This study was supported by grants from the National Research Foundation of Korea (NRF) funded by the Korean government (NRF-2017R1C1B2008406 and NRF-2021R1F1A1064350). Dr. Hee-Yeon Bae kindly provided the DF for primary culture.

This study was approved by the Institutional Review Board of Kyung Hee University Hospital at Gangdong (IRB approval no. KHNMC 2017-06-009).
1. Collect DF
NOTE: Patients gave informed consent before surgery for the removal of a mature or immature impacted third molar. Patients with the following disease were excluded: diabetes, hypertension, tuberculosis, hepatitis, acquired immunodeficiency syndrome. Pregnant women were also excluded.
<ol>
	<li>H...

<ol>
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S., 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=Directing+the+differentiation+of+human+dental+follicle+cells+into+cementoblasts+and/or+osteoblasts+by+a+combination+of+HERS+and+pulp+cells.">Directing the differentiation of human dental follicle cells into cementoblasts and/or osteoblasts by a combination of HERS and pulp cells.</a> Journal of Molecular Histology. 42 (3), 227-235 (2011).</li><li>Fang, J., Tang, L., Liu, X. H., Wen, L. Y., Jin, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+of+the+unique+odontogenic+properties+of+rat+apical+bud+cells+under+the+developing+apical+complex+microenvironment.">Changes of the unique odontogenic properties of rat apical bud cells under the developing apical complex microenvironment.</a> International Journal of Oral Science. 1 (1), 26-33 (2009).</li><li>Oh, J. E., Yi, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+dental+follicle-derived+Hertwig's+epithelial+root+sheath+cells.">Isolation and characterization of dental follicle-derived Hertwig's epithelial root sheath cells.</a> Clinical Oral Investigations. 25 (4), 1787-1796 (2021).</li><li>Oh, J. E., Kim, R. H., Shin, K. H., Park, N. H., Kang, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delta+Np63+alpha+protein+triggers+epithelial-mesenchymal+transition+and+confers+stem+cell+properties+in+normal+human+keratinocytes.">Delta Np63 alpha protein triggers epithelial-mesenchymal transition and confers stem cell properties in normal human keratinocytes.</a> Journal of Biological Chemistry. 286 (44), 38757-38767 (2011).</li><li>Morsczeck, C., 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=Isolation+of+precursor+cells+(PCs)+from+human+dental+follicle+of+wisdom+teeth.">Isolation of precursor cells (PCs) from human dental follicle of wisdom teeth.</a> Matrix Biology. 24 (2), 155-165 (2005).</li><li>Nam, H., 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=Expression+profile+of+the+stem+cell+markers+in+human+Hertwig's+epithelial+root+sheath/Epithelial+rests+of+Malassez+cells.">Expression profile of the stem cell markers in human Hertwig's epithelial root sheath/Epithelial rests of Malassez cells.</a> Molecular and Cells. 31 (4), 355-360 (2011).</li><li>Lee, J. H., 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=Dental+follicle+cells+and+cementoblasts+induce+apoptosis+of+ameloblast-lineage+and+Hertwig's+epithelial+root+sheath/epithelial+rests+of+Malassez+cells+through+the+Fas-Fas+ligand+pathway.">Dental follicle cells and cementoblasts induce apoptosis of ameloblast-lineage and Hertwig's epithelial root sheath/epithelial rests of Malassez cells through the Fas-Fas ligand pathway.</a> European Journal of Oral Sciences. 120 (1), 29-37 (2012).</li><li>Wan, A. C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recapitulating+cell-cell+Interactions+for+organoid+construction+-+are+biomaterials+dispensable.">Recapitulating cell-cell Interactions for organoid construction - are biomaterials dispensable.</a> Trends in Biotechnology. 34 (9), 711-721 (2016).</li><li>Guo, Y., 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=Are+Hertwig's+epithelial+root+sheath+cells+necessary+for+periodontal+formation+by+dental+follicle+cells.">Are Hertwig's epithelial root sheath cells necessary for periodontal formation by dental follicle cells.</a> Archives of Oral Biology. 94, 1-9 (2018).</li><li>Sugaya, T., Tomita, M., Motoki, Y., Miyaji, H., Kawamami, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+enamel+matrix+derivative+on+healing+of+root+surfaces+after+bonding+treatment+and+intentional+replantation+of+vertically+fractured+roots.">Influence of enamel matrix derivative on healing of root surfaces after bonding treatment and intentional replantation of vertically fractured roots.</a> Dental Traumatology. 32 (5), 397-401 (2016).</li><li>Fong, H. K., Foster, B. L., Popowics, T. E., Somerman, 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=The+crowning+achievement:+getting+to+the+root+of+the+problem.">The crowning achievement: getting to the root of the problem.</a> Journal of Dental Education. 69 (5), 555-570 (2005).</li><li>Bonczek, O., 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=Tooth+agenesis:+What+do+we+know+and+is+there+a+connection+to+cancer.">Tooth agenesis: What do we know and is there a connection to cancer.</a> Clinical Genetics. 99 (4), 493-502 (2021).</li></ol>

This protocol includes critical steps. Harvesting single-cell populations is essential for the successful isolation of epithelial cells from DF. We sought to isolate epithelial cells from the DF based on our hypothesis that there are more epithelial cells in the DF. The mincing procedure enhances the detachment and release of cells from the DF. The mincing procedure was improved, and mincing repeated until the DF appeared pulpy to facilitate the release of single cells. Obtaining the maximum number of single cells increa...

Tooth formation begins with the invagination of the oral epithelium1. According to the tooth developmental stage, the oral epithelium has different names, including inner and outer enamel epithelium, cervical loop, and Hertwig&#39;s epithelial root sheath (HERS). The epithelial compartments communicate with the surrounding mesenchymal cells. Epithelial-mesenchymal interactions regulate tooth formation and tissue regeneration. Procuring dental epithelial cells, such as oral keratinocytes and Hertwig&#39;s epithelial root sheath cells (HERSCs), is crucial for the study of dental epithelial-mesenchymal interactions2.
Rodent-derived dental epithelial cells are isolated from the epithelial structure, such as the HERS. Li and colleagues isolated and immortalized rat molar-derived HERSCs after harvesting the apical portion of the developing tooth germs from 8-day-old rats3. The HERS was separated from apical tissue under magnification. Considering the tooth developmental stage and age, harvesting the HERS from humans is nearly impossible because of ethical issues: a developing tooth germ needs to be removed from a young child to harvest the human HERS. Immature tooth germs are rarely extracted. Human dental epithelial cells can be isolated from the gingiva and periodontal ligament (PDL). Epithelial structure-derived cells participate in tooth formation together with mesenchymal components and might be more suitable for the study of dental epithelial-mesenchymal interactions than oral keratinocytes. Epithelial cell rests of Malassez (ERM) are HERS-derived epithelial remnants and reside in small numbers in the PDL4. Studies report the isolation of human HERSCs from PDL5. However, harvesting human HERSCs from PDL tissue is not always successful because of the scarcity of the epithelial population in this location5,6.
Although rodent-derived HERSCs are maintained in serum-containing media3,7, human DF-derived epithelial cells are cultured with serum-free media similar to other human epithelial cells, such as normal human epidermal keratinocytes and normal human oral keratinocytes8,9. This implies physiologic or functional differences between rodent dental epithelial cells and human dental epithelial cells. Understanding the mechanism regarding dental epithelial-mesenchymal interactions might contribute to the development of clinical applications, including periodontal reattachment during replantation, periodontal regeneration in periodontal disease, pulp-dentin complex regeneration, and bio-tooth generation. Considering the characteristics of translational research, human dental epithelial cells may be more appropriate than rodent dental epithelial cells for the study of epithelial-mesenchymal interactions.
The human DF is a loose connective tissue and often resides in an impacted tooth. The DF contains mesenchymal precursors10. However, to our knowledge, no study has reported the isolation of epithelial cells from dental follicles before 2021. Oh and Yi reported the isolation of epithelial cells from human DF in 20218. The epithelial phenotype was confirmed by western blotting and morphologic analysis. Analysis of the origin of DF-derived epithelial cells demonstrated similar results with other studies. DF-derived epithelial cells were neither endothelial nor hematopoietic5,11, and Oh and Yi suggested naming these cells as DF-HERSCs. The DF has a larger volume than the PDL, and more epithelial cells can be isolated from the DF. This enhances the emergence of epithelial colonies and results in a high success rate in harvesting epithelial cells from DF. This study suggests using the DF as a tissue source for the isolation of dental epithelial cells.
In the present study, single cells were isolated from the DF according to previously described procedures10,12. The DF contains heterogeneous cell populations, and several cell types could be present at the early stage of the procedure. Morsczek and colleagues isolated DF-derived mesenchymal stem cells10. We hypothesized that the DF contains epithelial cells and that only epithelial cells can survive under serum-free conditions. This study differs from that of Morsczek et al. in terms of the selection of the epithelial population and the inhibition of mesenchymal cells. The selection was performed using keratinocyte serum-free media (SFM), which allows epithelial cell proliferation and inhibits mesenchymal cell proliferation. This study originated from a report by Oh and Yi8. The aim of this study was to describe details of the method used in that report for the isolation of epithelial cells from human DF.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>EMSURE ACS,ISO,Reag. Ph Eur 2-Propanol</td>
			<td>EMD millipore Co., MA, USA</td>
			<td>1096341011</td>
			<td>1 L</td>
		</tr>
		<tr>
			<td>0.05% trypsin-EDTA</td>
			<td>Gibco, Grand island, NY, USA</td>
			<td>25300054</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>40 &#956;m cell strainer</td>
			<td>Falcon, NC, USA</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture dish (100 x 20 mm)</td>
			<td>Thermo Fisher Scientific Inc., Waltham, USA</td>
			<td>172958</td>
			<td>Discontinued</td>
		</tr>
		<tr>
			<td>Cell culture dish (60 x 15 mm)</td>
			<td>Thermo Fisher Scientific Inc., Waltham, USA</td>
			<td>150326</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase type 1</td>
			<td>Gibco, Grand island, NY, USA</td>
			<td>17100017</td>
			<td>1 g</td>
		</tr>
		<tr>
			<td>Combi-514R / Refrigerated large capacity centrifuge</td>
			<td>Hanil science industrial Co., LTD., Daejeon, South Korea</td>
			<td>CB-514R</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tube</td>
			<td>SPL Life Sciences Co., Ltd., Gyeonggi-do, South Korea</td>
			<td>50015 
			50050</td>
			<td>15 mL 
			50 mL</td>
		</tr>
		<tr>
			<td>Cryogenic vial</td>
			<td>Corning Inc., NY, US</td>
			<td>430488</td>
			<td>2 mL</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich, Missouri, US</td>
			<td>D2650-100ml</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Gibco, Grand island, NY, USA</td>
			<td>17105041</td>
			<td>1 g</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate-Buffered Saline</td>
			<td>Welgene Inc., Gyengsangbuk-do, South Korea</td>
			<td>&#160;LB 001-02</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco, Grand island, NY, USA</td>
			<td>16000044</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Heraeus BB 15 / CO2 incubator</td>
			<td>Thermo Fisher Scientific Inc., Waltham, USA</td>
			<td>51023121</td>
			<td></td>
		</tr>
		<tr>
			<td>Keratinocyte serum-free medium</td>
			<td>Gibco, Grand island, NY, USA</td>
			<td>10724-011</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>MVE CryoSystem 2000</td>
			<td>MVE Biological Solutions Co., GA, USA</td>
			<td>CryoSystem 2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Mr. Frosty Freezing Container</td>
			<td>Thermo Fisher Scientific Inc., Waltham, USA</td>
			<td>5100-0001</td>
			<td>for 1.2-2 mL CryoVials</td>
		</tr>
		<tr>
			<td>Olympus CKX41 / Inverted cell culture microscope</td>
			<td>Olympus Life Science, 
			Waltham, Massachusetts</td>
			<td>22-00723-01</td>
			<td>Discontinued</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Strep</td>
			<td>Gibco, Grand island, NY, USA</td>
			<td>15140122</td>
			<td>100 mL (10,000 U/mL)</td>
		</tr>
		<tr>
			<td>Pipet aid XP</td>
			<td>Drummond scientific Co., PA, USA</td>
			<td>HDR-4-000-201</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetman Classic P1000</td>
			<td>Gilson, Villiers le Bel, France</td>
			<td>F123602</td>
			<td>100-1000 &#181;L</td>
		</tr>
		<tr>
			<td>Refrigerant</td>
			<td>Nihon freezer Co. Ltd., Tokyo, Japan</td>
			<td>CLN 540U</td>
			<td>~-80 &#176;C / Discontinued</td>
		</tr>
		<tr>
			<td>Serological pipet</td>
			<td>SPL Life Sciences Co., Ltd., Gyeonggi-do, South Korea</td>
			<td>91010</td>
			<td>10ml</td>
		</tr>
		<tr>
			<td>TrypLE Express Enzyme (1x), phenol red</td>
			<td>Thermo Fisher Scientific Inc., Waltham, USA</td>
			<td>12605010</td>
			<td>cell dissociation protease</td>
		</tr>
	</tbody>
</table>

isolation of epithelial cells from human dental follicle

The dental follicle (DF) was harvested during the removal of an impacted third molar by an oral maxillofacial surgeon. Epithelial cell isolation was performed on the day of DF harvest. The DF was washed three times with DPBS and then dissected with tissue scissors until the tissue had a pulpy or squishy consistency. Single-cell populations were pelleted by centrifugation and washed with keratinocyte serum-free medium. Heterogeneous cell populations were distributed in a culture dish. Keratinocyte serum-free medium was used to select the epithelial cells. The culture medium was changed daily until no floating debris or dead cells were observed. Epithelial cells appeared within 7-10 days after cell population distribution. Epithelial cells survived in serum-free medium, while &#945;-modification minimal essential medium supplemented with 10% fetal bovine serum allowed the proliferation of mesenchymal-type cells. The DF is a tissue source for the isolation of dental epithelial cells.
The purpose of this study was to establish a method for the isolation of epithelial cells from human DF. Periodontal ligament (PDL) was used for the isolation of human dental epithelial cells. Procuring epithelial cells from human PDL is not always successful due to the small tissue volume, leading to low numbers of epithelial cells. DF has a larger volume than PDL and contains more cells. DF can be a tissue source for the primary culture of human dental epithelial cells. This protocol is easier and more efficient than the isolation method using PDL. Procuring human dental epithelial cells may facilitate further studies of dental epithelial-mesenchymal interactions.

DF harvesting 
Surgery was performed by an oral maxillofacial surgeon. Human-derived materials, including the tooth fragment, gingival tissue, and DF, were collected by a surgeon (Figure 1A). The DF might be attached to the tooth fragment. An oral maxillofacial surgeon will be able to identify the DF. Cooperation and communication with the surgeon are required for tissue collection. The DF is an irregularly shaped membrane-like tissue. Gingival tissue has a keratinized ...

Watch this Scientific Journal Video about Isolation of Epithelial Cells from Human Dental Follicle at JoVE.com

Isolation of Epithelial Cells from Human Dental Follicle

The dental follicle contains an epithelial population and mesenchymal cells. The epithelial population was selected from the heterogeneous dental follicle cell population by providing a distinct culture medium. Epithelial cells survived and formed colonies in a serum-free medium.

The dental follicle (DF) was harvested during the removal of an impacted third molar by an oral maxillofacial surgeon. Epithelial cell isolation was ...

This protocol demonstrate an enhanced method for procuring human dental epithelial cells. It is difficult to identify the aggregated cell pellet during the procedures. Be careful when you handle supernatant to prevent the loss cell populations.To begin, harvest dental follicles during the surgical extraction of impacted third molars. Start cell isolation procedures or store the dental follicles at four degrees Celsius in DPBS with 3%penicillin-streptomycin. To wash the dental follicles, hold the dental follicle with tweezers and place it in washing tube 1.Gently shake it 10 to 15 times. Take it out and place it in tube 2. Again, shake it 10 to 15 times.Finally, take it out and place it in tube 3, then shake it. After washing three times, place the dental follicle in a 60 millimeter culture dish. Mince the tissue with tissue scissors until the dental follicle has a pulpy or squishy appearance.Use a small side section of the culture dish to minimize tissue loss. Mix one milliliter each of collagenase type 1 and protease solution in a 15 milliliter conical tube. Transfer the minced tissue into the 15 milliliter conical tube, then gently shake it and incubate it for one hour at 37 degrees Celsius.Add five milliliters of 0.05%trypsin-EDTA to the tube. Shake it and incubate for 15 minutes at 37 degrees Celsius. Prepare keratinocyte medium containing keratinocyte serum-free medium and 10%fetal bovine serum.Add three milliliters of keratinocyte medium into a new 50 milliliter conical tube. Place a 40 micrometer strainer on top of this tube. Next, aspirate the supernatant from the tube containing the tissue with a 10 milliliter serological pipette and pass it through the strainer.Transfer the collected suspension to a 15 milliliter conical tube. Centrifuge the tube and remove the supernatant. Then add three milliliters of keratinocyte serum-free medium.Centrifuge for another three minutes and remove the supernatant. Plate the single cell population into a 60 millimeter culture dish using the keratinocyte serum-free medium. Maintain the culture dish with a final volume of five milliliters in a humidified atmosphere of 5%carbon dioxide at 37 degrees Celsius.Cells with epithelial morphology appeared within seven to 10 days after plating single cell populations. The number of cobblestone-shaped epithelial cells ranged from one to 10 at the time of emergence and the cells expanded into colonies over time. The most crucial step is mincing the dental follicle into small particles.It enhances the separation of single cells from follicle tissue. Mesenchymal cells can be isolated from human dental follicle. Serum containing culture environment selects mesenchymal cells and inhibits epithelial growth from single cell populations.This protocol provide the method of procuring the human dental epithelial cells. Dental follicle-derived epithelial cells can be utilized for the study of dental epithelial-mesenchymal interactions.

isolation-of-epithelial-cells-from-human-dental-follicle

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JoVE Journal

Biology

2.7K 次观看.  Kyung Hee University Dental Hospital at Gangdong. 该协议展示了一种用于获取人类牙齿上皮细胞的增强方法。在手术过程中很难识别聚集的细胞沉淀。处理上清液时要小心，以防止细胞群丢失。首先，在手术拔除受影响的第三磨牙期间收获牙囊。开始细胞分离程序或将牙科卵泡储存在4摄氏度的DPBS中，含3%青霉素 - 链霉素。要清洗牙囊，请用镊子握住牙囊并将其放入洗涤管1中。轻轻摇晃10至15次。取出并将其放入管...