculature.
Mesenchymal Stromal Cells (MSCs) are extensively investigated candidates for vascular regeneration due to their paracrine effects and their ability to detect and home to ischemic or inflamed tissues. In particular, first trimester human umbilical cord perivascular cells (FTM HUCPVCs) are a highly promising candidate due to their pericyte-like properties, high proliferative and multilineage potential, immune-privileged properties, and robust paracrine profile. To effectively evaluate potentially angiogenic regenerative cells, it is a requisite to test them in reliable and &#34;translatable&#34; pre-clinical assays. The aortic ring assay is an ex vivo angiogenesis model that allows for easy quantification of tubular endothelial structures, provides accessory supportive cells and extracellular matrix (ECM) from the host, excludes inflammatory components, and is fast and inexpensive to set up. This is advantageous when compared to in vivo models (e.g., corneal assay, Matrigel plug assay); the aortic ring assay can track the administered cells and observe intercellular interactions while avoiding xeno-immune rejection.
We present a protocol for a novel application of the aortic ring assay, which includes human MSCs in co-cultures with developing rat aortic endothelial networks. This assay allows for the analysis of the MSC contribution to tube formation and development through physical pericyte-like interactions and of their potency for actively migrating to sites of angiogenesis, and for evaluating their ability to perform and mediate ECM processing. This protocol provides further information on changes in MSC phenotype and gene expression following co-culture.

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Here, we present a novel application of the aortic ring assay where prelabelled mesenchymal cells are co-cultured with rat aorta-derived endothelial networks. This novel method allows visualization of Mesenchymal Stromal Cells (MSCs) homing and integration with endothelial networks, quantification of network properties, and evaluation of MSC immunophenotypes and gene expression.

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by Sertoli cell junctions at the blood-testis barrier (BTB), which is the tightest tissue barrier in the mammalian body and segregates the seminiferous epithelium into two compartments, a basal and an adluminal. The BTB creates a unique microenvironment for germ cells in meiosis I/II and for the development of postmeiotic spermatids into spermatozoa via spermiogenesis. Here, we describe a reliable assay to monitor BTB integrity of mouse testis in vivo. An intact BTB blocks the diffusion of FITC-conjugated inulin from the basal to the apical compartment of the seminiferous tubules. This technique is suitable for studying gene candidates, viruses, or environmental toxicants that may affect BTB function or integrity, with an easy procedure and a minimal requirement of surgical skills compared to alternative methods.

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Here, we present a protocol to assess the blood-testis barrier integrity by injecting inulin-FITC into testes. This is an efficient in vivo method to study blood-testis barrier integrity that can be compromised by genetic and environmental elements.

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by ...

Isolation, Propagation, and Prion Protein Expression During Neuronal Differentiation of Human Dental Pulp Stem Cells

Bioethical issues related to the manipulation of embryonic stem cells have hindered advances in the field of medical research. For this reason, it is very important to obtain adult stem cells from different tissues such as adipose, umbilical cord, bone marrow and blood. Among the possible sources, dental pulp is particularly interesting because it is easy to obtain in respect of bioethical considerations. Indeed, human Dental Pulp Stem Cells (hDPSCs) are a type of adult stem cells able to differentiate in neuronal-like cells and can be obtained from the third molar of healthy patients (13-19 ages). In particular, the dental pulp was removed with an excavator, cut into small slices, treated with collagenase IV and cultured in a flask. To induce the neuronal differentiation, hDPSCs were stimulated with EGF/bFGF for 2 weeks. Previously, we have demonstrated that during the differentiation process the content of cellular prion Protein (PrPC) in hDPSCs increased. The cytofluorimetric analysis showed an early expression of PrPC that increased after neuronal differentiation process. Ablation of PrPC by siRNA PrP prevented neuronal differentiation induced by EGF/bFGF. In this paper, we illustrate that as we enhanced the isolation, separation and in vitro cultivation methods of hDPSCs with several easy procedures, more efficient cell clones were obtained and large-scale expansion of the mesenchymal stem cells (MSCs) was observed. We also show how the hDPSCs, obtained with methods detailed in the protocol, are an excellent experimental model to study the neuronal differentiation process of MSCs and subsequent cellular and molecular processes.

Here we present a protocol for human Dental Pulp Stem Cells isolation and propagation in order to evaluate the prion protein expression during neuronal differentiation process.

Bioethical issues related to the manipulation of embryonic stem cells have hindered advances in the field of medical research. For this reason, it is very ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

An Enhanced Green Fluorescence Protein-based Assay for Studying Neurite Outgrowth in Primary Neurons

Neurite outgrowth is a fundamental event in the formation of the neural circuits during nervous system development. Severe neurite damage and synaptic dysfunction occur in various neurodegenerative diseases and age-related degeneration. Investigation of the mechanisms that regulate neurite outgrowth would not only shed valuable light on brain developmental processes but also on such neurological disorders. Due to the low transfection efficiency, it is currently challenging to study the effect of a specific protein on neurite outgrowth in primary mammalian neurons. Here, we describe a simple method for the investigation of neurite outgrowth by the co-transfection of primary rat cortical neurons with EGFP and a protein of interest (POI). This method allows the identification of POI transfected neurons through the EGFP signal, and thus the effect of the POI on neurite outgrowth can be determined precisely. This EGFP-based assay provides a convenient approach for the investigation of pathways regulating neurite outgrowth.

In this report, we describe a simple protocol for studying neurite outgrowth in embryonic rat cortical neurons by co-transfecting with EGFP and the protein of interest.

Neurite outgrowth is a fundamental event in the formation of the neural circuits during nervous system development. Severe neurite damage and synaptic ...