Name: a simplified and efficient method to isolate primary human keratinocytes from adult skin tissue
Uploaded: 2018-08-25T13:00:00
Description: Watch this Scientific Journal Video about A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue at JoVE.com

这项工作得到国家重点研究开发项目 (2017YFA0104604)、中国国家自然科学基金总计划 (81772093)、苏州科技发展规划 (ZXL2015128) 的支持,江苏省自然科学基金 (BK20161241), 山东泰山学者奖 (tshw201502065)。

本议定书所使用的人体组织已根据该机构的人类研究道德委员会的指导方针进行处理 (2015120401, 日期: 2015年5月12日)。
1. 筹备工作
<ol><li>收集在医院的整形手术丢弃的新鲜成人腹部皮肤组织50毫升管与10毫升冰冷 Dulbecco 的改良鹰培养基 (DMEM)。标本可以保持在4°c, 可达72小时, 而不会显著影响细胞的生存能力。</li>
	<li>准备试剂和培养基, 如下所述。<ol>
<li>添加1毫升青霉素 (100 U/?...

<ol>
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R., Neisi, N., Hashemitabar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin-derived+precursors+possess+the+ability+of+differentiation+into+the+epidermal+progeny+and+accelerate+burn+wound+healing.">Skin-derived precursors possess the ability of differentiation into the epidermal progeny and accelerate burn wound healing.</a> Cell Biology International. 41 (2), 187-196 (2017).</li><li>Hirsch, 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=Regeneration+of+the+entire+human+epidermis+using+transgenic+stem+cells.">Regeneration of the entire human epidermis using transgenic stem cells.</a> Nature. 551 (7680), 327-332 (2017).</li><li>Hentzer, B., Kobayasi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+human+epidermal+cells+from+fibroblasts+in+primary+skin+culture.">Separation of human epidermal cells from fibroblasts in primary skin culture.</a> Archiv f&#252;r dermatologische Forschung. 252 (1), 39-46 (1975).</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>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>Kurosawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+Rho-associated+protein+kinase+(ROCK)+inhibitor+to+human+pluripotent+stem+cells.">Application of Rho-associated protein kinase (ROCK) inhibitor to human pluripotent stem cells.</a> Journal of Bioscience and Bioengineering. 114 (6), 577-581 (2012).</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), e1251-e1255 (2018).</li><li>Zou, D., Pan, J., Zhang, P., 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=A+new+method+to+isolate+human+epidermal+keratinocytes.">A new method to isolate human epidermal keratinocytes.</a> Journal of Clinical Dermatology (in Chinese). 6, 424-429 (2016).</li><li>Cerqueira, M. 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培养的初级好客精神已被广泛用于治疗创伤的诊所超过三年, 并从那时起, 一直重要的是有效地获得足够数量的细胞, 为临床应用及时的方式。因此, 在实践中, 传统的隔离方法, 需要分离的表皮与真皮, 使难以满足这些要求, 由于细胞的低产量和低的能力, 通过成人细胞。在这里, 我们描述了一个新的简单方法, 我们开发了以有效地获得好客精神从成人组织的基础上报告13,<sup...

目的是制定一个简单有效的协议, 以隔离主要的人角质细胞 (好客精神) 从成人组织, 特别是在临床应用。皮肤表皮干细胞, 局部在皮肤的基底层, 具有很高的潜能增殖和分化, 并提供角质细胞维持皮肤的功能1,2,3, 4. 从皮肤组织中分离出来的好客精神广泛用于皮肤组织工程和再生目的, 特别是在修复受损皮肤和用于临床应用的基因治疗5,6。HKC 应用的关键问题是有效地分离和扩展大量的好客精神, 其体外7、8具有很高的潜力。虽然不同的研究小组已经开发出了培养茎样好客精神的方法, 但这些方法有时耗时且复杂, 并有其他限制, 如低细胞产量和受皮肤标本类型的限制。使用9。例如, 传统的方法分离好客精神从皮肤组织涉及两步酶消化分离的表皮从真皮6。这种方法通常适用于新生儿组织, 但它变得非常困难, 当用于隔离细胞从成人组织。
Y-27632, 一种蛋白激酶 (岩石) 抑制剂, 据报道, 大大提高了表皮干细胞分离和菌落生长的效率10,11,12。在以前的研究中, 我们发现 Y-27632 促进表皮细胞的克隆生长, 但通过差异控制黏附分子的表达来降低真皮细胞的产量13。我们还建立了一个新的条件接种培养基, 称为 G 培养基, 它支持的生长和产量的主要表皮细胞。采用 G-培养基与 Y-27632 相结合的方法, 可以在酶消化后自发分离表皮和真皮细胞, 从而去除表皮-真皮分离13、14的步骤。根据以前的报告, 我们现在描述了这个新方法的详细程序, 以隔离好客精神从成人皮肤组织。

<table border="0" cellpadding="0" cellspacing="0" style="width:1344px;" width="1342">
	<tbody>
		<tr height="23">
			<td height="23" style="height:23px;width:263px;">Countess automated cell counter</td>
			<td style="width:291px;">Invitrogen Inc.&#160;</td>
			<td style="width:264px;">C10227</td>
			<td style="width:527px;">Automatic cell counting&#160;</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">CO2 Incubator</td>
			<td>Thermo Scientific</td>
			<td>51026333</td>
			<td>For cell incubating</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Sorvall ST 16R Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75004380</td>
			<td>Cell centrifuge</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Constant Temperature Shaker</td>
			<td>Shanghai Boxun</td>
			<td>150036</td>
			<td>For water bath</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Electronic Scale</td>
			<td>Harbin Zhonghui</td>
			<td>1171193</td>
			<td>For tissue weighing</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Cell Culture Dish</td>
			<td>Eppendorf</td>
			<td>30702115</td>
			<td>For cell culture</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">50ml Centrifuge Tube</td>
			<td>KIRGEN</td>
			<td>171003</td>
			<td>For cell centrifuge</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Cell Strainer</td>
			<td>Corning incorporated</td>
			<td>431792</td>
			<td>Cell filtration</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Phosphate buffered solution</td>
			<td>Solarbio Life Science&#160;</td>
			<td>P1020-500</td>
			<td>Washing solution</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">DMEM</td>
			<td>Thermo Scientific</td>
			<td>C11995500</td>
			<td>Component of neutralization medium</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Defined K-SFM</td>
			<td>Life Technologies</td>
			<td>10785-012</td>
			<td>Epidermal cells culture medium</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Penicillin Streptomycin</td>
			<td>Thermo Scientific</td>
			<td>15140-122</td>
			<td>Antibiotics</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Fetal Bovine Serum</td>
			<td>Biological Industries</td>
			<td>04-001-1AC5</td>
			<td>Component of neutralization medium</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">0.05% Trypsin</td>
			<td>Life Technologies</td>
			<td>25300-062</td>
			<td>For HKC dissociation</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">0.25% Trypsin&#160;</td>
			<td>Beijing Solarbio Science &#38; Technology</td>
			<td>T1350-100</td>
			<td>For HKC dissociation</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Coating Matrix Kit</td>
			<td>Life Technologies</td>
			<td>R-011-K</td>
			<td>For coating matrix</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Dispase</td>
			<td>Gibco</td>
			<td>17105-041</td>
			<td>For HKC isolation</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Collagenase Type I</td>
			<td>Life Technologies</td>
			<td>17100-017</td>
			<td>For HKC isolation</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Deoxyribonuclease I</td>
			<td>Sigma</td>
			<td>9003-98-9</td>
			<td>For HKC isolation</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">F12 Nutrient Mix, Hams</td>
			<td>Life Technologies</td>
			<td>31765035</td>
			<td>Component of G-medium</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">B27 Supplement</td>
			<td>Life Technologies</td>
			<td>17504044</td>
			<td>Growth factor in G-medium</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">FGF-2 Millipore</td>
			<td>Merck Biosciences</td>
			<td>341595</td>
			<td>Growth factor in G-medium</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Y-27632</td>
			<td>Sigma-Aldrich</td>
			<td>Y0503</td>
			<td>ROCK inhibitor</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Fungizone</td>
			<td>Gibco</td>
			<td>15290026</td>
			<td>Preparation for G-medium</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">EGF Recombinant Human Protein</td>
			<td>Gibco</td>
			<td>PHG0311</td>
			<td>Growth factor in G-medium</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Cell Counting Kit-8</td>
			<td>Thermo Scientific</td>
			<td>NC9864731</td>
			<td>cell proliferation and cytotoxicity assays</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">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 height="23">
			<td height="23" style="height:23px;">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 height="23">
			<td height="23" style="height:23px;">Mouse anti-human Vimentin</td>
			<td>Cell Signaling Technology</td>
			<td>3390</td>
			<td>For immunofluorescence staining of dermal fibroblasts</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Integrin &#945;6(GOH3)</td>
			<td>Santa Cruz&#160;</td>
			<td>SC-19622</td>
			<td>flow cytometry analysis of HKCs</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:263px;">Rat IgG2a FITC</td>
			<td>Santa Cruz&#160;</td>
			<td>SC-2831</td>
			<td>negative control antibody of &#945;6-integrin&#160; in flow cytometry analysis&#160;</td>
		</tr>
	</tbody>
</table>

a simplified and efficient method to isolate primary human keratinocytes from adult skin tissue

从新鲜皮肤组织中分离出来的人角质形成细胞及其体外扩张已被广泛应用于实验室研究和临床应用。传统的人角质形成细胞的分离方法涉及两步序贯酶消化法, 由于细胞回收率低, 细胞减少, 在成年组织中产生原发体细胞效率低下。可行性。我们最近报告了一种先进的方法, 从皮肤组织中分离人类主要表皮祖细胞, 利用 Y-27632 激酶抑制剂在培养基中。与传统的协议相比, 该方法简单、简便、耗时少, 增加了上皮干细胞的产量, 增强了干细胞的特性。此外, 新的方法不需要分离表皮与真皮, 因此, 适合隔离细胞从不同类型的成人组织。这种新的隔离方法克服了传统方法的主要缺点, 更适合在实验室和临床应用中生产大量具有高效能的表皮细胞。在这里, 我们详细描述了新的方法。

图 1给出了新方法 (图 1A) 和常规方法 (图 1B) 的示意图。传统的方法是两步消化, 这需要2天的过程。相比之下, 新的方法是一步消化, 这需要大约3小时的时间来完成。重要的是, 一步新的方法可以同时获得两个种群 (表皮和真皮细胞), 这取决于接种培养基中 Y-27632 的存在。此外, 如果在隔离后接种相同数量的?...

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

在这里, 我们提出了一个协议, 以有效地隔离主要的人角质细胞从成人皮肤组织。该方法利用接种培养基中的岩石抑制剂 Y-27632, 将表皮细胞与真皮细胞自发分离, 从而简化了常规程序。

一种简便有效的成人皮肤组织中人角质形成细胞的分离方法

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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Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such ...

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

从背小鼠皮肤分离毛囊干细胞和表皮角质形成细胞

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

An Efficient Method to Obtain Dedifferentiated Fat Cells

一个有效的方法获取反分化脂肪细胞

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

从人胎盘组织源分离和间充质干扩张/基质细胞

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Generation and Culturing of Primary Human Keratinocytes from Adult Skin

成人皮肤角质形成细胞的生成与培养

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. ...

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

人成体和胎心标本中原发性心外膜细胞的分离培养

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of ...

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

一种直接而简单的方法，用于评估从胚胎到成人的果蝇黑色素的生存能力

In Drosophila melanogaster, viability assays are used to determine the fitness of certain genetic backgrounds. Allelic variations can result in partial or ...