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EoE Sub Articles

1. Plate Preparation
NOTE: 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>Coating filters
	<ol>
		<li>Dilute laminin to 10 &#956;g/mL. Filter the diluted laminin under the hood.</li>
		<li>Pipette 450-500 &#956;L of 10 &#956;g/mL laminin into each well of a 24-well plate.</li>
		<li>Place a transwell filter, 3 &#956;m porosity, in a well so that it contacts the laminin solution and leave it in a 37 &#176;C incubator for either 2 h or overnight. Filter pores should allow some of the solution to diffuse and coat both the top and bottom of the filter. These filters should be used within 48 h or be refrigerated at 4 &#176;C for up to 1 week.</li>
	</ol>
	</li>
</ol>
2. Cell Plating 
<ol>
	<li>Mice: Genetic alteration of dental pulp (DP) cells can be performed (but is not required) to study mesenchymal-neuronal interactions.</li>
	<li>DP dissection, dispersion, and plating (Figure 1) 
	NOTE: For DP dissection, use ultra-fine, straight-edge forceps. The ultra-fine edges will allow the user to wedge the forceps edge between the mineralized structure and DP tissue.
	<ol>
		<li>Harvest P5-P8 mice. At this stage, the teeth should be mineralizing, and the root is open.</li>
		<li>Anesthetize the neonates via hypothermia by placing them in a dish in the 4 &#176;C refrigerator until they no longer move or respond to touch. Euthanize neonates by decapitation and in accordance with Institutional Animal Care &#38; Use Committee (IACUC) procedures at the designated facility.</li>
		<li>Prepare a 3-5 mL aliquot of 0.25% trypsin-ethylenediamine tetraacetic acid (trypsin-EDTA) in a 50 mL conical tube to collect the DP from each mouse. This will facilitate the digestion of dental pulp from postnatal mice. Use more than 3 mL if digesting tissue from more than 10 postnatal mice.</li>
		<li>Place the head on a disposable underpad so that the mouth is toward the ceiling and the base of the neck is flat on the work surface. Use a razor blade in a sawing motion to separate the mandible from the maxilla.</li>
		<li>Optionally remove the tongue either with scissors or with forceps to allow easier access to the molars.</li>
		<li>Place the opened head in a dish atop a sterile gauze pad and place the specimen under a dissecting microscope (Figure 1D).</li>
		<li>Remove the alveolar bone tissue surrounding the first molars. Submandibular teeth are not fully erupted at this point. Insert forceps into alveolar opening and tease the tissue away from the tooth toward the buccal (cheek) or lingual (tongue) side of the mouth. Maxillary teeth will require full removal of the cleft around the tooth for exposure and removal.</li>
		<li>Gently transfer the submandibular and maxillary first molars (M1s) to a separate cell culture dish with 1x phosphate-buffered saline (PBS).</li>
		<li>Repeat 2.2.4-2.2.8 until all M1s are collected. Keep the dish containing the M1s on ice during the harvesting.</li>
		<li>Remove the Enamel Outer Organ (EOE) surrounding the outside of each M1. This can alternatively be done after step 2.2.11.</li>
		<li>With a set of forceps, rotate the M1 so the cusps are down and the open root is exposed. There will be an oval opening on the bottom of the tooth, and opaque DP tissue encapsulated by a thin layer of dentin and enamel.</li>
		<li>Using the tip of the forceps, gently loosen the DP by running one arm of the forceps around the internal circumference of the mineralized tissue. Remove DP tissue out of the mineralized structure and transfer it to a third dish containing 1x PBS. Remove the EOE if it was not already separated (Figure 1E).</li>
		<li>Transfer all DP tissue to 0.25% trypsin-EDTA in a 50 mL conical tube. Vortex the mixture and place in a 37 &#176;C warm water bath for 10 min. This can be done with the same forceps or with long, vial forceps. The tissue will be difficult to disperse and will require vortexing every 3-4 min. Do NOT exceed 10 min trypsinization since the trypsin can damage cell membranes.</li>
		<li>Under a sterile hood, add warmed co-culture media (Table 1) to a final ratio of at least 1:1 media to trypsin to inactivate the enzyme. Larger ratios are acceptable if more tissue dispersion is desired.</li>
		<li>Pipette the media up and down multiple times with a 10 mL pipette to further disperse the DP in the media. Be careful to avoid large bubbles. Complete dispersion is nearly impossible due to the sticky nature of the tissue. However, it is also not necessary since cells will migrate outward from the tissue once plated.</li>
		<li>Transfer 1 mL of the dispersed DP to each well of a 24-well tissue culture plate (Figure 1F).</li>
		<li>Place the plate in an incubator at 37 &#176;C and allow cells to attach and migrate out from the undispersed tissue for 48 h before changing media. Primary cells need to be plated at relatively high concentrations in order to reach 85-90% confluence within 1 week. If this is not achieved after 1 week, discard the plate.</li>
	</ol>
	</li>
	<li>Trigeminal neuron dissection, dispersion and plating 
	NOTE: In this protocol, the imaging of neurite outgrowth in co-culture with DP cells was optimized using adolescent (6-week-old) B6.Cg-Tg(Thy1-YFP)16Jrs/J mice. Central and peripheral nervous systems of Thy1-YFP mice have a yellow fluorescent protein (YFP) tag whose expression begins around P6-P10 in neurons and increases exponentially throughout the nervous system during postnatal and adult life. YFP and GFP have conserved sequences that allow these nerves to be stained with anti-GFP antibodies, resulting in a pan-neuronal stain. Ultimately, these mice allow for better visualization and quantification of the neurons used and grown in cell culture.&#160; &#160; &#160;&#160;
	<ol>
		<li>Euthanize adolescent mice with carbon dioxide followed by cervical dislocation.</li>
		<li>Decapitate the mice and remove the skin from the skull. Be sure to include equivalent numbers of males and females.</li>
		<li>Insert the tip of a pair of micro-dissecting scissors into the base of the skull. Cut along the sagittal suture of the skull (Figure 1A).</li>
		<li>Make four small horizontal cuts: two along the coronal sutures by the ears, and two along the lambdoid sutures at the base of the skull. This should create two flaps of bone.</li>
		<li>Use the forceps to peel back the two flaps of bone. This should reveal the brain.</li>
		<li>Remove the brain. Transfer the head to a tissue culture dish with 1x PBS and put under the microscope.</li>
		<li>Locate the trigeminal (TG) ganglia, which is easily visible in rodents13, housed in the dura mater between the brain and bone of the maxillary process (Figure 1B).</li>
		<li>Cut the three branches that travel to the eyes, maxilla and mandible and transfer the ganglia to cold 1x PBS using straight-edge fine forceps. Keep the dish containing the TG ganglia on ice during harvesting.</li>
		<li>Once all TG bundles are harvested, transfer ganglia to a 50 mL conical tube containing 5 mg/mL sterile-filtered collagenase type II using vial forceps.</li>
		<li>Vortex the collagenase with the TG bundle and place the tube in a 37 &#176;C water bath for 25-30 min. During this time, take the conical tube out of the water bath, vortex, and return to the bath every 5-10 min.</li>
		<li>Centrifuge the collagenase-TG neuron solution for 2 min at 643 x&#160;g.</li>
		<li>Under a tissue culture hood, gently aspirate the collagenase with a micropipette.</li>
		<li>Add 5 mL of 1% sterile-filtered trypsin type II and vortex. Place the conical tube in a 37 &#176;C water bath for 5 min.</li>
		<li>Centrifuge the trypsin-TG mix for 5 min at 643 x&#160;g. Remove the top portion of trypsin with a micropipette, so that the TG neurons are not removed. There will still be liquid in the tube.</li>
		<li>Add enough media to deactivate the remaining trypsin (at a 1:1 or lower ratio of trypsin to media).</li>
		<li>Count the number of cells and dilute the solution to 200,000 cells/mL (250 &#956;L of cell-containing 50,000 cells).</li>
		<li>Place coated transwell filters from section 1.2 into wells with DP.</li>
		<li>Dilute the cell-containing solution so that there are 200,000 cells/mL. Pipette 250 &#956;L onto the transwell filter, and culture the cells at 37 &#176;C overnight (Figure 1F).</li>
		<li>The next day, replace the media with 1 mL of Co-culture media with 1 &#956;M uridine and 15 &#956;M 5&#39;-fluoro-2 deoxyuridine to stop the over-proliferation of mesenchymal cells that may prevent neurite outgrowth. Optional: Add growth factors or inhibitors in this media if attempting further manipulation.</li>
	</ol>
	</li>
</ol>
Table 1: Co-culture media.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Component</td>
			<td>Volume</td>
			<td>Concentration</td>
		</tr>
		<tr>
			<td>MEM &#945;</td>
			<td>440 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat inactivated fetal bovine serume</td>
			<td>50 mL</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>100x L-glutamine</td>
			<td>5 mL</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin 100 x</td>
			<td>5 mL</td>
			<td>1x</td>
		</tr>
		<tr>
			<td colspan="3">Change media on day 2 with mitotic inhibitors at these final concentrations</td>
		</tr>
		<tr>
			<td>Uridine</td>
			<td></td>
			<td>1 &#956;M</td>
		</tr>
		<tr>
			<td>5&#39;-Fluor-2&#39;deoxyuridine</td>
			<td></td>
			<td>15 &#956;M</td>
		</tr>
	</tbody>
</table>

<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>B6;129- Tgfbr2tm1Karl/J</td>
			<td>The Jackson Laboratory</td>
			<td>12603</td>
			<td>Tgfbr2f/f&#160;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>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>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>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>Vial forceps</td>
			<td>Fine Science Tools</td>
			<td>110006-15</td>
			<td>Long forceps for tissue transfer to conicals</td>
		</tr>
	</tbody>
</table>

establishing a co-culture of dental pulp cells and trigeminal neurons for cross-communication

Source: Barkley, C. et al., <a href="https://app.jove.com/v/60809">A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals.</a>&#160;J. Vis. Exp. (2020).
This video showcases a technique for establishing a co-culture of trigeminal neurons and primary dental pulp cells using a transwell filter. In this physically separated condition, mesenchymal cells obtained from dental pulp tissue secrete paracrine signaling molecules that traverse through the pores, promoting the differentiation and migration of neurons towards the mesenchymal cells establishing a co-culture system.

<img alt="Figure 1" src="/files/ftp_upload/22382/22382_Figure1.jpg" /> 
Figure 1: A schematic of the mouse dissection to obtain cells for co-culture.&#160;(A) A diagram of where to cut to open the mouse skull and locate TG nerves, shown in black in the last depiction. Scissors indicate where to insert the scissor tips to cut along the dotted lines. (B) A combined darkfield and GFP image showing Thy1-YFP+ TG nerves circled in white. (C) Dissected TG ganglia can then be dispersed and cultured, as shown in&#160;F. (D) The mandible of a P7 mouse, with forceps holding the mandible on the left and alveolar bone ridges containing unerupted teeth on each side of the tongue. (E) DP tissue (circled) extracted from the mineralized structure (top), and the enamel outer epithelium (bottom) that was removed to disperse and plate in a tissue culture-treated plate, as shown in&#160;F. Images are not shown to scale. DP cells were dispersed and grown to confluence before adding TG neurons.&#160;

Establishing a Co-Culture of Dental Pulp Cells and Trigeminal Neurons for Cross-Communication

1. Plate Preparation
NOTE: Be sure the plate lid is on during all incubation and rinsing steps outside of the sterile tissue culture hood to prevent ...

Begin with a mesenchymal stem cell-rich suspension obtained from mouse dental pulp tissue.
Incubate to allow cell adhesion and proliferation, forming a monolayer.
Place a porous membrane insert coated with laminin into the well containing the dental pulp cells.
Seed the insert with a co-culture medium containing trigeminal neurons derived from the mouse brain. Incubate to allow neuron attachment.
Replace the medium with a co-culture medium containing growth regulators such as uridine and 5-fluoro-2 deoxyuridine.
These molecules enter the cells and control the growth of actively proliferating mesenchymal stem cells by inhibiting DNA synthesis.
Over time, the mesenchymal cells secrete small signaling molecules that pass through the porous membrane to stimulate the trigeminal neurons.
This stimulation causes the neuronal processes of the trigeminal neurons to elongate toward the mesenchymal cells. Additionally, the neurons secrete signaling molecules, enabling cross-communication.&#160;

establishing-co-culture-dental-pulp-cells-trigeminal-neurons-for

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Co-culture and Interaction Studies

42 Views. Source: Barkley, C. et al., A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals. J. Vis. Exp. (2020). This video showcases a technique for establishing a co-culture of trigeminal neurons and primary dental pulp cells using a transwell filter. In this physically separated condition, mesenchymal cells obtained from dental pulp tissue secrete paracrine signaling molecules that traverse through the pores, promoting the differentiation and migration of ne...

Video: Establishing a Co-Culture of Dental Pulp Cells and Trigeminal Neurons for Cross-Communication - Concept

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased understanding of the molecular events that give rise to glioblastomas, this cancer still remains highly refractory to conventional treatment. Surgical resection of high grade brain tumors is rarely complete due to the highly infiltrative nature of glioblastoma cells. Therapeutic approaches which attenuate glioblastoma cell invasion therefore is an attractive option. Our laboratory and others have shown that tumor associated macrophages and microglia (resident brain macrophages) strongly stimulate glioblastoma invasion. The protocol described in this paper is used to model glioblastoma-macrophage/microglia interaction using in vitro culture assays. This approach can greatly facilitate the development and/or discovery of drugs that disrupt the communication with the macrophages that enables this malignant behavior. We have established two robust coculture invasion assays where microglia/macrophages stimulate glioma cell invasion by 5 - 10 fold. Glioblastoma cells labelled with a fluorescent marker or constitutively expressing a fluorescent protein are plated without and with macrophages/microglia on matrix-coated polycarbonate chamber inserts or embedded in a three dimensional matrix. Cell invasion is assessed by using fluorescent microscopy to image and count only invasive cells on the underside of the filter. Using these assays, several pharmacological inhibitors (JNJ-28312141, PLX3397, Gefitinib, and Semapimod), have been identified which block macrophage/microglia stimulated glioblastoma invasion.

Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed.

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

JoVE Journal

Cancer Research

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 ...

Developmental Biology

In Vitro Myelination of Peripheral Axons in a Coculture of Rat Dorsal Root Ganglion Explants and Schwann Cells

The process of myelination is essential to enable rapid and sufficient signal transduction in the nervous system. In the peripheral nervous system, neurons and Schwann cells engage in a complex interaction to control the myelination of axons. Disturbances of this interaction and breakdown of the myelin sheath are hallmarks of inflammatory neuropathies and occur secondarily in neurodegenerative disorders. Here, we present a coculture model of dorsal root ganglion explants and Schwann cells, which develops a robust myelination of peripheral axons to investigate the process of myelination in the peripheral nervous system, study axon-Schwann cell interactions, and evaluate the potential effects of therapeutic agents on each cell type separately. Methodologically, dorsal root ganglions of embryonic rats (E13.5) were harvested, dissociated from their surrounding tissue, and cultured as whole explants for 3 days. Schwann cells were isolated from 3-week-old adult rats, and sciatic nerves were enzymatically digested. The resulting Schwann cells were purified by magnetic-activated cell sorting and cultured under neuregulin and forskolin-enriched conditions. After 3 days of dorsal root ganglion explant culture, 30,000 Schwann cells were added to one dorsal root ganglion explant in a medium containing ascorbic acid. The first signs of myelination were detected on day 10 of coculture, through scattered signals for myelin basic protein in immunocytochemical staining. From day 14 onward, myelin sheaths were formed and propagated along the axons. Myelination can be quantified by myelin basic protein staining as a ratio of the myelination area and axon area, to account for the differences in axonal density. This model provides experimental opportunities to study various aspects of peripheral myelination in vitro, which is crucial for understanding the pathology of and possible treatment opportunities for demyelination and neurodegeneration in inflammatory and neurodegenerative diseases of the peripheral nervous system.

In the coculture system of dorsal root ganglia and Schwann cells, myelination of the peripheral nervous system can be studied. This model provides experimental opportunities to observe and quantify peripheral myelination and to study the effects of compounds of interest on the myelin sheath.

The process of myelination is essential to enable rapid and sufficient signal transduction in the nervous system. In the peripheral nervous system, ...

Establishing a Co-Culture of Dental Pulp Cells and Trigeminal Neurons for Cross-Communication

Transcript

Establishing a Co-Culture of Dental Pulp Cells and Trigeminal Neurons for Cross-Communication

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

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

In Vitro Myelination of Peripheral Axons in a Coculture of Rat Dorsal Root Ganglion Explants and Schwann Cells