Name: isolation and culture of primary human gingival epithelial cells using y-27632
Uploaded: 2021-11-06T13:00:00
Description: Watch this Scientific Journal Video about Isolation and Culture of Primary Human Gingival Epithelial Cells using Y-27632 at JoVE.com

This work was supported by the Key Program of Shandong Province Natural Science Foundation (ZR2019ZD36) and the Key Research and Development Program of Shandong Province (2019GSF108107) to X.W.; the Key Research and Development Program of Shandong Province (2018GSF118240) to J.G.; Medical and Health Science and Technology Development Project of Shandong Province (2018WS163) to Z.X., and the Medical and Health Science and Technology Development Project of Shandong Province (2019WS045) to J.S.

Human tissues used in this protocol are fresh adult gingival tissues discarded from extractions of impacted teeth in the Maxillofacial Surgery Department according to the guidelines of the Institution&#39;s Human Research Ethics Committee (Protocol No. GR201711, Date: 02-27-2017).
1. Preparations
<ol>
	<li>Collect fresh adult gingival tissues in 15 mL tubes containing 10 mL of phosphate-buffered saline (PBS) supplemented with 3% penicillin/streptomycin (P/S) and keep the tis...

<ol>
	<li>Takahashi, N., 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=Gingival+barrier:+regulation+by+beneficial+and+harmful+microbes.">Gingival barrier: regulation by beneficial and harmful microbes.</a> Tissue Barriers. 7, 1651158 (2019).</li><li>Michea, P., 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=Epithelial+control+of+the+human+pDC+response+to+extracellular+bacteria.">Epithelial control of the human pDC response to extracellular bacteria.</a> European Journal of Immunology. 43, 1264-1273 (2013).</li><li>Bedran, T. B., Mayer, M. P., Spolidorio, D. P., Grenier, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+anti-inflammatory+activity+of+the+antimicrobial+peptides+human+beta-defensin-3+(hBD-3)+and+cathelicidin+(LL-37)+in+a+three-dimensional+co-culture+model+of+gingival+epithelial+cells+and+fibroblasts.">Synergistic anti-inflammatory activity of the antimicrobial peptides human beta-defensin-3 (hBD-3) and cathelicidin (LL-37) in a three-dimensional co-culture model of gingival epithelial cells and fibroblasts.</a> PloS one. 9, 106766 (2014).</li><li>Sciezynska, A., 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+and+culture+of+human+primary+keratinocytes-a+methods+review.">Isolation and culture of human primary keratinocytes-a methods review.</a> Experimental Dermatology. 28, 107-112 (2019).</li><li>Bryja, A., 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=Overview+of+the+different+methods+used+in+the+primary+culture+of+oral+mucosa+cells.">Overview of the different methods used in the primary culture of oral mucosa cells.</a> Journal of Biological Regulators and Homeostatic Agents. 33, 397-401 (2019).</li><li>Kedjarune, U., Pongprerachok, S., Arpornmaeklong, P., Ungkusonmongkhon, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+primary+human+gingival+epithelial+cells:+comparison+of+two+isolation+techniques.">Culturing primary human gingival epithelial cells: comparison of two isolation techniques.</a> Journal of Cranio-Maxillo-Facial Surgery: Official Publication of the European Association for Cranio-Maxillo-Facial Surgery. 29, 224-231 (2001).</li><li>Klingbeil, M. F., 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=Comparison+of+two+cellular+harvesting+methods+for+primary+human+oral+culture+of+keratinocytes.">Comparison of two cellular harvesting methods for primary human oral culture of keratinocytes.</a> Cell and Tissue Banking. 10, 197-204 (2009).</li><li>Qian, 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=One-step+simple+isolation+method+to+obtain+both+epidermal+and+dermal+stem+cells+from+human+skin+specimen.">One-step simple isolation method to obtain both epidermal and dermal stem cells from human skin specimen.</a> Methods in Molecular Biology. 1879, 139-148 (2019).</li><li>Wen, J., Zu, T., Zhou, Q., Leng, X., Wu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Y-27632+simplifies+the+isolation+procedure+of+human+primary+epidermal+cells+by+selectively+blocking+focal+adhesion+of+dermal+cells.">Y-27632 simplifies the isolation procedure of human primary epidermal cells by selectively blocking focal adhesion of dermal cells.</a> Journal of Tissue Engineering and Regenerative Medicine. 12, 1251-1255 (2018).</li><li>Liu, Z., 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=A+simplified+and+efficient+method+to+isolate+primary+human+keratinocytes+from+adult+skin+tissue.">A simplified and efficient method to isolate primary human keratinocytes from adult skin tissue.</a> Journal of Visualized Experiments: JoVE. , (2018).</li><li>Lauer, G., Wiedmann-Al-Ahmad, M., Otten, J. 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Keratin expression in fetal and adult gingival cells.</a> In Vitro Cellular &amp; Developmental Biology: Journal of the Tissue Culture Association. 26, 596-603 (1990).</li><li>Guarino, M., Tosoni, A., Nebuloni, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+contribution+of+epithelium+to+organ+fibrosis:+epithelial-mesenchymal+transition.">Direct contribution of epithelium to organ fibrosis: epithelial-mesenchymal transition.</a> Human Pathology. 40, 1365-1376 (2009).</li><li>Hatakeyama, S., Yaegashi, T., Takeda, Y., Kunimatsu, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+bromodeoxyuridine-incorporating,+p63-+and+p75(NGFR)-+expressing+cells+in+the+human+gingival+epithelium.">Localization of bromodeoxyuridine-incorporating, p63- and p75(NGFR)- expressing cells in the human gingival epithelium.</a> Journal of Oral Science. 49, 287-291 (2007).</li><li>Bayar, G. 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G., Green, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+a+keratinizing+epithelium+in+culture+by+a+cloned+cell+line+derived+from+a+teratoma.">Formation of a keratinizing epithelium in culture by a cloned cell line derived from a teratoma.</a> Cell. 6, 317-330 (1975).</li><li>Oda, D., Watson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+oral+epithelial+cell+culture+I.+Improved+conditions+for+reproducible+culture+in+serum-free+medium.">Human oral epithelial cell culture I. Improved conditions for reproducible culture in serum-free medium.</a> In Vitro Cellular &amp; Developmental Biology: Journal of the Tissue Culture Association. 26, 589-595 (1990).</li><li>Boyce, S. T., Ham, R. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+of+human+keratinocyte+culture+from+skin+explants:+cell+expansion+is+linked+to+markers+of+activated+progenitor+cells.">An improved method of human keratinocyte culture from skin explants: cell expansion is linked to markers of activated progenitor cells.</a> Experimental Dermatology. 18, 720-726 (2009).</li><li>Smola, H., Krieg, T., Irene, M., Leigh, E., Lane, B., Watt, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The keratinocyte handbook. 375, 311 (1994).</li><li>Shwetha, H. 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The gingival tissue is a key structure that maintains periodontal integrity and health. Gingival epithelial cells have significant roles in the repair and regeneration of periodontal tissue and can be used in scientific research and clinical applications and related fields, including oral biology, pharmacology, toxicology, and oral mucosa deficiencies18. Therefore, it is necessary to develop a stable and efficient method to harvest oral epithelial cells19. Primary epithelia...

The human gingiva, the first line defense structure that protects periodontal tissue, is not only a physical and chemical barrier1, but also secretes different classes of inflammatory mediators that participate in immune responses and constitute an immune barrier2,3. The gingival epithelium plays an important role in the repair and regeneration of periodontal tissue. Therefore, studying the defense and immunity of the gingival epithelium is of great significance for understanding the occurrence, diagnosis, and treatment of periodontitis. The isolation and culture of gingival epithelial cells from human gingival tissue is the first step required for studying the gingival epithelium. Such a procedure requires basic operations such as producing seed cells for tissue engineering, in vitro models of periodontal associated diseases, and materials for repairing periodontal defects.
Primary gingival epithelial cells are characterized by a low division rate in vitro4, researchers have been looking for an optimal isolation and cultivation method for decades. To date, two different techniques, a direct explant method and an enzymatic method, are commonly used in laboratories to obtain primary gingival epithelium cells in vitro4,5. The direct explant method has advantages such as the requirement of a lower amount of tissue specimens and simple isolation procedure, but it has the disadvantages of longer culture time and susceptibility to contamination5. Although the enzymatic method shortens the culture time required, the efficiency is relatively low and varies depending on the enzymes and medium used. Kedjarune et al.6 showed that the direct explant method, which requires more time before subculture (2 weeks), appeared to be more successful for culturing gingival epithelial cells compared to the enzymatic method. However, comparing these two methods, Klingbeil et al.7 found that the enzymatic method had the best results for primary cultures of oral epithelial cells, and it was possible to obtain the optimal cell yield within the shortest time period (11.9 days versus 14.2 days).
Therefore, it was important to develop a more convenient and effective method for the isolation and culture of oral epithelial cells4. We previously reported that adding Y-27632, an inhibitor of Rho-associated protein kinase (ROCK), simplifies the isolation procedure of human primary epidermal cells and keratinocytes from adult skin tissues8,9,10. We developed G-medium, a new conditioned inoculation medium that spontaneously separates epidermal from dermal cells and supports the growth and yield of primary epidermal cells8,9,10. In the present study, we developed a new serum-free isolation and culture technique for gingival epithelial cells by combining G-medium with Y-27632. In essence, our method is based on a simplification of the traditional two-step enzymatic method, so we compared our new method with the direct explant method. This modified enzymatic method significantly shortens the time required to separate gingival epithelial cells from gingival tissue and increases the efficiency of culturing gingival epithelial cells.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Names</td>
			<td></td>
			<td></td>
			<td>Abbreviations &#38; Comments</td>
		</tr>
		<tr>
			<td>Countess automated cell counter</td>
			<td>Shanghai Ruiyu Bio-science&#38;Technology Co.Ltd.</td>
			<td>BBA0218AC</td>
			<td>Automatic cell counting</td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Thermo Scientific</td>
			<td>51026333</td>
			<td>For cell incubation</td>
		</tr>
		<tr>
			<td>Sorvall ST 16R Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75004380</td>
			<td>Cell centrifuge</td>
		</tr>
		<tr>
			<td>Cell Culture Dish</td>
			<td>Eppendorf</td>
			<td>30702115</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>50 ml Centrifuge Tube</td>
			<td>KIRGEN</td>
			<td>171003</td>
			<td>For cell centrifugation</td>
		</tr>
		<tr>
			<td>1.5 ml microcentrifuge Tubes</td>
			<td>KIRGEN</td>
			<td>190691J</td>
			<td>For cell digestion</td>
		</tr>
		<tr>
			<td>Cell Strainer</td>
			<td>Corning incorporated</td>
			<td>431792</td>
			<td>Cell filtration</td>
		</tr>
		<tr>
			<td>Phosphate buffered solution</td>
			<td>Solarbio Life Science</td>
			<td>P1020-500</td>
			<td>Washing solution</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Scientific</td>
			<td>C11995500</td>
			<td>Component of neutralization medium</td>
		</tr>
		<tr>
			<td>Defined K-SFM</td>
			<td>Life Technologies</td>
			<td>10785-012</td>
			<td>Gingival epithelial&#160; cells culture medium</td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Thermo Scientific</td>
			<td>15140-122</td>
			<td>Antibiotics</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Biological Industries</td>
			<td>04-001-1AC5</td>
			<td>Component of neutralization medium</td>
		</tr>
		<tr>
			<td>0.05% Trypsin</td>
			<td>Life Technologies</td>
			<td>25300-062</td>
			<td>For HGGEPCs dissociation</td>
		</tr>
		<tr>
			<td>Dilution Medium</td>
			<td>Life Technologies</td>
			<td>50-9701</td>
			<td>For coating matrix</td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Gibco</td>
			<td>17105-041</td>
			<td>For HGGEPCs isolation</td>
		</tr>
		<tr>
			<td>Collagenase Type I</td>
			<td>Life Technologies</td>
			<td>17100-017</td>
			<td>For HGGEPCs isolation</td>
		</tr>
		<tr>
			<td>F12 Nutrient Mix, Hams</td>
			<td>Life Technologies</td>
			<td>31765035</td>
			<td>Component of G-medium</td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>Life Technologies</td>
			<td>17504044</td>
			<td>Growth factor in G-medium</td>
		</tr>
		<tr>
			<td>FGF-2 Millipore</td>
			<td>Merck Biosciences</td>
			<td>341595</td>
			<td>Growth factor in G-medium</td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Gene Operation</td>
			<td>IAD1011</td>
			<td>ROCK inhibitor</td>
		</tr>
		<tr>
			<td>Fungizone</td>
			<td>Gibco</td>
			<td>15290026</td>
			<td>Preparation for G-medium</td>
		</tr>
		<tr>
			<td>EGF Recombinant Human Protein</td>
			<td>Gibco</td>
			<td>PHG0311</td>
			<td>Growth factor in G-medium</td>
		</tr>
		<tr>
			<td>Cell Counting Kit-8</td>
			<td>Dojindo Molecular Technologies</td>
			<td>CK04</td>
			<td>For Cell proliferation assay</td>
		</tr>
		<tr>
			<td>Rabbit Anti-Human CK18</td>
			<td>Abcam</td>
			<td>ab82254</td>
			<td>For immunofluorescence staining to check differentiation marker of HGGEPCs</td>
		</tr>
		<tr>
			<td>Rabbit Anti-Human Cytokeratin10</td>
			<td>Abcam</td>
			<td>ab76318</td>
			<td>For immunofluorescence staining to check differentiation marker of HGGEPCs</td>
		</tr>
		<tr>
			<td>Mouse anti-human Vimentin</td>
			<td>Cell Signaling Technology</td>
			<td>3390</td>
			<td>For immunofluorescence staining of Gingival fibroblasts</td>
		</tr>
		<tr>
			<td>Rabbit Anti-Human pan-ck</td>
			<td>BD</td>
			<td>550951</td>
			<td>For immunofluorescence staining to check differentiation marker of HGGEPCs</td>
		</tr>
		<tr>
			<td>rabbit anti-Ki67</td>
			<td>Abcam</td>
			<td>15580</td>
			<td>For immunofluorescence staining to check differentiation marker of HGGEPCs</td>
		</tr>
		<tr>
			<td>rabbit anti-p63</td>
			<td>Biolegend</td>
			<td>619002</td>
			<td>For immunofluorescence staining to check differentiation marker of HGGEPCs</td>
		</tr>
		<tr>
			<td>rabbit anti-p75NGFR</td>
			<td>Abcam</td>
			<td>ab52987</td>
			<td>For immunofluorescence staining to check differentiation marker of HGGEPCs</td>
		</tr>
	</tbody>
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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.

Figure 1 shows a schematic diagram of the direct explant method and the modified enzymatic method. The direct explant method does not need any digestive enzyme during the whole process. In contrast, the traditional enzymatic method usually needs two sets of digestive enzymes, dispase and collagenase, to separate the epithelial sheet from the underlying fibroblast layer, and then trypsin to release the epithelial cells into suspension. Our new method omits the step of separation and is a simp...

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

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 ...

So general goal of this cell-based protocol is to isolate and encounter Human Gingival Epithelial Cells in order to improve the shortcomings of direct eggplant method.From the new modified endothemic method, we can get larger numbers of primary gingival epithelial cells in a relatively short time, based on our materials and equipment.So human tissue used in this protocol were fresh adult gingival tissues discarded from extractions of impacted teeth in the Maxillofacial Surgery Department according to the guidelines of the Institution's Human Research Ethics Committee.The New Modified Enzymatic Method.Gingival Tissue Pretreatment.Wash the tissues with 75%ethanol for 30 seconds.And then, with washing solution twice for five minutes each.Digestion of Gingival Tissue.Cut the tissues into tiny pieces with two surgical blades.Add 2.5mg/mililitre dispase and collagenase-1 solution into a 1.5 mililitre centrifuge tube.Put the tissue pieces into the 1.5 mililitre centrifuge tube.Incubate for 15-20 minutes in a 37

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