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

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.