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.

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<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

5.9K vistas.  University of Alabama at Birmingham. Este protocolo proporciona un esquema detallado de cómo co-cultivar células madre de pulpa dental con neuronas trigéminas. Con él, podemos investigar múltiples respuestas impulsadas por su interferencia. La principal ventaja de esta técnica es la capacidad de estudiar y manipular cualquiera o ambas poblaciones celulares y medir con precisión los resultados.Este método tiene relevancia directa para la investigación neuronal y podría proporcionar información sobre la investiga...