We are grateful to all staff members of the Oral and Maxillofacial Surgery (MKA) of UZ Leuven, as well as the patients, for their invaluable help in collecting freshly extracted third molars. We would also like to thank Dr. Reinhilde Jacobs and Dr. Elisabeth Tijskens for their help with the sample collection. This work was supported by grants from KU Leuven (BOF) and FWO-Flanders (G061819N). L.H. is an FWO Ph.D. Fellow (1S84718N).

All methods described here have been approved by the Ethics Committee Research UZ/KU Leuven (13/0104U). Extracted third molars (wisdom teeth) were obtained after patients&#39; informed consent.
1. Preparations
<ol>
	<li>Prewarm a 48-well culture plate for 15-20 h in a 1.9% CO2 incubator at 37 &#176;C.</li>
	<li>Liquefy a Matrigel aliquot (growth factor-reduced; phenol red-free; further referred to as basement membrane matrix; BMM) on ice (4 &#176;C) for a minimum ...

<ol>
	<li>Yu, T., Klein, O. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+cellular+mechanisms+of+tooth+development,+homeostasis+and+repair.">Molecular and cellular mechanisms of tooth development, homeostasis and repair.</a> Development (Cambridge). 147 (2), (2020).</li><li>Arrow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dental+enamel+defects,+caries+experience+and+oral+health-related+quality+of+life:+a+cohort+study.">Dental enamel defects, caries experience and oral health-related quality of life: a cohort study.</a> Australian Dental Journal. 62 (2), 165-172 (2017).</li><li>Mitsiadis, T. A., Orsini, G., Jimenez-Rojo, L., Zavan, B., Bressan, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dental+Stem+Cells+for+Tooth+Regeneration.">Dental Stem Cells for Tooth Regeneration.</a> Dental Stem Cells: Regenerative Potential. Stem Cell Biology and Regenerative Potential. Stem Cell Biology and Regenerative Medicine. , (2016).</li><li>Mitsiadis, T. A., Orsini, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Editorial:+a+new+era+in+dentistry:+stem+cell-based+approaches+for+tooth+and+periodontal+tissue+regeneration.">Editorial: a new era in dentistry: stem cell-based approaches for tooth and periodontal tissue regeneration.</a> Frontiers in Physiology. 7, 357 (2016).</li><li>Miran, S., Mitsiadis, T. A., Pagella, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innovative+dental+stem+cell-based+research+approaches:+the+future+of+dentistry.">Innovative dental stem cell-based research approaches: the future of dentistry.</a> Stem Cells International. 2016, 7231038 (2016).</li><li>Shinmura, Y., Tsuchiya, S., Hata, K. I., Honda, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quiescent+epithelial+cell+rests+of+malassez+can+differentiate+into+ameloblast-like+cells.">Quiescent epithelial cell rests of malassez can differentiate into ameloblast-like cells.</a> Journal of Cellular Physiology. 217 (3), 728-738 (2008).</li><li>Davis, E. 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This protocol describes the efficient and reproducible generation of organoids starting from the human tooth. To our knowledge, this is the first methodology for establishing current-concept (epithelial) organoids starting from human dental tissue. The organoids are long-term expandable and display a tooth epithelial stemness phenotype, duplicating DESCs previously reported in the ERM compartment of the DF7. Moreover, the organoids replicate functional DESC/ERM characteristics, including the unfol...

Teeth have essential roles in food mastication, speech, and psychological well-being (self-image). The human tooth consists of highly mineralized tissues of varying density and hardness1. Dental enamel, the main component of the tooth crown, is the highest mineralized tissue in the human body. During enamel formation (amelogenesis), when teeth develop, dental epithelial stem cells (DESCs) differentiate into enamel-forming cells (ameloblasts). Once formed, the enamel is rarely repaired or renewed due to the apoptotic loss of the ameloblasts at the onset of tooth eruption1. Restoration of damaged enamel tissue, as caused by trauma or bacterial disease, is currently accomplished using synthetic materials; however, these are troubled with important shortcomings such as microleakage, inferior osseointegration and anchorage, finite life span, and lack of fully functional repair2. Hence, a robust and reliable culture of human DESCs with the capacity to generate ameloblasts and the potential to produce mineralized tissue would be a major step forward in the dental regenerative field.
Knowledge on human DESC phenotype and biological function are scarce3,4,5. Interestingly, DESCs of human teeth have been proposed to exist in the Epithelial Cell Rests of Malassez (ERM), cell clusters present within the dental follicle (DF), which surrounds unerupted teeth, and remains present in the periodontal ligament around the root once the tooth erupts1. ERM cells co-cultured with dental pulp have been found to differentiate into ameloblast-like cells and generate enamel-like tissue6. However, profound studies of the specific role of ERM cells in enamel (re-) generation have been limited due to the lack of reliable study models7. Current ERM in vitro culture systems are hampered by limited life span and quick loss of phenotype in the 2D conditions standardly used8,9,10,11,12. Hence, a tractable in vitro system to faithfully expand, study, and differentiate human DESCs is strongly needed.
During the last decade, a powerful technique to grow epithelial stem cells in vitro has been successfully applied to several types of (human) epithelial tissues to study their biology as well as disease13,14,15,16. This technology enables the tissue epithelial stem cells to self-develop into 3D cell constructions (i.e., organoids) when seeded into an extracellular matrix (ECM)-mimicking scaffold (typically, Matrigel) and cultured in a defined medium replicating the tissue&#39;s stem cell niche signaling and/or embryogenesis. Typical growth factors needed for organoid development include epidermal growth factor (EGF) and wingless-type MMTV integration site (WNT) activators14,15,16. The resultant organoids are characterized by lasting fidelity in mimicking the tissue&#39;s original epithelial stem cells, as well as high expandability while retaining their phenotype and functional properties, thereby overcoming the often-limited primary human tissue availability as acquired from the clinic. To establish organoids, isolation of the epithelial stem cells from the heterogeneous tissue (i.e., comprising other cell types such as mesenchymal cells) prior to culturing is not required as mesenchymal cells do not attach to, or thrive in, the ECM, eventually resulting in purely epithelial organoids13,16,17,18,19. This promising and versatile technology has led to the development of manifold organoid models from various human epithelial tissues. However, human tooth-derived organoids, valuable for deep study of tooth development, regeneration and disease, were not established yet20,21. We recently succeeded in developing such a new organoid model starting from DF tissue from third molars (wisdom teeth) extracted from adolescent patients19.
Here, we describe the protocol to develop epithelial organoid cultures from the adult human tooth (i.e., from the DF of third molars) (Figure 1A). The resultant organoids express ERM-associated stemness markers while being long-term expandable. Intriguingly, opposite to most other organoid models, the typically needed EGF is redundant for robust organoid development and growth. Interestingly, the stemness organoids show ameloblast differentiation properties, thereby mimicking ERM/DESC features and processes occurring in vivo. The new and unique organoid model described here allows exploring DESC biology, plasticity, and differentiation capacity and opens the door for taking the first steps toward tooth-regenerative approaches.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL Microcentrifuge tube</td>
			<td>Eppendorf</td>
			<td>30120.086</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Centrifuge tube</td>
			<td>Corning</td>
			<td>430052</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M-6250</td>
			<td></td>
		</tr>
		<tr>
			<td>48-well flat bottom plates</td>
			<td>Corning</td>
			<td>3548</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Centrifuge tube</td>
			<td>Corning</td>
			<td>430290</td>
			<td></td>
		</tr>
		<tr>
			<td>A83-01</td>
			<td>Sigma-Aldrich</td>
			<td>SML0788</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Lonza</td>
			<td>50004</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin Bovine (cell culture grade)</td>
			<td>Serva</td>
			<td>47330.03</td>
			<td></td>
		</tr>
		<tr>
			<td>AMELX antibody</td>
			<td>Santa Cruz</td>
			<td>sc-365284</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Gibco</td>
			<td>15200018</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 (without vitamin A)</td>
			<td>Gibco</td>
			<td>12587-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Cassette</td>
			<td>VWR</td>
			<td>7202191</td>
			<td></td>
		</tr>
		<tr>
			<td>Catalase from bovine liver</td>
			<td>Sigma-Aldrich</td>
			<td>C100</td>
			<td></td>
		</tr>
		<tr>
			<td>CD44 antibody</td>
			<td>Abcam</td>
			<td>ab34485</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer, 40 &#181;m</td>
			<td>Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholera Toxin</td>
			<td>Sigma-Aldrich</td>
			<td>C8052</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid</td>
			<td>Sigma-Aldrich</td>
			<td>C0759</td>
			<td></td>
		</tr>
		<tr>
			<td>CK14 antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>MA5-11599</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase IV</td>
			<td>Gibco</td>
			<td>17104-019</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>VWR</td>
			<td>6310146</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryobox</td>
			<td>Thermo Scientific</td>
			<td>5100-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryovial</td>
			<td>Thermo Fisher Scientific</td>
			<td>375353</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethylsulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Sigma-Aldrich</td>
			<td>D4693</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM 1:1 F12 without Fe</td>
			<td>Invitrogen</td>
			<td>074-90715A</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM powder high glucose</td>
			<td>Gibco</td>
			<td>52100039</td>
			<td></td>
		</tr>
		<tr>
			<td>Dnase</td>
			<td>Sigma-Aldrich</td>
			<td>D5025-15KU</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey serum</td>
			<td>Sigma-Aldrich</td>
			<td>D9663 - 10ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Embedding workstation, 220 to 240 Vac</td>
			<td>Thermo Fisher Scientific</td>
			<td>12587976</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute, &#8805;99.8% (EtOH)</td>
			<td>Fisher Chemical</td>
			<td>E/0650DF/15</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Sigma-Aldrich</td>
			<td>F7524</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF10</td>
			<td>Peprotech</td>
			<td>100-26</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF2 (= basic FGF)</td>
			<td>R&#38;D Systems</td>
			<td>234-FSE-025</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF8</td>
			<td>Peprotech</td>
			<td>AF-100-25</td>
			<td></td>
		</tr>
		<tr>
			<td>GenElute Mammaliam Total RNA Miniprep Kit</td>
			<td>Sigma-Aldrich</td>
			<td>RTN350-1KT</td>
			<td>Includes 1% &#946;-mercaptoethanol dissolved in lysis buffer</td>
		</tr>
		<tr>
			<td>Glass Pasteur pipette</td>
			<td>Niko Mechanisms</td>
			<td>170-40050</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>VWR</td>
			<td>101194M</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>IGF-1</td>
			<td>PeproTech</td>
			<td>100-11</td>
			<td></td>
		</tr>
		<tr>
			<td>InSolution Y-27632 (ROCK inhibitor, RI)</td>
			<td>Sigma-Aldrich</td>
			<td>688001</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin from bovine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td>I6634</td>
			<td></td>
		</tr>
		<tr>
			<td>ITGA6 antibody</td>
			<td>Sigma-Aldrich</td>
			<td>HPA012696</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Gibco</td>
			<td>25030024</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel (growth factor-reduced; phenol red-free)</td>
			<td>Corning</td>
			<td>15505739</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slide</td>
			<td>Thermo Fisher Scientific</td>
			<td>J1800AMNZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex-GV Syringe Filter Unit, 0.22&#160;&#956;m</td>
			<td>Millipore</td>
			<td>SLGV033R</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum essential medium eagle (&#945;MEM)</td>
			<td>Sigma-Aldrich</td>
			<td>M4526</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse IgG (Alexa 555) secondary antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-31570</td>
			<td></td>
		</tr>
		<tr>
			<td>N2</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetyl L-cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>A7250</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma-Aldrich</td>
			<td>N0636</td>
			<td></td>
		</tr>
		<tr>
			<td>Noggin</td>
			<td>PeproTech</td>
			<td>120-10C</td>
			<td></td>
		</tr>
		<tr>
			<td>P63 antibody</td>
			<td>Abcam</td>
			<td>ab124762</td>
			<td></td>
		</tr>
		<tr>
			<td>Pap Pen</td>
			<td>Sigma-Aldrich</td>
			<td>Z377821-1EA</td>
			<td>Marking pen</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA), 16%</td>
			<td>Merck</td>
			<td>8.18715</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin G sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>P3032</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin (Pen/Strep)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Corning</td>
			<td>353002</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Gibco</td>
			<td>10010-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette (P20, P200, P1000)</td>
			<td>Eppendorf or others</td>
			<td>2231300006</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic transfer pipette (3.5&#160;mL)</td>
			<td>Sarstedt</td>
			<td>86.1171.001</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit IgG (Alexa 488) secondary antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>A21206</td>
			<td></td>
		</tr>
		<tr>
			<td>RSPO1</td>
			<td>PeproTech</td>
			<td>120-38</td>
			<td></td>
		</tr>
		<tr>
			<td>SB202190 (p38i)</td>
			<td>Biotechne (Tocris)</td>
			<td>1264</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel (surgical blade)</td>
			<td>Swann-Morton</td>
			<td>207</td>
			<td></td>
		</tr>
		<tr>
			<td>SHH</td>
			<td>R&#38;D Systems</td>
			<td>464-SH-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone molds (Heating block)</td>
			<td>VWR</td>
			<td>720-1918</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>BDH</td>
			<td>102415K</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydrogen Carbonate (NaHCO3)</td>
			<td>Merck</td>
			<td>106329</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium-pyruvate (C3H3NaO3)</td>
			<td>Sigma-Aldrich</td>
			<td>P-5280</td>
			<td></td>
		</tr>
		<tr>
			<td>SOX2 antibody</td>
			<td>Abcam</td>
			<td>ab92494</td>
			<td></td>
		</tr>
		<tr>
			<td>StepOnePlus</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>Real-Time PCR System</td>
		</tr>
		<tr>
			<td>Stericup-GP, 0.22 &#181;m</td>
			<td>Millipore</td>
			<td>SCGPU02RE</td>
			<td></td>
		</tr>
		<tr>
			<td>Steriflip-GP Sterile Centrifuge Tube Top Filter Unit, 0.22 &#956;m</td>
			<td>Millipore</td>
			<td>SCGP00525</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 1000 &#956;L pipette tips with filter</td>
			<td>Greiner</td>
			<td>740288</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 20 &#956;L pipette tips with filter</td>
			<td>Greiner</td>
			<td>774288</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 200 &#956;L pipette tips with and without filter</td>
			<td>Greiner</td>
			<td>739288</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile H2O</td>
			<td>Fresenius</td>
			<td>B230531</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin sulfate salt</td>
			<td>Sigma-Aldrich</td>
			<td>S6501</td>
			<td></td>
		</tr>
		<tr>
			<td>Superscript III first-strand synthesis supermix</td>
			<td>Invitrogen</td>
			<td>11752-050</td>
			<td>Reverse transcription kit</td>
		</tr>
		<tr>
			<td>Tissue processor</td>
			<td>Thermo Scientific</td>
			<td>12505356</td>
			<td></td>
		</tr>
		<tr>
			<td>Transferrin</td>
			<td>Serva</td>
			<td>36760.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787-50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE express</td>
			<td>Gibco</td>
			<td>12605-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield mounting medium+DAPI</td>
			<td>Labconsult NV</td>
			<td>H-1200</td>
			<td>Antifade mounting medium with DAPI</td>
		</tr>
		<tr>
			<td>WNT3a</td>
			<td>Biotechne (Tocris)</td>
			<td>5036-WN-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylenes, 99%, for biochemistry and histology</td>
			<td>VWR</td>
			<td>2,89,75,325</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishing organoids from human tooth as a powerful tool toward mechanistic research and regenerative therapy

Teeth are of key importance in life not only for food mastication and speech but also for psychological well-being. Knowledge on human tooth development and biology is scarce. In particular, not much is known about the tooth's epithelial stem cells and their function. We succeeded in developing a novel organoid model starting from human tooth tissue (i.e., dental follicle, isolated from extracted wisdom teeth). The organoids are robustly and long-term expandable and recapitulate the proposed human tooth epithelial stem cell compartment in terms of marker expression as well as functional activity. In particular, the organoids are capable to unfold an ameloblast differentiation process as occurring in vivo during amelogenesis. This unique organoid model will provide a powerful tool to study not only human tooth development but also dental pathology, and may pave the way toward tooth-regenerative therapy. Replacing lost teeth with a biological tooth based on this new organoid model could be an appealing alternative to the current standard implantation of synthetic materials.

Tooth organoid development 
We provide a detailed protocol to establish organoid cultures from human DF tissue acquired following wisdom tooth extraction (Figure 1A). Isolated DF is enzymatically and mechanically dissociated. The obtained cells are cultured within BMM in media that were empirically defined for optimal organoid development and growth (tooth organoid medium; TOM)19.
The organoids typically develop within...

Watch this Scientific Journal Video about Establishing Organoids from Human Tooth as a Powerful Tool Toward Mechanistic Research and Regenerative Therapy at JoVE.com

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

We present a protocol to develop epithelial organoid cultures starting from human tooth. The organoids are robustly expandable and recapitulate the tooth's epithelial stem cells, including their ameloblast differentiation capacity. The unique organoid model provides a promising tool to study human dental (stem cell) biology with perspectives for tooth-regenerative approaches.

Teeth are of key importance in life not only for food mastication and speech but also for psychological well-being. Knowledge on human tooth development ...

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.

establishing-organoids-from-human-tooth-as-powerful-tool-toward

Research

JoVE Journal

Developmental Biology

3.0K ビュー.  KU Leuven (University of Leuven). この新しいヒト歯由来オルガノイドモデルは、歯の再生アプリケーションに向けた視点でヒト歯科幹細胞生物学を解読する最初の強力なツールを提供します。現在、この歯オルガノイドモデルは、ヒト歯上皮幹細胞を確実かつ強固に増殖および増殖させるための唯一の利用可能なツールである。このオルガノイドプロトコルは、健康な歯だけでなく、歯科腫瘍や細菌の?...