Establishing Organoids from Human Tooth as a Powerful Tool Toward Mechanistic Research and Regenerative Therapy

This new human tooth-derived organoid model provides the first powerful tool to decipher human dental stem cell biology with perspectives toward tooth regenerative applications. At present, this tooth organoid model is the only available tool to reliably and robustly grow and expand human tooth epithelial stem cells. This organoid protocol can be applied to establish research models, not only from healthy, but also diseased tooth such as dental tumors and bacterial impact.

Exploration of such disease models may uncover new therapeutic targets. This organoid model is highly instrumental in deciphering the enamel formation process in the human tooth, and may enable the construction of tooth parts such as enamel for a generative purposes. Demonstrating the procedure will be Lara Hemeryck, PhD student in my research group.

To begin, collect the third molars with associated dental follicles in the collection medium placed on ice and transfer the content of the tubes to a Petri dish. Hold the tooth with a tweezer and carefully isolate the dental follicles using a surgical blade. Wash the remaining blood off the dental follicles by briefly placing the dental follicle tissues in the first well of 70%ethanol for 20 seconds, then transfer it to the next death ethanol plate for 20 seconds and further to the third ethanol well.

Continue rinsing it in phosphate-buffered saline wells three times followed by rinsing the dental follicles in the three remaining dental follicles collection medium wells for a maximum of 20 minutes in total. Transfer the rinsed dental follicles to a new Petri dish. Cut a small piece of one of the dental follicles for a paraformaldehyde fixation and store it in a microcentrifuge tube containing 500 microliters of collection medium for a maximum six hours.

Mince the rest of the dental follicles into small pieces and transfer them to a 15-milliliters tube containing four milliliters of prewarmed dissociation medium, then incubate the tube at 37 degrees Celsius for two hours in water bath. Every 15 minutes, pipette the dental follicles dissociation medium and mix up and down using a glass pipette to speed the tissue disintegration. When pieces of dental follicles are no longer observed, proceed with the dissociation using a narrowed, fire-polished Pasteur pipette.

In the meantime, prepare 10 milliliters of medium A containing 100 microliters of DNase and add five milliliters of this medium to each tube with dissociated dental follicles. Incubate for one minute at room temperature. Rinse the filter with one milliliter of medium A.Combine the several tubes of dissociated dental follicles from one patient at this step and centrifuge the filtered cell suspension at 200 times g for 10 minutes at 4 degrees Celsius.

Remove the supernatant. Resuspend the pellet in one milliliter of serum-free defined medium and transfer the cell suspension to a 1.5 milliliter microcentrifuge tube. Calculate the cell concentration using an automated cell counter and calculate the number of wells that can be seeded.

The final mix is composed of cell suspension and basement membrane matrix at a ratio of 30 to 70. Remove the appropriate amount of supernatant and resuspend it slowly to obtain a 70-to-30 ratio of ice-cold basement membrane matrix to cell suspension for plating. Once resuspended in the basement membrane matrix, keep the microcentrifuge tube on ice to avoid basement membrane matrix solidification.

Pipette the 20 microliters of basement membrane matrix droplets in the center of the preheated 48-well culture plate. Flip the plate upside down and place it to solidify in a 1.9%carbon dioxide incubator at 37 degrees Celsius for 20 minutes. Add rho-associated kinase inhibitor and amphotericin B to tooth organoid medium and prewarm the medium in a 37-degrees Celsius water bath.

Take the 48-well plate from the incubator. Place it upright and add 250 microliters of the prepared prewarmed medium to each well with a basement membrane matrix droplet containing the cells and return the plate to the carbon dioxide incubator. To refresh the medium, tilt the plate at a 45-degrees angle.

Gently remove the previous medium, while avoiding touching the basement membrane matrix droplet and add 250 microliters of new prewarmed tooth organoid medium every two to three days. To carry out the passage of the organoids, remove the medium from the wells with organoids and pool up to four confluent wells. To collect the organoids, add 400 microliters of ice-cold serum-free defined medium per well directly onto the membrane matrix droplet and repeatedly pipette the medium up and down until the entire droplet is dislodged.

If wells are pooled, transfer the 400 microliters from the first well to the next to dislodge the organoid containing droplets. Transfer the dislodged organoid assembly to a 1.5 microliter microcentrifuge tube and repeat the addition of serum-free defined medium until all the organoid structures are collected from the wells. Centrifuge it at 200 times g for five minutes at 4 degrees Celsius.

Remove the supernatant from the centrifuge tube and resuspend the pellet in a prewarmed aliquot of TrypLE Express. Add 400 microliters of ice-cold serum-free defined medium to inactivate the enzyme and centrifuge it at 200 times g for five minutes at 4 degrees Celsius. Remove the supernatant.

Pre-coat a tip with ice-cold serum-free defined medium and re-suspend the organoid pellet in sterile condition. Re-suspend the pellet in 200 microliters of ice-cold serum free defined medium for the low passage method. Push the completely emptied pipette tip against the bottom of the micro centrifuge tube to reduce its diameter.

Pipette up and down for five minutes to mechanically disrupt the organoids. For the higher passage method, resuspend the pellet in 700 microliters of ice-cold serum-free defined medium with a P1000 tip. Add a P200 tip to this P1000 tip and pre-coat with ice-cold serum-free defined medium.

Prevent air bubble formation by adjusting the pipette's volume setting. Aspire at least 90%of the medium volume with organoids and pipette up and down for five minutes to mechanically disrupt the organoids. The organoids typically develop two weeks after dental follicle cell seeding.

The organoids are long-term expandable up to 11 passages. Seeding around 20, 000 cells per basement membrane matrix droplet yields an optimal density of organoids, whereas seeding higher cell numbers leads to suboptimal organoid outgrowth with similar organoids at too high density due to insufficient space to grow. The developed organoids show a dense appearance and contain cells displaying a high nucleocytoplasmic ratio.

Moreover, the organoids express the ERM marker cytokeratin 14 confirming their epithelial origin and other proposed ERM markers, such as P63, CD44, and integrin alpha-6. Organoids express SOX2, a well-known DESC marker in mice. Interestingly, the main component of the enamel matrix, amelogenin, also expressed in the organoids.

The organoids retain their ERMM stemness phenotype during passaging as shown by the stable expression of markers. For optimal long-term organoids growth, it is essential to use the recommended passage methods meaning or the low passage or the higher passage methods. Once formed, the organoids can be used to scrutinize the amelogenesis process further and enamel matrix deposition currently not achieved in dentistry research.

This technique enables the exploration of dental epithelial stem cell biology, the process of enamel formation, and the interaction with the tooth mesenchyme, an interplay essential for correct tooth formation.