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 (NSFC, 81772093), the Science and Technology Development Program of Suzhou (ZXL2015128), the Natural Science Foundation of Jiangsu Province (BK20161241), and a Shandong Taishan Scholar Award (tshw201502065).

Human tissues used in this protocol have been handled according to the guidelines of the Institution&#39;s human research ethics committee (NO.2015120401, date: May 12, 2015).
1. Preparations
<ol>
	<li>Collect fresh adult abdominal skin tissues discarded from plastic surgery at the hospital in a 50-mL tube with 10 mL of ice-cold Dulbecco&#39;s modified Eagle medium (DMEM). The specimen can be kept at 4 &#176;C for up to 72 h without significantly affecting the cell viability.</li>
	<li>Prepare the reagents and culture medium as described below.
	<ol>
		<li>Add 1 mL of penicillin (100 U/mL) and streptomycin (100 mg/L) to 50 mL of phosphate-buffered solution (PBS) to prepare the washing solution.</li>
		<li>Prepare two sets of enzyme digestion solutions: (1) prepare 50 mL of dispase (2.5 mg/mL in DMEM) and 50 mL of type I collagenase (2.5 mg/mL in DMEM) separately for the conventional method; and (2) prepare 50 mL of a mixture of dispase and type I collagenase (dissolve 125 mg of dispase powder and 125 mg of type I collagenase powder in 50 mL of DMEM) for the new method. 
		NOTE: All enzyme solutions should be filtered with a 0.22-&#181;m strainer and kept at4 &#176;C.</li>
		<li>Prepare digestion solutions for both methods: 0.05% trypsin and 0.25% trypsin were commercially purchased. Prepare a 10 mg/mL DNase I solution by dissolving 50 mg of DNase I powder in 5 mL of PBS, filter it with a 0.22-&#181;m strainer, and store it at -20 &#176;C.</li>
		<li>Prepare 500 mL of medium to neutralize the enzymatic digestion: DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin.</li>
		<li>Prepare 500 mL of inoculation medium (growth-factor-containing medium, called G-medium): DMEM/F12 (3:1) medium containing 1% penicillin/streptomycin, 40 &#181;g/mL fungizone, 40 ng/mL fibroblast growth factor 2 (FGF2), 20 ng/mL epidermal growth factor (EGF), and 2% B27 supplement.</li>
		<li>Pretreat 100-mm cell culture dishes with 2 mL of coating matrix containing type I collagen for 30 min at room temperature.</li>
	</ol>
	</li>
</ol>
2. Conventional Method
<ol>
	<li>Skin tissue pretreatment
	<ol>
		<li>Take 1.5 cm2 of skin tissue and wash it 1x with 10 mL of PBS; then, rinse it with 10 mL of 70% ethanol for 30 s and incubate it 2x with 10 mL of washing solution (PBS containing 2% penicillin and streptomycin), 5 min each. 
		NOTE: All washes are performed in 100-mm cell culture dishes inside a laminar flow hood.</li>
		<li>Trim the tissue with sterile scissors to remove the subcutaneous fat layer in a 100-mm cell culture dish and, then, weigh the skin tissue (the weight is 1 g in this protocol). 
		NOTE: Sufficient trimming is very crucial for the efficient separation of the epidermis from the dermis. The yellowish fat layer can be easily distinguished visually.</li>
		<li>Transfer the tissue to another 100-mm sterile dish with the dermis side down and, then, cut the skin tissue into 3- to 4-mm-wide strips using a scalpel blade. 
		NOTE: The dermis side with the fat layer is easily recognized visually.</li>
		<li>Add 10 mL of 2.5 mg/mL dispase solution to the skin tissues in a 100-mm culture dish and, then, incubate the dish overnight at 4 &#176;C (no more than 20 h). 
		NOTE: The amount of dispase solution can be calculated by using 10 mL of dispase solution for the digestion of each gram of tissue.</li>
	</ol>
	</li>
	<li>Separation of the epidermis from the skin tissue
	<ol>
		<li>On the second day, peel off the epidermis from the dermis using fine tweezers. 
		NOTE: If it is difficult to peel off the epidermis, the dispase digestion was not sufficient, which is usually due to insufficient trimming of the tissue. In this case, the tissue needs a longer incubation time.</li>
		<li>Mince the peeled epidermis with 500 &#181;L of cell culture medium into a tissue slurry using scalpel blades; then, resuspend it with 10 mL of 0.25% trypsin in a 50-mL tube and incubate it 20 min in a water bath at 37 &#176;C, with shaking.</li>
	</ol>
	</li>
	<li>Collection and culturing of primary cells
	<ol>
		<li>Neutralize the trypsin activity by adding an equal volume of neutralization solution (10 mL in this protocol).</li>
		<li>Dissociate the epidermal cells by pipetting the solution up and down 20x with a 10-mL serological pipette and pass the cell solution through a 100-&#181;m cell strainer to remove any residual tissue debris.</li>
		<li>Centrifuge the cell solution at 200 x g for 5 min after filtration; resuspend the cell pellet in neutralization medium for washing and, then, centrifuge it again at 200 x g to obtain the cell pellet.</li>
		<li>Resuspend the cell pellet in 10 mL of low-calcium, serum-free keratinocyte medium (SFM). Take 10 &#181;L of this solution to count the total cell number and, then, plate about 2 x 106 cells with 10 mL of SFM into 100-mm cell culture dishes pretreated with coating matrix (containing type I collagen). 
		NOTE: Coating is a necessary step for the conventional method.</li>
		<li>Change the culture medium every 2 d. 
		NOTE: Check the cell adhesion under a microscope before and after changing the culture medium.</li>
		<li>Passage the cells when they reach about 80% confluence. 
		NOTE: All culture dishes are pretreated with the coating matrix.</li>
	</ol>
	</li>
</ol>
3. The New Method
<ol>
	<li>Skin tissue pretreatment
	<ol>
		<li>Collect the tissue as described above (step 2.1) for the conventional method.</li>
		<li>Wash the skin tissue 1x with 10 mL of PBS and, then, weigh it in a sterile plastic container by an electronic scale (the weight is around 1 g in this protocol). 
		NOTE: The minimum weight of tissue needed for this protocol is 0.1 g, since it is difficult to obtain a sufficient number of cells if the tissue specimen used is too small. There is no maximum weight of tissue for this protocol, which ultimately depends on the handling capacity of the operator.</li>
		<li>Use forceps to rinse the skin tissue with 10 mL of 70% ethanol for 30 s.</li>
		<li>Incubate the tissue 2x with 10 mL of washing solution (prepared in step 2.1.1), for 5 min each time. 
		NOTE: All the washing steps noted above are performed in 100-mm cell culture dishes inside a laminar flow hood (a tissue culture hood).</li>
		<li>Transfer the tissue to another 100-mm cell culture sterile dish.</li>
		<li>Using scalpel blades, mince the tissue thoroughly into a tissue slurry.</li>
		<li>Add 200 &#181;L of PBS every 5 min to keep the tissues wet. 
		NOTE: Take around 15 min for steps 3.1.5 - 3.1.7. All the above steps are performed at room temperature.</li>
	</ol>
	</li>
	<li>Digestion of skin tissue
	<ol>
		<li>Transfer the homogenized tissue solution into a 50-mL tube. Add 10 mL of enzyme mixture for every 1 g of skin tissue. Mix the enzymes thoroughly with the homogenized tissues.</li>
		<li>Incubate the mixture with shaking in a water bath at 37 &#176;C for 1 h.</li>
		<li>Add a 1/5 volume of 0.25% trypsin (2.5 mL in this protocol) to the digestion mixture for another 30 min in a water bath at 37 &#176;C.</li>
		<li>Add DNase I solution to the enzyme mixture at a 1:100 (v/v) ratio (250 &#181;L in this protocol) and incubate it for 5 min at room temperature.</li>
	</ol>
	</li>
	<li>Collection and culturing of primary cells
	<ol>
		<li>Stop the digestion process by adding 12.5 mL of neutralization medium in a 1:1 ratio. Pipette the solution up and down for about 20x, using a 10-mL serological pipette to dissociate the cells.</li>
		<li>Filter the dissociated cells through a 100-&#181;m cell strainer to remove the tissue debris. Centrifuge the supernatant at 200 x g for 5 min. Observe the pellet at the bottom of the tube.</li>
		<li>Discard the supernatant and wash the cell pellet with 10 mL of neutralization medium, and centrifuge again at 200 x g for 5 min to collect the cells.</li>
		<li>Resuspend the cell pellet in 10 mL of inoculation medium (G-medium from step 1.2.5) with 10 &#181;M of Y-27632.</li>
		<li>Take 10 &#181;L of the solution to count the total cell number and, then, plate about 2 x 106 cells with 10 mL of G-medium into a 100-mm cell culture dish. 
		NOTE: The precoating step is not necessary for the new method, and no removal of the dermal layer is required either.</li>
		<li>After 3 d, replace the inoculation medium with low-calcium SFM medium. Change the culture medium every 2 d. 
		NOTE: Check the cell adhesion before and after changing the medium.</li>
		<li>Passage the cells when they reach about 80% confluence. 
		NOTE: If necessary, pretreat the cells with 0.05% trypsin for 2 min and wash the cells with PBS to remove contaminating dermal cells before trypsinizing the epidermal cells.</li>
	</ol>
	</li>
</ol>
4. Cell Passaging
<ol>
	<li>Remove the medium, wash the cells with PBS, and, then, add 2 mL of 0.05% trypsin (per each 100-mm dish).</li>
	<li>Incubate the cells in an incubator (37 &#176;C with 5% CO2) for 5 min.</li>
	<li>Check the cells using a microscope to make sure that all cells have detached from the culture dish.</li>
	<li>Add 8 mL of DMEM with 10% FBS to neutralize the enzymatic reaction and, then, collect the cells in a 15-mL tube.</li>
	<li>Centrifuge at 200 x g for 5 min to obtain the cell pellet.</li>
	<li>Remove the supernatant slowly and, then, resuspend the cell pellet with 10 mL of SFM medium and count the number of cells.</li>
	<li>Plate 1 x 106 cells with 10 mL of SFM into each 100-mm cell dish.</li>
	<li>Change the medium every 2 d.</li>
</ol>

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The authors have nothing to disclose.

Cultured primary HKCs have been widely utilized to treat wounds in clinics for more than three decades and, since that time, it has been always important to efficiently obtain sufficient numbers of cells for clinical applications in a timely manner. Therefore, in practice, the conventional isolation method, which requires the separation of the epidermis from the dermis, makes it difficult to meet these demands, due to the low yield of cells and the low ability to passage adult cells. Here, we describe a new simple method we developed to efficiently obtain HKCs from adult tissue based on previous reports13,14, which is more suitable both for laboratory and for clinical applications.
Attention must be paid to the following critical steps to achieve the best results for the new method. First, the homogenization procedure is a crucial step; insufficient homogenization will clearly affect the efficiency of enzymatic digestion and result in a low yield of dissociated cells. Second, the trypsin digestion step should not take more than 30 min; otherwise, the viability of the dissociated cells will decrease. Third, to efficiently collect the dissociated cells, the pore size of the cell strainer used for filtration should be 100 &#181;m, and a single strainer should be used to filter no more than 20 mL of the cell solution. Fourth, the density of the initial plating is an important factor to minimize contamination with dermal cells, because a high cell density will significantly reduce the inhibitory effect of Y-27632 on dermal cells. Therefore, it is crucial to accurately count the total number of dissociated cells at the end of the isolation procedure before plating.
First, the new method simplifies the isolation procedure: the two-step conventional isolation procedure usually takes 2 days to perform, and the trimming procedure is crucial to completely remove the fat tissue from the skin, especially for adult tissues (Figure 1); otherwise, there will be an inefficient separation of the epidermis from the dermis after overnight digestion with dispase. By taking advantage of Y-27632, which inhibits the growth of dermal cells after inoculation13, the new method does not need to separate the epidermis from the dermis and, therefore, the trimming procedure to remove the fat tissue is not necessary for the new method, which only needs half a day to be performed. Second, the new method also simplifies the culture procedure since, with the traditional method, the dissociated cells are usually cultured on a feeder layer of irradiated mouse fibroblasts in medium containing 10% FBS or in a collagen-precoated dish. Animal-derived components are always risk factors that should be avoided for clinical applications. We developed a serum-free conditioned inoculation medium (G-medium), which does not require a precoating step for culture dishes and also enhances the yield of primary keratinocytes13 because G-medium combined with Y-27632 promotes the attachment of epidermal cells to the culture dish13,17,18. Therefore, the new method facilitates the culture procedure and is also a relatively safe procedure for clinical applications without the addition of animal-derived components. Third, the new method improves the yield of primary epidermal cells and shortens the initial culture time needed to reach confluence. Compared with the traditional method, the yield of cells produced by the new method is around 3 times higher, and the initial passage of epidermal cells reached confluence at least 3 days earlier (Figure 2). Finally, the new method can be utilized for the isolation of epidermal cells from all types of skin specimens, such as hairy scalp skin and adult skin tissues with a thick fat layer, which have been tested in a previous report13.
High-potential primary HKCs have been widely used for laboratory research and for clinical applications. This new method enhances the potential of cultured cells with characteristics of epidermal stem cells13. Y-27632 enhances the &#945;6-integrin expression by HKCs, and after three passages, more than 90% of the cultured epidermal cells expressed the basal cell marker keratin 5, and fewer than 10% of the cells expressed the differentiation marker Loricrin, indicating that these cells keep the characteristics of skin basal cells after several passages. The culture-expanded epidermal cells can reconstitute skin in vivo after grafting13, suggesting that they possess regeneration potential after expansion in culture. Notably, the new method is ideal to isolate cells from adult tissues, as mentioned above. Therefore, epidermal cells prepared by this method can be used for in vitro and in vivo models to study skin diseases and can be developed into autologous cell-based products for clinical applications such as the treatment of skin wounds.
In summary, the new protocol provides a simplified and effective procedure to isolate and expand primary epidermal cells from adult skin tissue. This advanced method is more suitable for producing large numbers of high-potential epidermal cells both for laboratory and for clinical applications.

The goal was to develop a simple and efficient protocol to isolate primary human keratinocytes (HKCs) from adult tissues, especially for clinical applications. Skin epidermal stem cells, localized in the basal layer of the skin, possess a high potential to proliferate and differentiate and provide keratinocytes to maintain the functions of the skin1,2,3,4. HKCs isolated from skin tissues are widely used for skin tissue engineering and regeneration purposes, especially in the repair of damaged skin and in gene therapy for clinical applications5,6. The key issue for HKC-based applications is to efficiently isolate and expand large numbers of HKCs with high potential in vitro7,8. Although various research groups have developed methods to produce cultures of stem-like HKCs, these methods are sometimes time-consuming and complicated to perform and have other limitations, such as low cell yields and being limited by the type of skin specimen used9. For instance, the traditional method to isolate HKCs from skin tissues involves a two-step enzymatic digestion with a separation of the epidermis from the dermis6. That method usually works well for neonatal tissues, but it becomes very difficult when used to isolate cells from adult tissues.
Y-27632, an inhibitor of Rho-associated protein kinase (ROCK), has been reported to significantly enhance the efficiency of epidermal stem cell isolation and colony growth10,11,12. In a previous study, we discovered that Y-27632 facilitates the clonal growth of epidermal cells but reduces the yield of dermal cells by differentially controlling the expression of adhesion molecules13. We also established a new conditioned inoculation medium, called G-medium, which supports the growth and yield of primary epidermal cells. By combining G-medium with Y-27632, this novel method can spontaneously separate epidermal and dermal cells after enzyme digestion, thus removing the step of epidermis-dermis separation13,14. Based on previous reports, we now describe the detailed procedure of this new method to isolate HKCs from adult skin tissue.

<table><tbody><tr><td>Countess automated cell counter</td><td>Invitrogen Inc.&amp;nbsp;</td><td>C10227</td><td>Automatic cell counting&amp;nbsp;</td></tr><tr><td>CO2 Incubator</td><td>Thermo Scientific</td><td>51026333</td><td>For cell incubating</td></tr><tr><td>Sorvall ST 16R Centrifuge</td><td>Thermo Scientific</td><td>75004380</td><td>Cell centrifuge</td></tr><tr><td>Constant Temperature Shaker</td><td>Shanghai Boxun</td><td>150036</td><td>For water bath</td></tr><tr><td>Electronic Scale</td><td>Harbin Zhonghui</td><td>1171193</td><td>For tissue weighing</td></tr><tr><td>Cell Culture Dish</td><td>Eppendorf</td><td>30702115</td><td>For cell culture</td></tr><tr><td>50ml Centrifuge Tube</td><td>KIRGEN</td><td>171003</td><td>For cell centrifuge</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&amp;nbsp;</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>Epidermal 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 HKC dissociation</td></tr><tr><td>0.25% Trypsin&amp;nbsp;</td><td>Beijing Solarbio Science &amp;amp; Technology</td><td>T1350-100</td><td>For HKC dissociation</td></tr><tr><td>Coating Matrix Kit</td><td>Life Technologies</td><td>R-011-K</td><td>For coating matrix</td></tr><tr><td>Dispase</td><td>Gibco</td><td>17105-041</td><td>For HKC isolation</td></tr><tr><td>Collagenase Type I</td><td>Life Technologies</td><td>17100-017</td><td>For HKC isolation</td></tr><tr><td>Deoxyribonuclease I</td><td>Sigma</td><td>9003-98-9</td><td>For HKC 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>Sigma-Aldrich</td><td>Y0503</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>Thermo Scientific</td><td>NC9864731</td><td>cell proliferation and cytotoxicity assays</td></tr><tr><td>Mouse Anti-Human Cytokeratin5</td><td>Hewlett-Packard Development Company</td><td>MA-20142</td><td>For immunofluorescence staining to check differentiation marker of HKCs</td></tr><tr><td>Rabbit Anti-Human Loricrin</td><td>Covance</td><td>PRB-145p</td><td>For immunofluorescence staining to check differentiation marker of HKCs</td></tr><tr><td>Mouse anti-human Vimentin</td><td>Cell Signaling Technology</td><td>3390</td><td>For immunofluorescence staining of dermal fibroblasts</td></tr><tr><td>Integrin &amp;alpha;6(GOH3)</td><td>Santa Cruz&amp;nbsp;</td><td>SC-19622</td><td>flow cytometry analysis of HKCs</td></tr><tr><td>Rat IgG2a FITC</td><td>Santa Cruz&amp;nbsp;</td><td>SC-2831</td><td>negative control antibody of &amp;alpha;6-integrin&amp;nbsp; in flow cytometry analysis&amp;nbsp;</td></tr></tbody></table>

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.

Schematic diagrams of the new method (Figure 1A) and the conventional method (Figure 1B) are presented in Figure 1. The conventional method is a two-step digestion, which requires a 2-day procedure. By contrast, the new method is a one-step digestion, which takes around 3 hours to perform. Importantly, the one-step new method can obtain two populations (epidermal and dermal cells) at the same time, which depends on the presence of Y-27632 in the inoculation medium. Moreover, the initial culture time of epidermal cells to reach around 80% confluence usually takes at least 3 days less than the conventional method if the same number of cells is inoculated after isolation.
The new method yields more primary epidermal cells, which grow in a colony morphology. The isolated HKCs from human skin tissues were inoculated with G-medium containing Y-27632. The same number of cells were plated following the conventional method as the control. Representative images of the epidermal cells at days 3 and 5 after the initial inoculation are shown in Figure 2A. The cells obtained from the new method tended to grow as a colony morphology. The growth curve after inoculation was assessed using a cell counting kit-8 (CCK-8) at different time points after inoculation (Figure 2B), and the doubling time is around 3 - 4 days for cells prepared by the new method but is around 6 - 8 days for the conventional method. At day 7, the yields of the cells were calculated. The results are shown in Figure 2C, which shows that the new method yielded three times more cells than the conventional method and that the time needed to reach confluence after the initial inoculation was at least 3 days less (Figure 2D). At day 7, the epidermal cells reached confluence with the new method, but not with the conventional method (Figure 2D).
The new method enhanced the expression of &#945;6-integrin by the primary cells. &#945;6-integrin has been shown to be highly expressed by epidermal stem cells15. Cells isolated by the new method maintained the characteristics of skin basal cells after several passages. Colony growth is one of the characteristics of epidermal stem cells derived from the basal layer of the skin, and a high level of &#945;6-integrin expression is another feature of these epidermal stem cells. We found that the addition of Y-27632 significantly increased the expression of &#945;6-integrin in epidermal cells at passage 3 (Figure 3A-B), and at passage 3, epidermal cells isolated using the new method show no contamination by dermal cells13. It is very important to maintain the basal cell features after several passages during in vitro expansion. Immunofluorescence (IF) analysis of the basal cell marker keratin 5 and the terminal differentiation marker loricrin16 was performed with those cells after three passages. We found that 90% of all cells were K5-positive but less than 10% of them expressed loricrin (Figure&#160;3C - 3D). These results indicate that primary HKCs isolated using the new method contain an epidermal stem cell population and can maintain the characteristic features of basal cells. But the longer that HKCs are passaged, the more they begin to differentiate. By the new method, we found that epidermal cells cultured in vitro could maintain their proliferation ability until passage 8. Finally, we tested the purity of epidermal cells isolated using the new method, since a mixture of epidermal and dermal cells was inoculated right after isolation. With a short-time (2 - 3 min) treatment with trypsin to remove a few dermal cells contaminating the initial culture, a relatively pure population of epidermal cells was obtained after one passage (Figure 4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57784/57784fig1.jpg" /> 
Figure 1: Comparison of the new and conventional isolation procedures. (A) This panel shows a schematic representation of the new isolation method. At day 1, the dissociated skin cells are inoculated in G-medium with or without Y-27632. In the presence of Y-27632 for 2 days, the epidermal cells selectively expand. At around day 6, the cells reach a density sufficient enough for passaging. (B) This panel shows a schematic representation of the conventional isolation method. The epidermis is separated from the dermis and the epidermal cells are isolated on day 2, and usually, the cells reach a density sufficient enough for passaging at around 10 days. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57784/57784fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57784/57784fig2.jpg" /> 
Figure 2: The new isolation method increases the yield of primary epidermal cells. (A) This panel shows representative images of epidermal cells prepared by the new method (top row) and by the conventional method (bottom row) at days 3 and 5 after the initial inoculation. The scale bars = 200 &#181;m. (B) Primary HKCs prepared by the new and the conventional methods were collected at the indicated time points for the analysis of viable cells using a cell counting kit-8 (CCK-8). (C) The total number of cells prepared by the new method and by the conventional method were counted at day 7 after inoculation. (D) The average time to reach confluence of primary epidermal cells (passage 0) is shown for both the new isolation method and the conventional method. For panels B - D: Student&#39;s t-test was used; the error bars show the standard variation; n = 4; ** p &#60; 0.01, * p &#60; 0.05 when comparing the new method with the conventional method. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57784/57784fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57784/57784fig3.jpg" /> 
Figure 3: Primary epidermal cells obtained by the new method are able to maintain basal cell features during in vitro expansion. (A) This panel shows an FACS analysis of &#945;6-integrin expression of epidermal cells at passage 3 treated with Y-27632 (Y-27632, red) or without Y-27632 (control, grey) for 48 h. (B) This panel shows a quantification analysis of cells with high expression levels of &#945;6-integrin from panel A;&#160;high &#945;6-integrin expression is defined as a signal &#62; 103, ** p &#60; 0.01. (C) This panel shows representative images of immunofluorescence staining of K5 (red) and loricrin (red); DAPI (blue) was used to stain nuclei. The scale bars = 100 &#181;m. (D) This panel shows a quantification analysis of K5- and loricrin-positive cells. A total of 400 cells were counted to quantify each group, and the average numbers of K5- and loricrin-positive cells are shown. The experiment was independently repeated 4 times. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57784/57784fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57784/57784fig4.jpg" /> 
Figure 4: After the initial passage, relatively pure epidermal cells were obtained using the new method. (A) This panel shows representative images of immunofluorescence staining of vimentin (red) for epidermal cells at passages 0 (P0) and 3 (P3); DAPI (blue) was used to stain nuclei. The scale bars = 50 &#181;m. (B) This panel shows a quantification analysis of vimentin-positive cells at passages 0 (P0) and 1 (P1). A total of 400 cells were counted to quantify each group, and the average number of vimentin-positive cells is shown. The experiment was independently repeated 4 times.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57784/57784fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue at JoVE.com

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

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 ...

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Developmental Biology

10.0K Views.  Shandong University. 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.

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

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

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A 3D Organotypic Melanoma Spheroid Skin Model

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased metastatic potential. Recently, mono-chemotherapies continue to improve by melanoma specific combination therapies with targeted kinase inhibitors. Still, metastatic melanoma remains a life-threatening disease because tumors exhibit primary resistance or develop resistance to novel therapies, thereby regaining tumorigenic capacity. In order to improve the therapeutic success of malignant melanoma, the determination of molecular mechanisms conferring resistance against conventional treatment approaches is necessary; however, it requires innovative cellular in vitro models. Here, we introduce an in vitro three-dimensional (3D) organotypic melanoma spheroid model that can portray the in vivo architecture of malignant melanoma and may warrant new insights into intra-tumoral as well as tumor-host interactions. The model incorporates defined numbers of mature and differentiated melanoma spheroids in a 3D human full skin reconstruction model consisting of primary skin cells. The cellular composition and differentiation status of the embedded melanoma spheroids is similar to the one of cutaneous melanoma metastasis in vivo. Using this organotypic melanoma spheroid model as a drug screening platform may support the identification of responders to selected combination therapies, while sparing the unnecessary treatment burden for non-responders, thereby increasing the benefit of therapeutic interventions.

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Generation and Culturing of Primary Human Keratinocytes from Adult Skin

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. In addition, keratinocytes play an essential role in the initiation, maintenance, and regulation of epidermal immune responses by being part of the innate immune system responding to antigenic stimuli in a fast, nonspecific manner. Here, we describe a protocol for isolation of primary human keratinocytes from adult skin, and demonstrate that these cells respond to calcium-induced terminal differentiation, as measured by an increased expression of the differentiation marker involucrin. In addition, we show that the isolated keratinocytes are responsive to IL-1&#946;-induced activation of intracellular signaling pathways as measured by the activation of the p38 MAPK pathway. Taken together, we describe a method for isolation and culturing of primary human keratinocytes from adult skin. Because the keratinocytes are the predominant cell type in the epidermis, this method is useful to study molecular mechanisms in cutaneous biology in vitro.

The human skin acts as a first line of defense against the external environment. We present a method for isolating primary human keratinocytes from adult skin. These isolated keratinocytes are useful in numerous experimental setups, and are a highly suitable model for studying molecular mechanisms in cutaneous biology in vitro.

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Establishing a High Throughput Epidermal Spheroid Culture System to Model Keratinocyte Stem Cell Plasticity

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human cancers. As a lining tissue, the skin epithelium is often subject to injury and has evolutionarily adapted by acquiring the cellular plasticity necessary to repair damaged tissue. Over the years, several efforts have been made to study epithelial plasticity using in&#160;vitro and ex vivo cell-based models. However, these efforts have been limited in their capacity to recapitulate the various phases of epithelial cell plasticity. We describe here a protocol for generating 3D epidermal spheroids and epidermal spheroid-derived cells from primary neonatal human keratinocytes. This protocol outlines the capacity of epidermal spheroid cultures to functionally model distinct stages of keratinocyte generative plasticity and demonstrates that epidermal spheroid re-plating can enrich heterogenous normal human keratinocytes (NHKc) cultures for integrin&#945;6hi/EGFRlo keratinocyte subpopulations with enhanced stem-like characteristics. Our report describes the development and maintenance of a high throughput system for the study of skin keratinocyte plasticity and epidermal regeneration.

Here we describe a protocol for the systematic cultivation of epidermal spheroids in 3D suspension culture. This protocol has wide-ranging applications for use in a variety of epithelial tissue types and for the modeling of several human diseases and conditions.

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Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

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

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

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

Biology

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

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This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid enzymatic dissociation of skin. Primary keratinocytes, fibroblasts, and Schwann cells are all harvested from the human newborn foreskin, which is available following standard of care procedures. The removed skin is disinfected, and the subcutaneous fat and muscle are removed using a scalpel. The method consists of enzymatic and mechanical separation of epidermal and dermal layers, followed by additional enzymatic digestion to obtain single-cell suspensions from each of these skin layers. Finally, single cells are grown in appropriate cell culture media following standard cell culture protocols to maintain growth and viability over weeks. Together, this simple protocol allows isolation, culturing, and characterization of all three cell types from a single piece of skin for in vitro evaluation of skin-nerve models. Additionally, these cells can be used together in co-cultures to gauge their effects on each other and their responses to in vitro trauma in the form of scratches performed robotically in the culture associated with wound healing.

The study of wound healing associated with musculoskeletal injury often requires the assessment of in vitro interactions between Schwann cells (SCs), keratinocytes, and fibroblasts. This protocol describes the isolation, culturing, and characterization of these primary cells from the human foreskin.

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Neuroscience

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Isolation of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture

The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 &#177; 2 days old) yielding approximately 30 x 106 viable cells per mouse. Primary adult mouse keratinocytes are harvested from the dorsal skin of female mice. Male mice (~6 weeks old) can be used for keratinocyte harvesting depending on the requirements of the experiment. Euthanized mice are shaved and sterilized with serial washes in povidone iodine and ethanol solutions (70% alcohol). After disinfecting the mice, the dorsal skin is removed and the subcutaneous fat and muscle are removed with a scalpel and discarded. The skins are cut into small pieces and treated with a mild, low temperature trypsinization to detach the lower dermis from the epidermis. The scraped epidermises are stirred at low speed, filtered to remove the hairs, counted, and re-suspended in culture medium. This method provides an excellent single cell suspension of highly culturable cells for many downstream applications.

The goal of this protocol is to isolate epidermal keratinocytes from the dorsal skin of adult mice for a variety of downstream applications such as molecular biology, biochemistry, fluorescence activated cell sorting, and primary in vitro uses (e.g., clonogenic keratinocytes).

The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 &#177; 2 days old) yielding approximately ...

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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 ...