This work was supported by The National Key Research and Development Program of China (2017YFA0104604), The General Program of National Natural Science Foundation of China (81772093), Military Logistics Research Project (AWS17J005), the Key Program of Shandong Province Natural Science Foundation (ZR2019ZD36) and The Key Research and Development Program of Shandong Province (2019GSF108107) to X.W. Guangdong Basic and Applied Basic Research Foundation (2019A1515110833) and The Fundamental Research Funds of Shandong University (2019GN043) to J.M.

The use of adult foreskin tissues in this protocol has been approved by the Human Research Ethics Committee (No.2015120401, date: May 12, 2015).
NOTE: Perform all the following procedures in a sterile environment to prevent contaminations of cells and cultures.
1. Preparations
<ol>
	<li>Gather fresh adult foreskin tissues from circumcision surgeries in 15 mL tubes containing 10 mL of phosphate buffer saline (PBS) and store in 4 &#176;C. 
	NOTE: Separate the t...

<ol>
	<li>Costin, G. E., Hearing, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+skin+pigmentation:+melanocytes+modulate+skin+color+in+response+to+stress.">Human skin pigmentation: melanocytes modulate skin color in response to stress.</a> FASEB Journal. 21 (4), 976-994 (2007).</li><li>Battyani, Z., Xerri, L., Hassoun, J., Bonerandi, J. J., Grob, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tyrosinase+gene+expression+in+human+tissues.">Tyrosinase gene expression in human tissues.</a> Pigment Cell Research. 6 (6), 400-405 (1993).</li><li>Michaloglou, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BRAFE600-associated+senescence-like+cell+cycle+arrest+of+human+naevi.">BRAFE600-associated senescence-like cell cycle arrest of human naevi.</a> Nature. 436 (7051), 720-724 (2005).</li><li>Hu, F., Staricco, R. J., Pinkus, H., Fosnaugh, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+melanocytes+in+tissue+culture.">Human melanocytes in tissue culture.</a> Journal of Investigative Dermatology. 28 (1), 15-32 (1957).</li><li>Eisinger, M., Marko, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+proliferation+of+normal+human+melanocytes+in+vitro+in+the+presence+of+phorbol+ester+and+cholera+toxin.">Selective proliferation of normal human melanocytes in vitro in the presence of phorbol ester and cholera toxin.</a> Proceedings of the National Academy of Sciences of the United States of America. 79 (6), 2018-2022 (1982).</li><li>Hirobe, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+keratinocyte-derived+factors+involved+in+regulating+the+proliferation+and+differentiation+of+mammalian+epidermal+melanocytes.">Role of keratinocyte-derived factors involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes.</a> Pigment Cell Research. 18 (1), 2-12 (2005).</li><li>Halaban, 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=Basic+fibroblast+growth+factor+from+human+keratinocytes+is+a+natural+mitogen+for+melanocytes.">Basic fibroblast growth factor from human keratinocytes is a natural mitogen for melanocytes.</a> Journal of Cell Biology. 107 (4), 1611-1619 (1988).</li><li>Kunisada, T., 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=Keratinocyte+expression+of+transgenic+hepatocyte+growth+factor+affects+melanocyte+development,+leading+to+dermal+melanocytosis.">Keratinocyte expression of transgenic hepatocyte growth factor affects melanocyte development, leading to dermal melanocytosis.</a> Mechanisms of Development. 94 (1-2), 67-78 (2000).</li><li>Hirobe, T., Shinpo, T., Higuchi, K., Sano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+cycle+of+human+melanocytes+is+regulated+by+endothelin-1+and+stem+cell+factor+in+synergy+with+cyclic+AMP+and+basic+fibroblast+growth+factor.">Life cycle of human melanocytes is regulated by endothelin-1 and stem cell factor in synergy with cyclic AMP and basic fibroblast growth factor.</a> Journal of Dermatological Science. 57 (2), 123-131 (2010).</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 (2), 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. (138), e7862 (2018).</li><li>Terunuma, A., Limgala, R. P., Park, C. J., Choudhary, I., Vogel, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+procurement+of+epithelial+stem+cells+from+human+tissue+specimens+using+a+Rho-associated+protein+kinase+inhibitor+Y-27632.">Efficient procurement of epithelial stem cells from human tissue specimens using a Rho-associated protein kinase inhibitor Y-27632.</a> Tissue Engineering Part A. 16 (4), 1363-1368 (2010).</li><li>Cerqueira, M. T., Frias, A. M., Reis, R. L., Marques, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interfollicular+epidermal+stem+cells:+boosting+and+rescuing+from+adult+skin.">Interfollicular epidermal stem cells: boosting and rescuing from adult skin.</a> Methods in Molecular Biology. 989, 1-9 (2013).</li><li>Zhou, Q., 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=ROCK+inhibitor+Y-27632+increases+the+cloning+efficiency+of+limbal+stem/progenitor+cells+by+improving+their+adherence+and+ROS-scavenging+capacity.">ROCK inhibitor Y-27632 increases the cloning efficiency of limbal stem/progenitor cells by improving their adherence and ROS-scavenging capacity.</a> Tissue Engineering Part C, Methods. 19 (7), 531-537 (2013).</li><li>Yokoyama, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacologic+suppression+of+MITF+expression+via+HDAC+inhibitors+in+the+melanocyte+lineage.">Pharmacologic suppression of MITF expression via HDAC inhibitors in the melanocyte lineage.</a> Pigment Cell &amp; Melanoma Rresearch. 21 (4), 457-463 (2008).</li><li>Mi, J., 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+ROCK+inhibitor+promotes+keratinocyte+survival+and+paracrine+secretion,+enhancing+establishment+of+primary+human+melanocytes+and+melanocyte-keratinocyte+co-cultures.">A ROCK inhibitor promotes keratinocyte survival and paracrine secretion, enhancing establishment of primary human melanocytes and melanocyte-keratinocyte co-cultures.</a> Pigment Cell &amp; Melanoma Research. 33 (1), 16-19 (2020).</li></ol>

The protocol described here was based on a recent publication16. Some attention should be paid to the following critical steps to achieve the best results with the new method. First, successful separation of the epidermis and the dermis is crucial. Cut the adult foreskin tissues into 3-4 mm wide strips using a scalpel blade to make the dispase work more thoroughly and easily to separate the epidermis from the dermis. Second, when separating those two layers, contamination of the dermis should be a...

The goal of this study was to develop a simple and effective protocol to isolate melanocytes from the epidermis of adult skin for biological research and clinical applications. Melanocytes, which are located in the basal epidermis of the skin and in hair follicles, play an important role in pigmentation of the skin and hair by producing melanin1. The resulting skin pigmentation from epidermal melanocytes acts as an ultraviolet radiation &#64257;lter that reduces/prevents DNA damage to underlying cells in the skin2. The abnormal proliferation of melanocytes in the skin is quite common, such as in the formation of benign nevi (moles) in which melanocytes potentially transform to oncogenic growth followed by cellular senescence3.
Since 1957, the isolation and subsequent culture of human primary melanocytes has been possible4, but only since 1982 has there been an efficient method that can reproducibly establish cultures of human melanocytes from the epidermis5. The conventional method to isolate primary melanocytes from the epidermis involves a two-step enzymatic digestion. Briefly, the skin is initially digested with dispase to separate the epidermis from the dermis, after which the epidermis is digested with trypsin to produce suspensions containing melanocytes and keratinocytes, which can then be selectively grown in different media. Currently, the initial culture usually takes about 3 to 4 weeks for melanocytes to reach confluency using the conventional method, which is likely due to the low efficiency of their isolation. Therefore, increasing the initial production of melanocytes from adult skin tissues would be quite helpful for both laboratory research and clinical applications.
Many growth factors, most of which have been shown to be secreted by keratinocytes (e.g., &#945;-MSH, ACTH, bFGF, NGF, ET-1, GM-CSF, HGF, LIF and SCF)5-8, regulate the differentiation and proliferation of mammalian melanocytes. In the absence of feeder layers, the lack of those growth factors leads to the decreased proliferation of melanocytes and their increased apoptosis9. The co-culture of keratinocytes as feeder cells and melanocytes could lead to accelerated melanocyte proliferation and reduced apoptosis. However, the co-culture method not only demands more skin tissues to prepare keratinocytes, which is not practical, but also could not work efficiently because the culture conditions required by melanocytes does not favor keratinocyte growth, and vice-versa.
Previous studies have reported that the addition of Y-27632, a ROCK inhibitor, into the growth medium can enhance the yield of human primary epidermal cells from skin tissues10-14. Therefore, it would be interesting to test whether the isolation of human primary melanocytes would benefit from the presence of Y-27632. Indeed, the addition of Y-27632 into the inoculation TIVA medium15, which contains TPA, IBMX, Na3VO4 and dbcAMP, significantly increased the yield of primary melanocytes isolated from foreskin tissues in the initial cultures16. Compared to the conventional protocol, this new protocol is significantly more efficient in increasing the yield of melanocytes16. Therefore, this protocol describes the new method in detail using adult skin tissues based on a previous study16 in order to promote its application in basic and applied biological research.

<table>
	<tbody>
		<tr>
			<td>Alexa fluor-594 donkey anti-mouse IgG</td>
			<td>Thermo Fisher Scientific</td>
			<td>A21203</td>
			<td>For Immunofluorescence</td>
		</tr>
		<tr>
			<td>Alexa fluor-594 donkey anti-rabbit IgG</td>
			<td>Thermo Fisher Scientific</td>
			<td>R37119</td>
			<td>For Immunofluorescence</td>
		</tr>
		<tr>
			<td>Cell Culture Dish</td>
			<td>Eppendorf</td>
			<td>30702115</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>Cell Strainer</td>
			<td>Corning incorporated</td>
			<td>431792</td>
			<td>Cell filtration</td>
		</tr>
		<tr>
			<td>15 mL Centrifuge Tube</td>
			<td>KIRGEN</td>
			<td>171003</td>
			<td>For cell centrifuge</td>
		</tr>
		<tr>
			<td>50 mL Centrifuge Tube</td>
			<td>KIRGEN</td>
			<td>171003</td>
			<td>For cell centrifuge</td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Thermo Scientific</td>
			<td>51026333</td>
			<td>For cell incubating</td>
		</tr>
		<tr>
			<td>Constant Temperature Shaker</td>
			<td>Shanghai Boxun</td>
			<td>150036</td>
			<td>For water bath</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Abcam</td>
			<td>ab104139</td>
			<td>For Immunofluorescence</td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Gibco</td>
			<td>17105-041</td>
			<td>For melanocyte isolation</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Scientific</td>
			<td>C11995500</td>
			<td>Component of neutralization medium</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>Fluorescence microscope</td>
			<td>Olympus</td>
			<td>5E44316</td>
			<td>For Immunofluorescence</td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>MCE</td>
			<td>HY-15371</td>
			<td>Induce pigmentation</td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Thermo Fisher Scientific</td>
			<td>25030081</td>
			<td>melanocyte culture medium</td>
		</tr>
		<tr>
			<td>Ham&#39;s F12</td>
			<td>Thermo Scientific</td>
			<td>11330032</td>
			<td>melanocyte culture medium</td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Olympus</td>
			<td>5C42258</td>
			<td>For cell microscopic observation</td>
		</tr>
		<tr>
			<td>3-isobutyl-1-methyl xanthine (IBMX)</td>
			<td>Sigma</td>
			<td>17018</td>
			<td>melanocyte culture medium</td>
		</tr>
		<tr>
			<td>mouse anti-human MITF</td>
			<td>Abcam</td>
			<td>ab12039</td>
			<td>For Immunofluorescence</td>
		</tr>
		<tr>
			<td>Na3VO4</td>
			<td>Sigma</td>
			<td>S6508</td>
			<td>melanocytes culture medium</td>
		</tr>
		<tr>
			<td>N6,2&#39;-O-dibutyryladeno-sine 3&#39;,5&#39;-cyclic monophosphate (dbcAMP)</td>
			<td>Sigma</td>
			<td>D0627</td>
			<td>melanocyte culture medium</td>
		</tr>
		<tr>
			<td>12-O-tetradecanoyl phorbol-13-acetate (TPA&#65289;</td>
			<td>Sigma</td>
			<td>79346</td>
			<td>melanocyte culture medium</td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Thermo Scientific</td>
			<td>15140-122</td>
			<td>Antibiotics</td>
		</tr>
		<tr>
			<td>rabbit anti-human Ki-67</td>
			<td>Abcam</td>
			<td>ab15580</td>
			<td>For Immunofluorescence</td>
		</tr>
		<tr>
			<td>Phosphate buffered solution</td>
			<td>Solarbio Life Science</td>
			<td>P1020-500</td>
			<td>Washing solution</td>
		</tr>
		<tr>
			<td>Sorvall ST 16R Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75004380</td>
			<td>Cell centrifugation</td>
		</tr>
		<tr>
			<td>TC20TM automated cell counter</td>
			<td>Bio-Rad</td>
			<td>1450102</td>
			<td>Automatic cell counting</td>
		</tr>
		<tr>
			<td>0.05% Trypsin</td>
			<td>Life Technologies</td>
			<td>25300-062</td>
			<td>For melanocyte dissociation</td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Sigma</td>
			<td>Y0503</td>
			<td>For melanocyte isolation</td>
		</tr>
	</tbody>
</table>

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.

Figure 1 shows a schematic diagram comparing the conventional and the new methods. The procedure for tissue preparation and digestion with the new method is the same as the procedure for the conventional method, the only difference being that the isolated cells are resuspended in 10 mL of TIVA medium in the presence of 10 &#181;M Y-27632. Two days after seeding, replace the media with normal TIVA medium without Y-27632. The conventional method of isolating primary melanocytes from skin tissu...

Watch this Scientific Journal Video about Y-27632 Enriches the Yield of Human Melanocytes from Adult Skin Tissues at JoVE.com

Y-27632 Enriches the Yield of Human Melanocytes from 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 ...

The general goal of the cells based protocol is to isolate and culture human melanocytes cells, improving the shortcomings of conventional methods. From the new method we can get larger amounts of primary melanocytes cells in a relatively short time, based on materials and equipments. Human tissues used in this protocol are fresh adult for skin tissues discarded from circumcision surgery at the hospital and handled according to the guidelines of the Institution's Human Research Ethics Committee number 2015120401, date May 12 2015.This is a display of all required reagents. Prepare sterile instruments and materials in advance and set the tissue into a dish, wash the tissue with 75%ethanol for 30 seconds and with washes solution ties five minutes each cuts the tissue into convenient pieces and scrap the tissue with surgical blade to remove the fat layer. Flatten the tissues with the dermis side down add 2.5 milligram per milliliter the space solution to the skin tissues.Cover and store the pieces at four degrees centigrade overnight. On the second day, peel off the epidermis from the dermis and immediately transfer the epidermis into another three dishes with washing solution in turn submerges 0.05%trypsin in the tube incubated for 30 minutes in a water bath at 37 degrees centigrade. Add an equal volume of the neutralization solution.Pipettes the solution up and down for 10 to 15 times. Pass the solution through a 100 micrometer mash filter. centrifuge at 200 times g for five minutes remove the supernatant re-suspend the cell pallet in 10 milliliter of tiva medium.Add 10 micrometer of Y-27632. Mark the information this is the field of view under the microscope. It shows cells floating in the medium incubate in a 37 degrees centigrade incubator.This view shows the view on day two the cells have started to attach the bottom of the dish. And the view on day nine shows obvious deposits of melanocytes exist. Remove the supernatant wash the cells with PBS add two millimeter of 0.05%trypsin incubate the cells for two minutes add two milliliter of the neutralization solution.centrifuge at 200 times g for five minutes. Remove the supernatant slowly re-suspend the cell pallet with 10 millimeter of tiva medium. Change the medium every two days.These pictures show the views of passage one schematic diagram so the new method and the conventional method are presented in this figure. The procedure for tissue preparation and the digestion of new method is the same as the conventional method. The only difference is that the isolated fat cells inoculated in 10 minute in her tiva medium with the presence of 10 micrometer Y-27632.After two days replace the media with tiva medium without supplements at Y-27632. This panel shows representative images of melanocytes prepared by the conventional method top row and by the new method bottom row at day three, day six and day eight after the initial inoculation we call the number of melanocytes at day three, day six and eight, we found that the new method increased the melanocytes yield by about five times. After eight days counter we did dozen and count the number of melanocytes in each single dish.The results showed that the number of melanocytes isolated by new method is much more than the conventional method. Ki67 standing confirm that melanocytes isolated by new method can normally proliferate more importantly we obtained normal function of melanocytes with the new method. We checked the purity of melanocytes after initial passage from the isolation.The passage one melanocytes for stand for MITF expression which expressed only in melanocytes. And we found nearly 100%cells were positive for MITF indicating pure melanocytes obtained after first passage. To test melanocytes isolated from the new methods can function normally.We treated melanocytes with force cooling for over 90 in vitro and observe that force cooling can induce the pigmentation of melanocytes. Taking these data together suggests that the melanocytes isolated from the new method can normally produce pigmentation. Primary melanocytes, as stated above skin and extended in culture have been readily used for laboratory research and the clinical applications.Having reported that ID Y-27632 into the initial culture medium before two days can dramatically increases the out of melanocytes. In summary, we successfully established a new simple and highly efficient method to isolate pure primary melanocytes from adult tissues.

y-27632-enriches-yield-human-melanocytes-from-adult-skin

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JoVE Journal

Biology

5.4K visualizações.  Shandong University & Shandong Key Laboratory of Oral Tissue Regeneration & Shandong Engineering Laboratory for Dental Materials and Oral Tissue Regeneration. O objetivo geral do protocolo baseado em células é isolar e cultivar células de melanócitos humanos, melhorando as deficiências dos métodos convencionais. A partir do novo método podemos obter maiores quantidades de células melanócitos primárias em um tempo relativamente curto, com base em materiais e equipamentos. Os tecidos humanos utilizados neste protocolo são adultos frescos para tecidos de pele descartados da cirurgia de circuncisão no hospital e manuseados de acordo com as ...