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>Harvest DF during the surgical extraction of impacted third molars (Figure 1A). 
	NOTE: Surgery was performed by an oral maxillofacial surgeon. Cooperation is required with a dentist for tissue collection.</li>
	<li>Store DF in Dulbecco&#39;s phosphate-buffered saline (DPBS) with 3% penicillin-streptomycin at 4 &#176;C before use. Perform isolation procedures on the day of DF harvest (Figure 1B).</li>
</ol>
2. Isolate single-cell populations from DF
<ol>
	<li>Prepare stock solutions of collagenase type I and protease in DPBS so that final concentrations of collagenase type I and protease are 1 mg/mL and 2.4 mg/mL, respectively.</li>
	<li>Prepare three 50 mL washing tubes with 20 mL of DPBS per tube. Label the tubes as 1, 2, and 3.</li>
	<li>Wash the DF in the washing tubes sequentially. Hold the DF with tweezers and place the DF in tube 1. Take the DF out of tube 1 and place it in tube 2. Take the DF out of tube 2 and place it in tube 3. Gently shake DF 10-15 times in each tube. After washing three times, place the DF in a 60 mm culture dish (Figure 2A).</li>
	<li>Mince the DF with tissue scissors (Figure 2B). Transfer only a small portion to the culture dish to minimize tissue loss. Repeat the cutting until the DF has a pulpy or squishy appearance (Figure 2C).</li>
	<li>Mix 1 mL of collagenase type I and 1 mL of protease solution in a 15 mL conical tube to obtain final concentrations of 1 mg/mL and 2.4 mg/mL, respectively.</li>
	<li>Transfer the minced DF into the 15 mL conical tube from step 2.5. Gently shake and incubate the tube for 1 h at 37 &#176;C.</li>
	<li>Add 5 mL of 0.05% trypsin-EDTA to the tube, shake, and incubate the tube for 15 min at 37 &#176;C.</li>
	<li>Prepare keratinocyte medium containing keratinocyte SFM 10% fetal bovine serum (FBS). Add 3 mL of keratinocyte medium into a new 50 mL conical tube. Place a 40 &#181;m strainer on top of this tube and use a 10 mL serological pipette to aspirate the supernatant from step 2.6 and pass it through the strainer. 
	NOTE: Minimize the incorporation of digested tissue remnants while taking the supernatant. Remove the tissue remnants with a 40 &#181;m strainer to minimize failure. Tissue remnants may pass through 70 &#181;m strainers and hinder colony formation. FBS in the keratinocyte medium will inactivate the enzymes.</li>
	<li>Repeat steps 2.7 and 2.8.</li>
	<li>Transfer the collected suspension to a 15 mL conical tube.</li>
	<li>Centrifuge the 15 mL conical tube at 288 &#215; g for 3 min at 4 &#176;C. Remove the supernatant. 
	NOTE: Be careful to avoid any accidental removal of pelleted cells, which are mostly invisible.</li>
	<li>Add 3 mL of keratinocyte SFM and wash the cells twice with a 1 mL pipette with centrifugation as in step 2.11.</li>
	<li>Plate the single-cell population into a 60 mm culture dish using keratinocyte SFM. Maintain the culture dish with a final volume of 5 mL in a humidified atmosphere of 5% CO2 at 37 &#176;C. Change the culture medium daily until no tissue or cell debris are observed. 
	NOTE: Remove floating debris in the culture medium by changing the medium. Delayed removal of tissue debris or dead cells has an unfavorable effect on the primary culture.</li>
	<li>Examine the plate from step 2.13 to check the growth of epithelial cells (Figure 3A). 
	NOTE: Epithelial cells grow within 7-10 days after plating single-cell populations.</li>
	<li>Expand the epithelial colonies and subculture the cells.
	<ol>
		<li>Aspirate the medium and wash the bottom of the culture plate once with 3 mL of keratinocyte SFM.</li>
		<li>Add 2 mL of cell dissociation protease and incubate in a humidified atmosphere of 5% CO2 at 37 &#176;C for 3 min. 
		NOTE: Repeat step 2.15.2 if cell detachment is not effective.</li>
		<li>Add 2 mL of keratinocyte medium to the culture plate and transfer the contents in a 15 mL conical tube. 
		NOTE: Adding more keratinocyte medium is not required if step 2.15.2 is repeated.</li>
		<li>Centrifuge the 15 mL conical tube at 288 &#215; g for 3 min at 4 &#176;C.</li>
		<li>Remove the supernatant and wash the cell pellet two times with 3 mL of keratinocyte SFM.</li>
		<li>Plate the cells in a 100 mm culture dish using keratinocyte SFM (final volume of 10 mL per 100 mm dish).</li>
	</ol>
	</li>
	<li>Prepare and store cell stocks in a liquid nitrogen tank.
	<ol>
		<li>Harvest cells as described in steps 2.15.1-2.15.5.</li>
		<li>Distribute the cells into cryovials in 1 mL of freezing medium (80% keratinocyte SFM, 10% dimethylsulfoxide, 10% FBS).</li>
		<li>Place the vials in a freezing container and store them at -80 &#176;C for 24 h.</li>
		<li>Transfer the vials from the deep freezer to a liquid nitrogen tank.</li>
	</ol>
	</li>
</ol>

<ol><li>Tucker, A., Sharpe, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+cutting-edge+of+mammalian+development%3B+How+the+embryo+makes+teeth%5BTitle%5D)+AND+%22Nature+Reviews+Genetics%22%5BJournal%5D)">The cutting-edge of mammalian development; How the embryo makes teeth.</a> Nature Reviews Genetics. 5 (7), 499-508 (2004).</li>
<li>Choung, H. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effect+of+CPNE7+on+periodontal+regeneration%5BTitle%5D)+AND+%22Connective+Tissue+Research%22%5BJournal%5D)">The effect of CPNE7 on periodontal regeneration.</a> Connective Tissue Research. 60 (5), 419-430 (2019).</li>
<li>Li, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+immortalized+Hertwig%27s+epithelial+root+sheath+cell+lines+for+cementum+and+dentin+regeneration%5BTitle%5D)+AND+%22Stem+Cell+Research+and+Therapy%22%5BJournal%5D)">Development of immortalized Hertwig's epithelial root sheath cell lines for cementum and dentin regeneration.</a> Stem Cell Research and Therapy. 10 (1), 3(2019).</li>
<li>Huang, X. F., Bringas, P., Slavkin, H. C., Chai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fate+of+HERS+during+tooth+root+development%5BTitle%5D)+AND+%22Developmental+Biology%22%5BJournal%5D)">Fate of HERS during tooth root development.</a> Developmental Biology. 334 (1), 22-30 (2009).</li>
<li>Farea, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+enhancement+of+a+homogenous+in+vitro+human+Hertwig%27s+epithelial+root+sheath+cell+population%5BTitle%5D)+AND+%22International+Journal+of+Molecular+Sciences%22%5BJournal%5D)">Isolation and enhancement of a homogenous in vitro human Hertwig's epithelial root sheath cell population.</a> International Journal of Molecular Sciences. 14 (6), 11157-11170 (2013).</li>
<li>Jung, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Directing+the+differentiation+of+human+dental+follicle+cells+into+cementoblasts+and%2For+osteoblasts+by+a+combination+of+HERS+and+pulp+cells%5BTitle%5D)+AND+%22Journal+of+Molecular+Histology%22%5BJournal%5D)">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/pubmed?term=((Changes+of+the+unique+odontogenic+properties+of+rat+apical+bud+cells+under+the+developing+apical+complex+microenvironment%5BTitle%5D)+AND+%22International+Journal+of+Oral+Science%22%5BJournal%5D)">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/pubmed?term=((Isolation+and+characterization+of+dental+follicle-derived+Hertwig%27s+epithelial+root+sheath+cells%5BTitle%5D)+AND+%22Clinical+Oral+Investigations%22%5BJournal%5D)">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/pubmed?term=((Delta+Np63+alpha+protein+triggers+epithelial-mesenchymal+transition+and+confers+stem+cell+properties+in+normal+human+keratinocytes%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">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/pubmed?term=((Isolation+of+precursor+cells+%28PCs%29+from+human+dental+follicle+of+wisdom+teeth%5BTitle%5D)+AND+%22Matrix+Biology%22%5BJournal%5D)">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/pubmed?term=((Expression+profile+of+the+stem+cell+markers+in+human+Hertwig%27s+epithelial+root+sheath%2FEpithelial+rests+of+Malassez+cells%5BTitle%5D)+AND+%22Molecular+and+Cells%22%5BJournal%5D)">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/pubmed?term=((Dental+follicle+cells+and+cementoblasts+induce+apoptosis+of+ameloblast-lineage+and+Hertwig%27s+epithelial+root+sheath%2Fepithelial+rests+of+Malassez+cells+through+the+Fas-Fas+ligand+pathway%5BTitle%5D)+AND+%22European+Journal+of+Oral+Sciences%22%5BJournal%5D)">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/pubmed?term=((Recapitulating+cell-cell+Interactions+for+organoid+construction+-+are+biomaterials+dispensable%5BTitle%5D)+AND+%22Trends+in+Biotechnology%22%5BJournal%5D)">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/pubmed?term=((Are+Hertwig%27s+epithelial+root+sheath+cells+necessary+for+periodontal+formation+by+dental+follicle+cells%5BTitle%5D)+AND+%22Archives+of+Oral+Biology%22%5BJournal%5D)">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/pubmed?term=((Influence+of+enamel+matrix+derivative+on+healing+of+root+surfaces+after+bonding+treatment+and+intentional+replantation+of+vertically+fractured+roots%5BTitle%5D)+AND+%22Dental+Traumatology%22%5BJournal%5D)">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/pubmed?term=((The+crowning+achievement%3A+getting+to+the+root+of+the+problem%5BTitle%5D)+AND+%22Journal+of+Dental+Education%22%5BJournal%5D)">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/pubmed?term=((Tooth+agenesis%3A+What+do+we+know+and+is+there+a+connection+to+cancer%5BTitle%5D)+AND+%22Clinical+Genetics%22%5BJournal%5D)">Tooth agenesis: What do we know and is there a connection to cancer.</a> Clinical Genetics. 99 (4), 493-502 (2021).</li>
</ol>

The authors declare that they have no conflicts of interest.

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 increases the probability of the emergence of the epithelial population. Contamination frequently occurs during the isolation of epithelial cells from the DF. The use of 40 &#181;m strainers minimizes the inclusion of tissue debris in single-cell populations, as tissue remnants can pass through 70 &#181;m strainers and inhibit epithelial cell colonization. 
Epithelial cells seem to be more vulnerable to certain environments than mesenchymal cells, and the daily changing of the culture medium also provided a clean environment for the emergence of the epithelial population. Losing cells due to frequent medium change may be a limitation of this protocol. However, daily changing of the medium was performed after troubleshooting for this protocol, and consequently, epithelial cells appeared and expanded. Delaying the medium change can affect the cultures. As the cell pellet after centrifugation is nearly invisible, the narrow tip of a 15 mL conical tube increases the ease of supernatant removal without losing the cell pellet. Therefore, this protocol suggests transferring the contents of the 50 mL conical tube to a 15 mL conical tube.
The DF provides a larger volume of tissue and more single cells than the PDL. Hence, the DF can be used instead of the PDL as a tissue source for the isolation of human dental epithelial cells. Selection and inhibition of cell types in the DF can be done simply through a selective culture medium. SFM induces epithelial growth, whereas serum-containing medium selects for mesenchymal cells. This methodology allows easy accessibility to human dental epithelial cells. Morsczek and colleagues previously isolated DF-derived mesenchymal precursors. This study differs from theirs by the selection of epithelial populations and the inhibition of mesenchymal cells. 
The regulatory role of dental epithelial cells during tooth formation is of great interest in dental research. Studying epithelial-mesenchymal interactions during tooth development may suggest clues for the resolution of clinical problems13. Tissue regeneration by combining epithelial and mesenchymal components has been attempted14. Dental epithelial cell-derived factors, such as an enamel matrix derivative, have been evaluated as therapeutic targets aiming at periodontal regeneration, pulp-dentin complex regeneration, and periodontal reattachment2,15. The combination of regeneration factors, including stem cells, epithelial-mesenchymal cell sheets, cell-derived factors, and biomaterials, may generate bio-teeth in case of tooth loss or agenesis16. Tooth agenesis-associated genes, such as PAX9, MSX1, AXIN2, EDA, EDAR, and WNT10A genes, are potential candidate key regulatory genes for epithelial-mesenchymal interactions17. Further, next-generation sequencing method-derived data from tooth agenesis-associated studies may similarly propose putative novel target genes regulating molecular control during dental epithelial-mesenchymal interactions.

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><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 &amp;mu;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&lt;br/&gt; 50050</td><td>15 mL&lt;br/&gt; 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&#039;s Phosphate-Buffered Saline</td><td>Welgene Inc., Gyengsangbuk-do, South Korea</td><td>&amp;nbsp;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 / CO&lt;sub&gt;2&lt;/sub&gt; 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,&lt;br/&gt; 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 &amp;micro;L</td></tr><tr><td>Refrigerant</td><td>Nihon freezer Co. Ltd., Tokyo, Japan</td><td>CLN 540U</td><td>~-80 &amp;deg;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 surface and can be distinguished from the DF. Pulp tissue resides in the pulp chamber and is rarely separated from teeth during surgery. The DF was stored in DPBS with 3% penicillin-streptomycin at 4 &#176;C before use (Figure 1B).
Mechanical treatment of DF 
Tissue debris and blood were removed by washing the DF with DPBS. Three conical tubes were prepared with washing solution. After washing, the DF was transferred onto a 60 mm or 100 mm culture dish (Figure 2A). The DF should be minced with scissors until the tissue has a pulpy or mushy appearance (Figure 2B, C).
Emergence of epithelial population 
After plating heterogeneous cell populations, many cells and small debris floated in the culture medium. The number of floating cells gradually decreased with successive medium changes. Delayed medium change may cause a turbid culture environment. At the initial stage, different cell types appear at the bottom of the culture dish but disappear when maintained in keratinocyte SFM. Cells with epithelial morphology appear within 7-10 days after plating single-cell populations (Figure 3A). The number of cobblestone-shaped cells ranges from 1 to 10 at the time of the emergence of epithelial cells, which expand into colonies over time (Figure 3B).
The mincing procedure was repeated to increase DF contact area with 0.05% trypsin-EDTA to promote cell dissociation. More single cells were harvested from DF pulp than intact DF. The stickiness of the DF pulp indicated the probability of success of the isolation of epithelial cells. Daily medium changes prevented the keratinocyte SFM from becoming turbid, which is important for epithelial cell survival.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63104/63104fig01.jpg" /> 
Figure 1: Human-derived materials. (A) Human-derived materials were collected by an oral maxillofacial surgeon. The yellow arrow indicates the DF. Black arrows indicate the tooth fragment and tooth-attached soft tissue. (B) The DF was stored in PBS supplemented with 3% penicillin-streptomycin at 4 &#176;C. Isolation procedures were performed within 24 h of collection. Abbreviations: DF = dental follicle; PBS = phosphate-buffered saline. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63104/63104fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63104/63104fig02.jpg" /> 
Figure 2: Treatment of dental follicles. (A) The DF was transferred to a 60 mm culture dish after washing with DPBS three times. (B) The DF was minced with tissue scissors. (C) The minced DF had a pulpy or mushy appearance. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63104/63104fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63104/63104fig03.jpg" /> 
Figure 3: Emergence of epithelial cells. (A) Cells with epithelial morphology appear within 7-10 days after plating single-cell populations. (B) Ex vivo expansion of DF-derived epithelial cells by subculture. Scale bars = 100 &#181;m. Abbreviation: DF = dental follicle. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63104/63104fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Universidad San Sebastian

Research

JoVE Journal

Biology

2.8K Views.  Kyung Hee University Dental Hospital at Gangdong. 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. 

Video: Isolation of Epithelial Cells from Human Dental Follicle

Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by distinct pools of SCs, which are located in the permanent portion of the HF, a region known as the bulge. These multipotent bulge SCs were initially identified as slow cycling label retaining cells; however, their isolation has been made feasible after identification of specific cell markers, such as CD34 and keratin 15 (K15). Here, we describe a robust method for isolating bulge SCs and epidermal keratinocytes from mouse HFs utilizing fluorescence activated cell-sorting (FACS) technology. Isolated hair follicle SCs (HFSCs) can be utilized in various in vivo grafting models and are a valuable in vitro model for studying the mechanisms that govern multipotency, quiescence and activation.

An ideal model for studying adult stem cell biology is the mouse hair follicle. Here we present a protocol for isolating different populations of hair follicles stem cells and epidermal keratinocytes, employing enzymatic digestion of mouse dorsal skin followed by FACS analysis.

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by ...

Developmental Biology

Isolation, Characterization and MicroRNA-based Genetic Modification of Human Dental Follicle Stem Cells

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such as initial massive cell death and low therapeutic effects, impaired their broad clinical translation. Genetic engineering of stem cells prior to transplantation emerged as a promising method to optimize therapeutic stem cell effects. However, safe and efficient gene delivery systems are still lacking. Therefore, the development of suitable methods may provide an approach to resolve current challenges in stem cell-based therapies.
The present protocol describes the extraction and characterization of human dental follicle stem cells (hDFSCs) as well as their non-viral genetic modification. The postnatal dental follicle unveiled as a promising and easily accessible source for harvesting adult multipotent stem cells possessing high proliferation potential. The described isolation procedure presents a simple and reliable method to harvest hDFSCs from impacted wisdom teeth. Also this protocol comprises methods to define stem cell characteristics of isolated cells. For genetic engineering of hDFSCs, an optimized cationic lipid-based transfection strategy is presented enabling highly efficient microRNA introduction without causing cytotoxic effects. MicroRNAs are suitable candidates for transient cell manipulation, as these small translational regulators control the fate and behavior of stem cells without the hazard of stable genome integration. Thus, this protocol represents a safe and efficient procedure for engineering of hDFSCs that may become important for optimizing their therapeutic efficacy.

This protocol describes the transient genetic engineering of dental stem cells extracted from the human dental follicle. The applied non-viral modification strategy may become a basis for the improvement of therapeutic stem cell products.

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such ...

Genetics

Isolation of Papillary and Reticular Fibroblasts from Human Skin by Fluorescence-activated Cell Sorting

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have traditionally been attributed to the superficial papillary or lower reticular dermis according to their histological localization. In mouse dermis, papillary and reticular fibroblasts originate from two different lineages with diverging functions regarding physiological and pathological processes and a distinct cell surface marker expression profile by which they can be distinguished.
Importantly, evidence from explant cultures from superficial and lower dermal layers suggest that at least two functionally distinct dermal fibroblasts lineages exist in human skin dermis as well. However, unlike for mouse skin, cell surface markers enabling the discrimination of different fibroblast subsets have not yet been established for human skin. We developed a novel protocol for the isolation of human papillary and reticular fibroblast populations via fluorescence-activated cell sorting (FACS) using the two cell surface markers Fibroblast Activation Protein (FAP) and Thymocyte antigen 1 (Thy1)/CD90. This method enables the isolation of pure fibroblast subsets without in vitro manipulation, which was shown to affect gene expression, thus permitting accurate functional analysis of human dermal fibroblast subsets in regard to tissue homeostasis or disease pathology.

This manuscript describes a FACS-based protocol for isolation of papillary and reticular fibroblasts from human skin. It circumvents in vitro culture which was inevitable with the commonly used isolation protocol via explant cultures. The emanating fibroblast subsets are functionally distinct and display differential gene expression and localization within the dermis.

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Medicine

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Immunology and Infection

Rapid Isolation of Single Cells from Mouse and Human Teeth

Mouse and human teeth represent challenging organs for quick and efficient cell isolation for single-cell transcriptomic or other applications. The dental pulp tissue, rich in the extracellular matrix, requires a long and tedious dissociation process that is typically beyond the reasonable time for single-cell transcriptomics. For avoiding artificial changes in gene expression, the time elapsed from euthanizing an animal until the analysis of single cells needs to be minimized. This work presents a fast protocol enabling to obtain single-cell suspension from mouse and human teeth in an excellent quality suitable for scRNA-seq (single-cell RNA-sequencing). This protocol is based on accelerated tissue isolation steps, enzymatic digestion, and subsequent preparation of final single-cell suspension. This enables fast and gentle processing of tissues and allows using more animal or human samples for obtaining cell suspensions with high viability and minimal transcriptional changes. It is anticipated that this protocol might guide researchers interested in performing the scRNA-seq not only on the mouse or human teeth but also on other extracellular matrix-rich tissues, including cartilage, dense connective tissue, and dermis.

The current protocol presents a fast, efficient, and gentle method for isolating single cells suitable for single-cell RNA-seq analysis from a continuously growing mouse incisor, mouse molar, and human teeth.

Mouse and human teeth represent challenging organs for quick and efficient cell isolation for single-cell transcriptomic or other applications. The dental ...

An Efficient Method for Extracting Human Fallopian Tube Epithelia for Single-cell Analyses

Human fallopian tubes are intrinsic to reproduction. The purpose of the fallopian tubes is to allow for the transit of sperm, the ovum, and if fertilization is successful, the embryo. The epithelial cells that line the inner surface of fallopian tubes are integral to both normal and abnormal fallopian tube processes, including disease initiation. After menopause, fallopian tubes cease to have a significant role in the body and the intraepithelial cell makeup changes. We describe a method in which these epithelial cells can be isolated from fresh fallopian tubes with minimal stromal cell contamination into a single-cell suspension. These cells can be grown in culture or used for further analysis such as flow cytometry and single-cell RNA sequencing. This isolation protocol can be achieved in 4-6 h and yields viable cells that can be used for immediate downstream analysis. This efficient protocol facilitates the isolation of fallopian tube epithelial cells with an enriched epithelial population.

This protocol describes a one-day method for the isolation of human fallopian tube epithelial cells. Isolated epithelial cells can be plated in 2-dimensional (2D) culture or dissociated into single-cell suspensions and utilized in downstream experiments, including flow cytometry and single-cell RNA sequencing.

Human fallopian tubes are intrinsic to reproduction. The purpose of the fallopian tubes is to allow for the transit of sperm, the ovum, and if ...

Cancer Research

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Isolation of Salivary Epithelial Cells from Human Salivary Glands for In Vitro Growth as Salispheres or Monolayers

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to restore function of glands damaged by radiation therapies or due to pathologies associated with Sj&#246;gren's syndrome. A second goal of researchers is to define the mechanisms by which viruses replicate within glandular tissue where they can then gain access to salivary fluids important for horizontal transmission. These goals highlight the need for a robust and accessible in vitro salivary gland model that can be utilized by researchers interested in the above mentioned as well as related research areas. Here we discuss a simple protocol to isolate epithelial cells from human salivary glands and propagate them in vitro. Our protocol can be further optimized to meet the needs of individual studies. Briefly, salivary tissue is mechanically and enzymatically separated to isolate single cells or small clusters of cells. Selection for epithelial cells occurs by plating onto a basement membrane matrix in the presence of media optimized to promote epithelial cell growth. These resulting cultures can be maintained as three-dimensional clusters, termed "salispheres", or grown as a monolayer on treated plastic tissue culture dishes. This protocol results in the outgrowth of a heterogenous population of mainly epithelial cells that can be propagated for 5-8 passages (15-20 population doublings) before undergoing cellular senescence.

We present a method for isolating and cultivating primary human salivary gland-derived epithelial cells. These cells exhibit gene expression patterns consistent with them being of salivary epithelial origin and can be grown as salispheres on basement membrane matrices derived from Engelbreth-Holm-Swarm tumor cells or as monolayers on treated culture dishes.

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to ...

Isolation and Culture of Primary Human Gingival Epithelial Cells using Y-27632

The gingival tissue is the first structure that protects periodontal tissues and plays meaningful roles in many oral functions. The gingival epithelium is an important structure of gingival tissue, especially in the repair and regeneration of periodontal tissue. Studying the functions of gingival epithelial cells has crucial scientific value, such as repairing oral defects and detecting the compatibility of biomaterials. As human gingival epithelial cells are highly differentiated keratinized cells, their lifespan is short, and they are difficult to passage. So far, there are only two ways to isolate and culture gingival epithelial cells, a direct explant method and an enzymatic method. However, the time required to obtain epithelial cells using the direct explant method is longer, and the cell survival rate of the enzymatic method is lower. Clinically, the acquisition of gingival tissue is limited, so a stable, efficient, and simple in vitro isolation and culture system is needed. We improved the traditional enzymatic method by adding Y-27632, a Rho-associated kinase (ROCK) inhibitor, which can selectively promote the growth of epithelial cells. Our modified enzymatic method simplifies the steps of the traditional enzymatic method and increases the efficiency of culturing epithelial cells, which has significant advantages over the direct explant method and the enzymatic method.

Here we present a modified method for the isolation and culture of human gingival epithelial cells by adding the Rock inhibitor, Y-27632, to the traditional method. This method is easier, less time-consuming, enhances stem cell properties, and produces larger numbers of high-potential epithelial cells both for the laboratory and for clinical applications.

The gingival tissue is the first structure that protects periodontal tissues and plays meaningful roles in many oral functions. The gingival epithelium is ...