Name: isolation and culture of primary human gingival epithelial cells using y-27632
Uploaded: 2021-11-06T13:00:00.000+00:00
Duration: 6 min 47 s
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 tissues at 4 &#176;C. 
	NOTE: Treat the tissues as detailed below within 24 h after excision.</li>
</ol>
2. Prepare reagents and culture medium
<ol>
	<li>Prepare the washing solution: 3% P/S. Mix 50 mL of PBS with 1.5 mL of penicillin (100 U/mL) and 1.5 mL of streptomycin (100 mg/L).</li>
	<li>Prepare 500 mL of growth-factor-containing medium (called G-medium): DMEM/F12 (3:1) medium containing 1% P/S, 2% B27 supplement, 20 ng/mL of epidermal growth factor (EGF), 40 ng/mL of fibroblast growth factor 2 (FGF2), and 40 &#181;g/mL of fungizone.</li>
	<li>Prepare enzyme digestion solutions for the new method: Dissolve 125 mg of Dispase powder and 125 mg of Type I collagenase powder in 50 mL of DMEM. 
	NOTE: Filter all the enzyme solutions through a 0.22 &#181;m strainer and store at 4 &#176;C.</li>
	<li>Prepare 500 mL of the medium to neutralize the enzymatic digestion: 10% fetal bovine serum (FBS) and 1% P/S in the DMEM medium.</li>
</ol>
3. Direct explant method
<ol>
	<li>Gingival tissue pretreatment
	<ol>
		<li>Wash gingival tissue with 5 mL of 75% ethanol for 30 s. Then, rinse twice with 5 mL of the washing solution (step 2.1) for 5 min. 
		NOTE: Perform all washes in cell culture dishes with a diameter of 50 mm.</li>
	</ol>
	</li>
	<li>Gingival epithelial cell culture
	<ol>
		<li>Cut the gingival tissue into 1 mm3 pieces and scatter them in a 100 mm cell culture dish. Use fine pointed forceps and ophthalmic scissors for the cutting operations.</li>
		<li>Add the G-medium (step 2.2) to the culture dish. Then, incubate the culture dish in a 5% CO2 incubator at 37 &#176;C. Use an inverted microscope (40x) to examine the tissues every 24 h.</li>
		<li>Remove the gingival tissue pieces when the epithelial cells have propagated to a 2-5 mm in diameter around the tissue pieces. Change the G-medium every 2-4 days.</li>
	</ol>
	</li>
</ol>
4. The new modified enzymatic method
<ol>
	<li>Gingival tissue pretreatment
	<ol>
		<li>For this, follow the same steps as described for the direct explant method, step 3.1.1.</li>
	</ol>
	</li>
	<li>Digestion of gingival tissue
	<ol>
		<li>Cut the gingival tissue into tiny pieces, approximately &#60;1 mm in size, with two sterilized blades. Repeat the process of shredding with two surgical blades.</li>
		<li>Add 1 mL of 2.5 mg/mL dispase + collagenase solution to a 1.5 mL centrifuge tube containing the gingival tissue pieces, and then incubate the tube for 15-20 min in an incubator at 37 &#176;C.</li>
		<li>When the tissue becomes transparent and flocculent, add 1 mL of the neutralization solution to terminate the digestion. Thoroughly mix the solution by pipetting up and down 10-15 times. Pass the solution through a 100 &#181;m mesh filter.</li>
		<li>Centrifuge at 200 x g for 5 min.</li>
		<li>Remove the supernatant and resuspend the cell pellet at the bottom in 10 mL of growth-factor-containing medium (step 2.2). Transfer the cell suspension to a 100 mm cell culture dish. Add 10 &#181;M of Y-27632 to the cell culture dish.</li>
		<li>Culture the cells at 37 &#176;C in a 5% CO2 incubator. Replace the old G-medium with the fresh G-medium every 2 days. Observe the cells on Day 1 and Day 3.</li>
	</ol>
	</li>
</ol>
5. Cell passaging
<ol>
	<li>Take the dish out of the incubator, remove the spent medium and wash twice with PBS. Add 2 mL of 0.05% trypsin for each 100 mm dish. 
	NOTE: Shake the dish to make sure there is enough contact between the Trypsin solution and the dish bottom.</li>
	<li>Leave the dish in an incubator around 5 min at 37 &#176;C for the digestion process.</li>
	<li>Use a microscope (40x) to examine the cells and make sure that most cells have separated from the bottom of the dish.</li>
	<li>Add 2 mL of the neutralization solution to stop the digestion and transfer the cells into a 15 mL tube. Pipette up and down 10-15 times. Centrifuge the cells at 200 x g for 5 min.</li>
	<li>Remove the supernatant slowly. Resuspend the cells with 10 mL of G-medium and count the number of cells.</li>
	<li>Add about 1 x 106 cells in 10 mL of G-medium and 10 &#181;M Y-27632 to each 100 mm dish.</li>
	<li>Renew the G-medium and Y-27632 every 2 days. View the cells on Day 1 and Day 5.</li>
</ol>

<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. E., Hubner, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+study+during+healing+of+free+palatal+mucosa+grafts+on+plastic-embedded+samples.">Immunohistochemical study during healing of free palatal mucosa grafts on plastic-embedded samples.</a> Journal of Oral Pathology &amp; Medicine: Official Publication of the International Association of Oral Pathologists and the American Academy of Oral Pathology. 30, 104-112 (2001).</li><li>Locke, M., Hyland, P. L., Irwin, C. R., Mackenzie, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+gingival+epithelial+phenotypes+by+interactions+with+regionally+defined+populations+of+fibroblasts.">Modulation of gingival epithelial phenotypes by interactions with regionally defined populations of fibroblasts.</a> Journal of Periodontal Research. 43, 279-289 (2008).</li><li>Shabana, A. H., Ouhayoun, J. P., Sawaf, M. H., Forest, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokeratin+patterns+of+human+oral+mucosae+in+histiotypic+culture.">Cytokeratin patterns of human oral mucosae in histiotypic culture.</a> Archives of Oral Biology. 36, 747-758 (1991).</li><li>Kasai, 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=biopsy+of+human+oral+mucosal+epithelial+cells+as+a+quality+control+of+the+cell+source+for+fabrication+of+transplantable+epithelial+cell+sheets+for+regenerative+medicine.">biopsy of human oral mucosal epithelial cells as a quality control of the cell source for fabrication of transplantable epithelial cell sheets for regenerative medicine.</a> Regenerative Therapy. 4, 71-77 (2016).</li><li>Oda, D., Dale, B. A., Bourekis, G. <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.+II.+Keratin+expression+in+fetal+and+adult+gingival+cells.">Human oral epithelial cell culture. II. 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. R., Aydintug, Y. S., Gunhan, O., Ozturk, K., Gulses, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+produced+oral+mucosa+equivalent+by+using+the+direct+explant+cell+culture+technique.">Ex vivo produced oral mucosa equivalent by using the direct explant cell culture technique.</a> Balkan Medical Journal. 29, 295-300 (2012).</li><li>Daniels, J. T., Kearney, J. N., Ingham, 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+keratinocyte+isolation+and+cell+culture:+a+survey+of+current+practices+in+the+UK.">Human keratinocyte isolation and cell culture: a survey of current practices in the UK.</a> Burns: Journal of the International Society for Burn Injuries. 22, 35-39 (1996).</li><li>Carrel, A., Burrows, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultivation+of+adult+tissues+and+organs+outside+the+body.">Cultivation of adult tissues and organs outside the body.</a> Journal of the American Medical Association. 55, 1379-1381 (1910).</li><li>Rheinwald, J. 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. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-regulated+differentiation+of+normal+human+epidermal+keratinocytes+in+chemically+defined+clonal+culture+and+serum-free+serial+culture.">Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum-free serial culture.</a> The Journal of Investigative Dermatology. 81, 33-40 (1983).</li><li>Guo, A., Jahoda, 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=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. R., 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=Ex+vivo+culture+of+oral+keratinocytes+using+direct+explant+cell+culture+technique.">Ex vivo culture of oral keratinocytes using direct explant cell culture technique.</a> Journal of Oral and Maxillofacial Pathology: JOMFP. 23, 243-247 (2019).</li><li>Yin, J., Yu, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rho+kinases+regulate+corneal+epithelial+wound+healing.">Rho kinases regulate corneal epithelial wound healing.</a> American Journal of Physiology. Cell Physiology. 295, 378-387 (2008).</li><li>Chapman, S., Liu, X., Meyers, C., Schlegel, R., McBride, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+keratinocytes+are+efficiently+immortalized+by+a+Rho+kinase+inhibitor.">Human keratinocytes are efficiently immortalized by a Rho kinase inhibitor.</a> The Journal of Clinical Investigation. 120, 2619-2626 (2010).</li><li>Strudwick, X. L., Lang, D. L., Smith, L. E., Cowin, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+of+low+calcium+with+Y-27632+rock+inhibitor+increases+the+proliferative+capacity,+expansion+potential+and+lifespan+of+primary+human+keratinocytes+while+retaining+their+capacity+to+differentiate+into+stratified+epidermis+in+a+3D+skin+model.">Combination of low calcium with Y-27632 rock inhibitor increases the proliferative capacity, expansion potential and lifespan of primary human keratinocytes while retaining their capacity to differentiate into stratified epidermis in a 3D skin model.</a> PloS One. 10, 0123651 (2015).</li><li>Orazizadeh, M., Hashemitabar, M., Bahramzadeh, S., Dehbashi, F. N., Saremy, S. <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+the+enzymatic+and+explant+methods+for+the+culture+of+keratinocytes+isolated+from+human+foreskin.">Comparison of the enzymatic and explant methods for the culture of keratinocytes isolated from human foreskin.</a> Biomedical Reports. 3, 304-308 (2015).</li><li>Rosin, F. C. 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=Keratin+expression+in+gingival+tissue+and+primary+cultured+gingival+keratinocytes:+Are+there+differences.">Keratin expression in gingival tissue and primary cultured gingival keratinocytes: Are there differences.</a> Archives of Oral Biology. 117, 104780 (2020).</li><li>Wang, T., Kang, W., Du, L., Ge, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rho-kinase+inhibitor+Y-27632+facilitates+the+proliferation,+migration+and+pluripotency+of+human+periodontal+ligament+stem+cells.">Rho-kinase inhibitor Y-27632 facilitates the proliferation, migration and pluripotency of human periodontal ligament stem cells.</a> Journal of Cellular and Molecular Medicine. 21, 3100-3112 (2017).</li></ol>

All the authors declare no conflicts of interest.

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 epithelial cells are a kind of fully differentiated cells with few passages and a short lifespan. Culturing epithelial cells has proven to be more complex than culturing fibroblasts5.
At present, the published literature shows that various protocols exist for the isolation and culture of epithelial cells. However, two methods, the direct explant method and the enzymatic method, are the most frequently used among those protocols. In 1910, Carrel and Burrows first described a method to obtain gingival and buccal epithelial cells called the direct explant method20. The direct explant method has the advantages of low-weight requirements for tissue specimens, less complicated procedures, less variation, and less involvement in the steps, but it has the disadvantages of requiring a long culture time and is highly susceptible to contamination5. In 1975, Rheinwald and Green first reported the enzymatic method using irradiated 3T3 mouse fibroblasts as a feeder layer to culture oral epithelial cells in vitro21. Although that method greatly improved the yield of keratinocytes, the irradiated mouse feeder cell layer had potential biological risks22. After that, culture systems without feeder-cells22 and without serum23,24 were developed which proved that 3T3 cells were not necessary for epithelial cell cultures. Although the enzymatic method requires less culture time, the efficiency is relatively low and varies depending on the enzymes and medium used25. Several laboratories compared the two methods and Kedjarune et al.6 observed the direct explant technique to be more successful than the enzymatic method and found a higher rate of cell proliferation. Shwetha et al.26 concluded that the direct explant method appears to be a simple and successful technique for the isolation of oral mucosal keratinocytes. However, Klingbeil et al.7 concluded just the opposite, i.e., the enzymatic method showed the best results in less time with a good life span. A survey conducted in the UK by Daniels et al.19 reviewed the isolation and cell culture methods of human keratinocytes and reported that 21 out of 34 laboratories that responded used the enzymatic method with some variations. Although the direct explant method requires less tissue and has fewer handling steps than the enzymatic method, it has been suggested that the enzymatic method is faster and easier to manage6. Therefore, it was necessary to develop a more convenient and effective method for the isolation and culture of oral epithelial cells4. Our new method, a simplified enzymatic method that uses Y-27632 significantly increased the production of gingival epithelial cells and shortened the time required to separate gingival epithelial cells from gingival tissue.
Figure 2A shows that the new method significantly increased the production of gingival epithelial cells on the third and seventh days. Figure 2B shows that the doubling time of cells prepared using the new enzymatic method was about 1-2 days but using the direct explant method took about 5-6 days. The number of cells produced using the new enzymatic method (9 x 106) was more than three times greater than the direct explant method (3 x 106) on the seventh day (Figure 2C). Cells prepared using the new enzymatic method took 13 days to become 80% confluent, which is consistent with previous studies, while the direct explant method took about 2 weeks6, 20.25 &#177; 1.05 days18 and 14.2 &#177; 2.76 days7 for the cells to become fully confluent before subculture. However, the new enzymatic method took only 6 days to become 80% confluent, which is much shorter than the direct explant method of 13 days in our laboratory and Klingbeil et al.&#39;s7 enzymatic method of 11.9 &#177; 2.36 days. We also found an interesting phenomenon, that is, the epithelial cell colonies grew in a multi-layered structure in the direct explant method, while a uniform monolayer structure was observed using the new enzymatic method. Obviously, thicker multi-layered colonies prolong the time required to become 80%-100% confluent. The reason why the new enzymatic method promotes cell proliferation is the addition of Y-27632, an inhibitor of ROCK1 and ROCK2. Y-27632 was initially reported for its positive effects on human epithelial cell proliferation and differentiation in 200827. Chapman et al.28 reported that treatment with Y-27632 greatly increased the proliferative capacity of keratinocytes and resulted in efficient immortalization without detectable cell crisis. In our previous studies, we discovered that Y-27632 simplifies the isolation procedure of human primary epidermal cells and keratinocytes from adult skin tissue8,9,10. Strudwick et al.29 reported that the use of 10 &#181;M Y-27632 together with low calcium allowed primary human keratinocytes to be cultured without a cell feeder layer for longer periods of time while retaining the ability to differentiate and form a stratified epithelium. In this study, the new enzymatic method using Y-27632 greatly increased the harvesting of epithelial cells by extending their lifespan and promoting their proliferative capacity.
The traditional enzymatic method contains two digestion operations. The first digestion is to separate epithelial tissue from connective tissue using dispase, which works from the stromal side4,5. The second digestion usually uses trypsin to separate epithelial cells from epithelial tissue4,5. Due to the destruction of epithelial cells by trypsin, the efficiency of the enzymatic method is affected4. The new enzymatic method described here is a simplified method that omits one digestion step. The main differences between the new and traditional enzymatic methods refer to the protocols of separating epithelial tissue from connective tissue5. Additionally, the new method uses dispase and collagenase instead of trypsin in the digestion step, and hence the integrity of the epithelial cells is well protected. Clinically, it is much more difficult to obtain the same amount of tissue from human gingiva than from the skin and buccal mucosa. The direct explant method required only small pieces of gingival tissue and cultivated a larger number of cells when compared with the enzymatic method18. We treated a small amount of gingival epithelial tissue using the new method and harvested a very satisfactory cell density. This shows that the new method does not depend on relatively large amounts of tissue to be initially provided. One possible limitation of the new method is that there could appear a small amount (&#60;5%) contamination of fibroblasts at initial culture (passage 0), but it can be easily removed by a short-term treatment of 0.05% trypsin as reported previsouly9.
In this study, CK5, CK18, and Pan-CK (CK14, CK15, CK16, and CK19) were positive (Figure 3A,C,D). This suggested that the isolated gingival epithelial cells were able to maintain their basement membrane function, which is consistent with the studies of Orazizadeh et al.30 and Kedjarune et al.6. CK5 and CK14 are expressed by undifferentiated cells localized in the epithelial-basal layer. CK19 is typically found in simple epithelium and in non-keratinized stratified epithelium31. Immunofluorescence analysis of gingival epithelial cells (P3) obtained using the new enzymatic method showed that the expression levels of ki67, p63, and p75NGFR were higher than in cells obtained using the direct explant method (Figure 4). This result indicates that the new method can maintain the stem cell properties of gingival epithelial cells and promotes the proliferation potential of gingival epithelial cells. Our previous study found that Y-27632 maintains the skin epidermal stem cell potential after expansion8. Wang et al. found that Y-27632 facilitates the proliferation, migration, and pluripotency of human periodontal ligament stem cells32.
In summary, the new modified enzymatic method provides a simplified and effective procedure to isolate and culture human primary epithelial cells from adult gingival tissue. This new method is easier, less time-consuming and enhances their stem cell properties, and thus has obvious advantages compared to the direct explant method in many parameters. This advanced method is more suitable for producing large numbers of high-potential epithelial cells both for laboratory and for clinical applications.

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><tbody><tr><td>Names</td><td></td><td></td><td>Abbreviations &amp;amp; Comments</td></tr><tr><td>Countess automated cell counter</td><td>Shanghai Ruiyu Bio-science&amp;amp;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&amp;nbsp; 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></table>

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 simplified enzymatic method. Moreover, adding Y-27632 to the G-medium effectively promotes epithelial cell growth. The direct explant method usually takes about 2 weeks for the gingival epithelial cells to grow sufficiently to passage and is easily contaminated. However, the number of gingival epithelial cells obtained by the new method is significantly larger than that obtained by the direct explant method and takes only an average of 8 days to meet the requirements for passaging.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62978/62978fig01.jpg" /> 
Figure 1: Comparison of the direct explant method and the new method. (A) Scheme showing the process of the direct explant method. The gingival tissue pieces are placed in a culture dish. The epithelial cells are cultured in G-medium and grow out from the tissue pieces. The direct explant method usually takes about 13 days for the epithelial cells to reach 80% confluence. (B) Scheme showing the process of the new enzymatic method. The gingival tissue fragments are digested by dispase and collagenase I, after which the cell pellets are cultured in G-medium with Y-27632. The new enzymatic method usually takes about 6 days to reach 80% confluence which is suitable for passaging. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62978/62978fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Adult gingival epithelial cells prepared by the direct explant method and by the new method were observed in a microscope on the third and seventh days (Figure 2A). It can be seen that the new method significantly increased the production of gingival epithelial cells on the third and seventh days. The growth curves of gingival epithelial cells obtained by the new method and by the direct explant method were measured using a cell counting kit (CCK-8) at different time points (1d, 2d, 3d, 4d, 5d, 6d) (Figure 2B). The doubling time of cells prepared using the new method was about 1-2 days, but the doubling time of cells prepared using the direct explant method was about 5-6 days. The cell yields by the two methods were calculated on the seventh day (Figure 2C) and showed that the number of cells produced by the new method (9 x 106) was more than three times higher than the number of cells produced by the direct explant method (3 x 106). Figure 2D shows that it took about half the time for the new method (6 days) to achieve 80% confluence compared with the direct explant method (13 days).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62978/62978fig02.jpg" /> 
Figure 2: The new enzymatic method increases the production of primary gingival epithelial cells. (A) Images of gingival epithelial cells prepared by the direct explant method (bottom row) and the new enzymatic method (top row) on the third and seventh day after initial inoculation. Scale bars = 200 &#181;m. (B) Gingival epithelial cells cultured by the direct method and the new enzymatic method were collected on days 1, 2, 3, 4, 5, and 6, and the number of gingival epithelial cells was analyzed using a CCK-8 kit. (C) After 7 days of culture, gingival epithelial cells were detached by trypsin and were collected. A cell counting plate was used to determine the number of gingival epithelial cells prepared separately by the two methods. The number of cells was calculated from the two methods, which were repeated three times separately, and the results are expressed as the average number of cells per 100 mm dish. (D) The average time to reach 80% confluence of primary gingival epithelial cells (passage 0) prepared by the new enzymatic method and by the direct explant method is compared. B, C, and D: Student&#39;s t-test was used; the error bars show the standard deviation; n = 4; **p &#60; 0.01, *p &#60; 0.05 when comparing the new enzymatic method with the direct explant method. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62978/62978fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Immunofluorescence analysis of gingival epithelial cells (at passage 3) obtained by the new method showed that the expression of CK5, CK18, and Pan-CK (CK14, CK15, CK16, and CK19) was positive (Figure 3A,C,D)11,12,13,14,15, while the expression of CK10 and vimentin was low (Figure 3B,E)11,16. CK5 and Pan-CK (CK14, CK15, CK16, and CK19) were mainly localized in the basal layer of epithelial cells, which is a sign of their high differentiation potential11,14,15. CK10 is a marker of differentiated epithelium11, and vimentin is a marker of gingival fibroblasts16. The keratin positive rate of gingival epithelial cells isolated by the new method was higher, especially CK5, CK18, and Pan-CK (Figure 3A,C,D), which indicated that the isolated gingival epithelial cells could maintain a good basement membrane function. The low expression of the differentiation marker CK10 (Figure 3B) indicated that the gingival epithelial cells had a high differentiation potential. In vitro cultures showed that the gingival epithelial cells isolated by the new method could be cultured to the eighth generation, and the low expression of vimentin indicated that the gingival epithelial cells had a high purity, that no gingival fibroblasts contaminated them and that the isolation efficiency was high (Figure 3E).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62978/62978fig03.jpg" /> 
Figure 3: Specific markers of gingival epithelial cells (P3) prepared by the new enzymatic method. (A-E) show CK5 (red), CK10 (red), CK18 (red), Pan-CK (red), and vimentin (red) positive cells, DAPI (blue) was used to stain nuclei. (A-C) scale bars = 100 &#181;m; (D) and (E) scale bars = 50 &#181;m; the experiment was repeated four times independently. (A), (C), and (D) show the positive expression of CK5, CK18, and pan-ck (CK14, 15, 16, and 19) in gingival epithelial cells, while (B) and (E) show the low expression of CK10 and vimentin in gingival epithelial cells. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62978/62978fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The new method increased the expression of ki67, p63, and p75NGFR (low affinity nerve growth factor receptor p75) in primary gingival epithelial cells. Passage 3 cells were selected for Immunofluorescence analysis, which revealed that the positive expression of ki67, p63, and p75NGFR was higher than that of gingival epithelial cells obtained by the direct explant method (Figure 4A,C,E). The positive expression rates of ki67, p63, and p75NGFR were 80%, 98%, and 96% in the new method, respectively, which were significantly higher than those of ki67 (35%), p63 (40%), and p75NGFR (47%) obtained by the direct method (Figure 4B,D,F). Ki67, p63, and p75NGFR are markers of oral epithelial stem cells17. These results indicated that the primary gingival epithelial cells isolated and cultured using the new method contained stem cell populations, had a high proliferation potential, and maintained the stem cell properties of gingival epithelial cells.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62978/62978fig04.jpg" /> 
Figure 4: Stem cell characteristics of gingival epithelial cells (P3) obtained by the direct explant method and the new enzymatic method. (A), (C), and (E) show representative immunofluorescence images of gingival epithelial cells at passage 3 (P3), respectively, showing ki67 (red), p63 (red), and p75NGFR (red) positive cells obtained by the direct explant method and the new enzymatic method. DAPI (blue) was used to stain nuclei. Scale bars = 50 &#181;m. (B), (D), and (F) show quantitative analysis of ki67-positive cells, p63-positive cells, and p75NGFR-positive cells, respectively, in gingival epithelial cells (P3) obtained by the direct explant method and by the new enzymatic method. A total of 400 cells were counted to quantify each group, and the average numbers of ki67, p63, and p75NGFR positive cells are shown. B, D, and F: Student&#39;s t-test was used; the error bars show the standard deviation; the experiment was repeated four times independently (n = 4); **p &#60; 0.01, when comparing the new enzymatic method with the direct explant method. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62978/62978fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Isolation and Culture of Primary Human Gingival Epithelial Cells using Y-27632 at JoVE.com

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 ...

isolation-culture-primary-human-gingival-epithelial-cells-using-y

Nanyang Technological University

Research

JoVE Journal

Bioengineering

4.9K Views.  Shandong University & Shandong Key Laboratory of Oral Tissue Regeneration & Shandong Engineering Laboratory for Dental Materials and Oral Tissue Regeneration. Dus het algemene doel van dit celgebaseerde protocol is om menselijke gingivale epitheelcellen te isoleren en tegen te komen om de tekortkomingen van de directe auberginemethode te verbeteren. Van de nieuwe gemodificeerde endothemische methode kunnen we in relatief korte tijd grotere aantallen primaire gingivale epitheelcellen krijgen, op basis van onze materialen en apparatuur. Dus menselijk weefsel dat in dit protocol werd gebruikt, waren verse volwassen gingivale weefsels die werden weggeg...

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

Isolation, Processing and Analysis of Murine Gingival Cells

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique and important tissue to study immune mechanisms because it is involved in host immune response against oral biofilm that might cause periodontal diseases. Furthermore, the close proximity of the gingiva to alveolar bone tissue enables also studying bone remodeling under inflammatory conditions. Our method yields large amount of immune cells that allows analysis of even rare cell populations such as Langerhans cells and T regulatory cells as we demonstrated previously 1. Employing mice to study local immune responses involved in alveolar bone loss during periodontal diseases is advantageous because of the availability of various immunological and experimental tools. Nevertheless, due to their small size and the relatively inconvenient access to the murine gingiva, many studies avoided examination of this critical tissue. The method described in this work could facilitate gingival analysis, which hopefully will increase our understating on the oral immune system and its role during periodontal diseases.

This study describes an efficient technique to isolate and process gingival tissues from the mouse oral cavity in order to produce a single-cell culture. The resulting cells can be further used for flow cytometry analysis and molecular studies.

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique ...

Immunology and Infection

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

Developmental Biology

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

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 Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Biology

Y-27632 Enriches the Yield of Human Melanocytes from Adult Skin Tissues

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical applications. Isolating primary melanocytes from skin tissues by the conventional method usually takes about 3 to 4 weeks to passage sufficiently. More importantly, the tissues used are usually newborn foreskins and it is still a challenge to efficiently isolate primary melanocytes from adult tissues. We recently developed a new isolation method for melanocytes that adds Y-27632, a Rho kinase inhibitor, to the initial culture medium for 48 h. Compared with the conventional protocol, this new method dramatically increases the yield of melanocytes and shortens the time required to isolate melanocytes from foreskin tissues. We now describe this new method in more detail using adult epidermis to efficiently culture primary melanocytes. Importantly, we show that melanocytes obtained from adult tissues prepared by this new method can function normally. This new protocol will significantly benefit studies of pigmentation defects and melanomas using primary melanocytes prepared from easily accessed adult skin tissues.

This paper reports that the addition of Y-27632 to TIVA medium can significantly increase the yield of melanocytes from adult skin tissues.

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...

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.

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 ...

Protocol for Isolating Mammary Epithelial Cells from Human Tissue

Isolation and Culture of Primary Human Mammary Epithelial Cells

The mammary gland is a fundamental structure of the breast and plays an essential role in reproduction. Human mammary epithelial cells (HMECs), which are the origin cells of breast cancer and other breast-related inflammatory diseases, have garnered considerable attention. However, isolating and culturing primary HMECs in vitro for research purposes has been challenging due to their highly differentiated, keratinized nature and their short lifespan. Therefore, developing a simple and efficient method to isolate and culture HMECs is of great scientific value for the study of breast biology and breast-related diseases. In this study, we successfully isolated primary HMECs from small amounts of mammary tissue by digestion with a mixture of enzymes combined with an initial culture in 5% fetal bovine serum-DMEM containing the Rho-associated kinase (ROCK) inhibitor Y-27632, followed by culture expansion in serum-free keratinocyte medium. This approach selectively promotes the growth of epithelial cells, resulting in an optimized cell yield. The simplicity and convenience of this method make it suitable for both laboratory and clinical research, which should provide valuable insights into these important areas of study.

Author Spotlight: Integrating Eastern and Western Medicine for Treatment of Granulomatous Mastitis

The present study reports an easier, time-saving, and economical protocol to efficiently isolate and grow primary human mammary epithelial cells (HMECs) from small amounts of mammary tissue. This protocol is suitable for quickly producing primary HMECs both for laboratory and clinical applications.

The mammary gland is a fundamental structure of the breast and plays an essential role in reproduction. Human mammary epithelial cells (HMECs), which are ...

A Bacterial Infection Assay for Studying Anaerobic Bacteria-Host Cell Interactions

Source: Wunsch, C. M., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/53408">Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells.</a> J. Vis. Exp. (2015)
This video demonstrates a technique for assessing the ability of Porphyromonas gingivalis, an anaerobic pathogenic bacterium, to adhere to and invade human umbilical vein endothelial cells (HUVECs). After infecting the host cells with the bacteria, the host cells are lysed to release both adhered and internalized bacteria. When the lysate is plated in an appropriate medium, the presence of colonies indicates successful bacteria-host cell interactions.

The following protocols will describe methods for culturing and studying the invasion by the anaerobic species, P. gingivalis; however, these protocols ...

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

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