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/毫升) 和链霉素 (100 毫克/升) 到50毫升的磷酸盐缓冲溶液 (PBS), 以准备洗涤液。</li>
		<li>制备两组酶消化液: (1) 分别制备50毫升的酶 (2.5 毫克/毫升 DMEM) 和50毫升的 I 型胶原酶 (2.5 毫克/毫升在 DMEM) 单独为常规方法;(2) 准备50毫升的酶和 i 型胶原酶 (溶解125毫克的酶粉和125毫克的 i 型胶原酶粉在50毫升的 DMEM) 的新方法。 注意: 所有的酶溶液都应用0.22 µm 过滤器过滤, 并保持 at4 °c。</li>
		<li>为两种方法准备消化液: 0.05% 胰蛋白酶和0.25% 胰蛋白酶被商业购买。准备一个10毫克/毫升 DNase i 溶液溶解50毫克 DNase I 粉末5毫升的 PBS, 过滤它与0.22 µm 过滤器, 并存储在-20 °c。</li>
		<li>制备500毫升培养基以中和酶消化: DMEM 培养基辅以10% 胎牛血清 (血清) 和1% 青霉素/链霉素。</li>
		<li>准备500毫升的接种培养基 (生长因子含培养基, 称为 G 培养基): DMEM/F12 (3:1) 培养基含有1% 青霉素/链霉素, 40 µg/毫升 fungizone, 40 ng/毫升成纤维细胞生长因子 2 (FGF2), 20 ng/毫升表皮生长因子 (EGF), 和2% B27 补充剂。</li>
		<li>预处理100毫米细胞培养皿, 2 毫升的涂层基质, 含有 i 型胶原蛋白, 在室温下为30分钟。</li>
	</ol>
</li>
</ol>2. 常规方法
<ol><li>
皮肤组织预处理
	<ol>
<li>采取 1.5 cm2皮肤组织和洗涤它1x 与10毫升 PBS;然后, 用10毫升70% 乙醇冲洗它三十年代和孵化2x 与10毫升的洗涤液 (PBS 含有2% 青霉素和链霉素), 每5分钟。 注: 所有洗涤都是在层流罩内的100毫米细胞培养皿中进行的。</li>
		<li>用无菌剪刀修剪组织, 去除皮下脂肪层在一个100毫米细胞培养皿, 然后, 体重的皮肤组织 (重量是1克在这个协议)。 注意: 足够的修剪对于有效地将表皮与真皮分离是非常关键的。黄色脂肪层可以很容易地区分视觉。</li>
		<li>将组织转移到另一100毫米无菌皿中, 真皮侧向下, 然后, 用手术刀刀片将皮肤组织切成3至4毫米宽的小条。 注意: 真皮侧与脂肪层很容易被识别视觉。</li>
		<li>在100毫米培养皿中加入10毫升的2.5 毫克/毫升酶溶液, 然后在4摄氏度 (不超过20小时) 一夜之间孵化盘子。 注: 酶溶液的用量可以通过使用10毫升的酶溶液来计算每克组织的消化。</li>
	</ol>
</li>
	<li>
表皮与皮肤组织的分离
	<ol>
<li>第二天, 用细镊子从真皮中剥离表皮。 注: 如果表皮不易脱落, 酶消化不足, 通常是由于组织的修剪不够。在这种情况下, 组织需要更长的孵化时间。</li>
		<li>用手术刀刀片将去皮的表皮与500µL 细胞培养基放入组织浆料中;然后, 并用重悬它与10毫升0.25% 胰蛋白酶在50毫升管和孵化它20分钟在水浴在37°c, 与震动。</li>
	</ol>
</li>
	<li>
原代细胞的采集与培养
	<ol>
<li>在本协议中加入等量的中和溶液 (10 毫升) 中和胰蛋白酶活性。</li>
		<li>通过用10毫升血清学吸管将溶液吹打和下 20x, 通过细胞溶液通过100µm 细胞过滤器, 去除任何残留的组织碎片, 使表皮细胞游离。</li>
		<li>过滤后将细胞溶液在 200 x g处离心, 5 分钟;并用重悬中中和介质中的细胞颗粒进行洗涤, 然后再将其离心在 200 x g上以获得细胞颗粒。</li>
		<li>并用重悬细胞颗粒在10毫升低钙, 无血清角质形成培养基 (可以持续森林)。用10µL 这个解决方案来计算总的细胞数, 然后, 将 2 x 106细胞与10毫升的可持续可持续性转化为100毫米细胞培养皿, 用涂层基质 (含 i 型胶原) 预处理。 注: 涂层是常规方法的必要步骤。</li>
		<li>每 2 d 改变培养基。 注: 在改变培养基前后, 检查显微镜下的细胞黏附力。</li>
		<li>通过细胞时, 他们达到约80% 汇合。 注意: 所有的养殖菜肴都经过预处理的涂层基质。</li>
	</ol>
</li>
</ol>3. 新方法
<ol><li>
皮肤组织预处理
	<ol>
<li>收集的组织如上文所述 (步骤 2.1) 的常规方法。</li>
		<li>用10毫升的 PBS 清洗皮肤组织, 然后用电子秤将其称量在无菌塑料容器中 (该协议的重量约为1克)。 注: 本协议所需组织的最小重量为0.1 克, 因为使用的组织标本太小, 很难获得足够数量的细胞。该协议没有最大的组织重量, 这最终取决于操作员的处理能力。</li>
		<li>使用镊子冲洗皮肤组织与10毫升70% 乙醇在三十年代。</li>
		<li>用10毫升洗涤液 (在步骤2.1.1 中制备) 培养组织 2x, 每次5分钟。 注意: 上面提到的所有洗涤步骤都是在层流罩 (组织培养罩) 内的100毫米细胞培养皿中进行的。</li>
		<li>将组织转移到另一种100毫米细胞培养无菌皿中。</li>
		<li>使用手术刀刀片, 将组织彻底剁碎成组织浆液。</li>
		<li>每5分钟添加200µL 的 PBS, 以保持组织湿润。 注: 采取步骤 3.1.5-3.1.7 约15分钟。以上步骤均在室温下进行。</li>
	</ol>
</li>
	<li>
皮肤组织的消化
	<ol>
<li>将匀质组织溶液转化为50毫升管。每1克皮肤组织添加10毫升的酶合剂。将酶与匀质组织彻底混合。</li>
		<li>在37摄氏度的水浴中以1小时的温度, 孵育混合物。</li>
		<li>增加1/5 容量0.25% 胰蛋白酶 (2.5 毫升在这个协议) 到消化混合物为另30分钟在水浴在37°c。</li>
		<li>添加 DNase I 溶液的酶混合物在 1:100 (v/v) 比 (250 µL 在本议定书) 和孵化它在室温下5分钟。</li>
	</ol>
</li>
	<li>
原代细胞的采集与培养
	<ol>
<li>在1:1 的比例中加入12.5 毫升中和介质, 停止消化过程。用10毫升的血清吸管将溶液中的液吸管向上和向下大约 20x, 以分离细胞。</li>
		<li>通过100µm 细胞滤网过滤分离细胞, 去除组织碎片。离心机在 200 x g 5 分钟内清液观察试管底部的颗粒。</li>
		<li>丢弃上清液, 用10毫升中和介质清洗细胞颗粒, 再以 200 x g为5分钟来收集细胞。</li>
		<li>并用重悬细胞颗粒在10毫升接种培养基 (G-培养基从步 1.2.5) 与10µM Y-27632。</li>
		<li>采取10µL 的解决方案计数的总细胞数, 然后, 板约 2 x 106细胞与10毫升的 G 培养基成100毫米细胞培养皿。 注意: 新方法不需要预涂步骤, 也不需要去除真皮层。</li>
		<li>3 d 后, 用低钙能持续的培养基取代接种培养基。每 2 d 改变培养基。 注意: 在改变培养基前后, 检查细胞黏附力。</li>
		<li>通过细胞时, 他们达到约80% 汇合。 注: 如有必要, 预处理细胞以0.05% 胰蛋白酶为2分钟, 并用 PBS 清洗细胞, 在 trypsinizing 表皮细胞之前清除污染的真皮细胞。</li>
	</ol>
</li>
</ol>4. 细胞传代
<ol><li>取出培养基, 用 PBS 冲洗细胞, 然后添加2毫升0.05% 胰蛋白酶 (每100毫米菜)。</li>
	<li>孵化细胞在孵化器 (37 °c 与 5% CO2) 为5分钟。</li>
	<li>用显微镜检查细胞, 以确保所有细胞都与培养皿分离。</li>
	<li>添加8毫升的 DMEM 与10% 的血清, 以中和酶反应, 然后, 收集细胞在15毫升管。</li>
	<li>离心机在 200 x g 5 分钟获得细胞颗粒。</li>
	<li>慢慢去除上清液, 然后用10毫升的并用重悬细胞颗粒, 计算细胞数。</li>
	<li>板 1 x 106细胞与10毫升的森林, 以每100毫米细胞盘。</li>
	<li>每2维更换一次介质。</li>
</ol>

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作者没有什么可透露的。

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13,14, 这是更适合于实验室和临床应用。
必须注意以下关键步骤, 以达到新方法的最佳效果。第一, 均匀化程序是关键步骤;均匀性不足会明显影响酶消化的效率, 导致分离细胞的低产量。第二, 胰蛋白酶消化步骤不应超过30分钟;否则, 离解细胞的生存能力将会减少。第三, 为了有效地收集分离出的细胞, 用于过滤的细胞过滤器的孔径大小应为100µm, 而单一滤网应用于过滤不超过20毫升的细胞溶液。第四, 初始电镀的密度是减少皮肤细胞污染的一个重要因素, 因为高细胞密度会显著降低 Y-27632 对真皮细胞的抑制作用。因此, 在电镀前, 在隔离过程结束时, 准确地计算出分离细胞的总数是至关重要的。
首先, 新方法简化了隔离过程: 两步常规隔离程序通常需要2天的时间来完成, 修剪过程对于彻底去除皮肤上的脂肪组织, 特别是成人组织 (图 1);否则, 在隔夜消化酶后, 表皮与真皮的分离将会有效率低下。利用 Y-27632, 抑制13接种后真皮细胞的生长, 新方法不需要将表皮与真皮分离, 因此, 去除脂肪组织的修剪过程并不需要新的方法, 只需要半天的时间来执行。其次, 新方法也简化了培养过程, 因为传统的方法是, 分离细胞通常在含有10% 血清或胶原膜的培养基中被辐照的小鼠成纤维细胞的进给层培养。动物衍生的成分总是危险因素, 应避免的临床应用。我们开发了无血清条件接种培养基 (G 培养基), 这不需要一个预涂步骤的培养皿, 也提高了原角质细胞13的产量, 因为 G 培养基结合 Y-27632 促进附着表皮细胞到文化盘13,17,18。因此, 新的方法有利于培养过程, 也是一个相对安全的程序, 在临床应用不添加动物衍生成分。第三, 新方法提高了原发表皮细胞的产量, 缩短了达到汇流所需的初始培养时间。与传统方法相比, 新方法产生的细胞产量约为3倍以上, 表皮细胞的初始通道至少在3天前达到汇合 (图 2)。最后, 新的方法可以用于从所有类型的皮肤标本, 如毛茸茸的头皮皮肤和成人皮肤组织与厚脂肪层的表皮细胞的分离, 这已经在以前的报告中测试13。
高电位初级好客精神已广泛用于实验室研究和临床应用。这种新方法提高了表皮干细胞特征的培养细胞的潜能13。Y-27632 通过好客精神增强α6-整合素表达, 三通道后, 超过90% 的培养表皮细胞表达基底细胞标记角蛋白 5, 而少于10% 的细胞表达分化标记 Loricrin, 表明这些细胞在几个段落以后保持皮肤基底细胞的特征。培养后的表皮细胞可以在13移植后重建皮肤, 提示在培养后具有再生潜能。值得注意的是, 新的方法是理想的分离细胞与成人组织, 如上文所述。因此, 这种方法制备的表皮细胞可用于体外和体内模型研究皮肤病, 并可发展成自体细胞为基础的产品, 用于临床应用, 如皮肤创面的治疗。
总之, 新的协议提供了一个简化和有效的程序, 以隔离和扩大主要表皮细胞从成人皮肤组织。这种先进的方法更适合生产大量的高潜能表皮细胞, 无论是实验室和临床应用。

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><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

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细胞时, 表皮细胞的初始培养时间达到大约80% 汇合通常要比常规方法少3天。
新方法产生更多的主要表皮细胞, 生长在菌落形态学。从人皮肤组织中分离好客精神, 用含 Y-27632 的 G 介质进行接种。相同数量的细胞被电镀后, 传统的方法作为控制。最初接种后3和5天表皮细胞的代表性图像如图 2A所示。从新方法获得的细胞倾向于作为菌落形态生长。接种后生长曲线使用细胞计数 kit-8 (CCK-8) 在不同的时间点接种 (图 2B), 并且加倍的时间是大约 3-4 天为被新的方法准备的细胞, 但大约 6-8 天为常规方法。在7天, 计算出细胞的产量。结果如图 2C所示, 这表明新方法产生的细胞比常规方法多出三倍, 初始接种后达到汇合的时间至少减少3天 (图 2D)。7天, 表皮细胞与新方法融合, 但不采用常规方法 (图 2D)。
新的方法增强了α6的表达, 由主细胞。α6-整合素已被证明是高度表达的表皮干细胞15。新方法分离的细胞在几个通道后维持皮肤基底细胞的特征。菌落生长是表皮干细胞从皮肤基底层中提取的特征之一, α6-整合素的表达是这些表皮干细胞的另一个特征。我们发现, 添加 Y-27632 显著增加了α6-整合素在表皮细胞中的表达在 3 (图 3A-B), 并在通道 3, 使用新方法隔离的表皮细胞不显示皮肤细胞污染13.在体外扩增过程中, 保持基底细胞特征是非常重要的。免疫荧光 (IF) 分析的基础细胞标记角蛋白5和终端分化标记 loricrin16是执行与这些细胞后三通道。我们发现, 90% 的细胞是 K5-positive 的, 但不到10% 表达 loricrin (图 3C - 3D)。这些结果表明, 采用新方法分离的原发性好客精神含有表皮干细胞种群, 可维持基底细胞的特征特征。但好客精神传代的时间越长, 他们开始区分的越多。通过新的方法, 我们发现体外培养的表皮细胞可以保持其增殖能力直到8。最后, 我们用新的方法对表皮细胞的纯度进行了测试, 因为在隔离后, 表皮和真皮细胞的混合物被接种。用胰蛋白酶进行短时间 (2-3 分钟) 的治疗, 除去少数真皮细胞污染最初的培养, 在一条通道后获得相对纯净的表皮细胞种群 (图 4)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57784/57784fig1.jpg"> 图 1: 比较新的和常规的隔离程序.(A) 此面板显示了新隔离方法的示意图表示形式。1天, 游离的皮肤细胞在 G 培养基中接种或不 Y-27632。在 Y-27632 2 天的存在下, 表皮细胞有选择性地扩张。在6天左右, 细胞达到足够的密度足够传代。(B) 本小组展示了传统隔离方法的示意图表示法。表皮与真皮分离, 表皮细胞在2天被隔离, 通常, 细胞达到足够的密度足以传代在10天左右。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57784/57784fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57784/57784fig2.jpg"> 图 2: 新的分离方法增加了原发表皮细胞的产量.(A) 本小组显示了新方法 (顶排) 和常规方法 (下排) 在初始接种后3和5天内所制备的表皮细胞的代表性图像。刻度条 = 200 µm. (B) 新的和常规方法编制的初级好客精神是在指定的时间点收集的, 用于用细胞计数 kit-8 (CCK-8) 分析可行细胞。(C) 接种后7天计算新方法和常规方法制备的细胞总数。(D) 在新的隔离方法和常规方法的基础上, 显示了主要表皮细胞的平均到达汇合时间 (通道 0)。为小组B D: 学生的t测试被使用了;误差条显示标准变化;n = 4;** p < 0.01, * p < 0.05 在比较新方法与传统方法。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57784/57784fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57784/57784fig3.jpg"> 图 3: 新方法获得的原发表皮细胞能够维持基底细胞的功能, 在体外扩张.(A) 本小组对 48 h (Y-27632、红) 或无 Y-27632 (控制、灰色) 治疗的3通道表皮细胞α6-整合素表达的方法进行了分析. (B) 本小组对高的细胞进行量化分析从A组α6-整合素的表达水平;高α6-整合素表达式被定义为信号 > 103, ** p < 0.01。(C) 本小组显示 K5 (红) 和 loricrin (红色) 的免疫荧光染色的代表性图像;DAPI (蓝色) 被用来染色细胞核。刻度条 = 100 µm. (D) 本小组对 K5 和 loricrin 阳性细胞进行了量化分析。共400个细胞计数, 以量化每组, 并显示 K5 和 loricrin 阳性细胞的平均数量。实验独立地重复了4次。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57784/57784fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57784/57784fig4.jpg"> 图 4: 在初始通道后, 采用新方法获得相对纯净的表皮细胞.(A) 本小组在 0 (P0) 和 3 (P3) 中显示了对表皮细胞波形 (红) 免疫荧光染色的代表性图像;DAPI (蓝色) 被用来染色细胞核。刻度条 = 50 µm. (B) 本小组对 0 (P0) 和 1 (P1) 通道的波形阳性细胞进行了定量分析。总计400个细胞数以量化每组, 并显示波形阳性细胞的平均数。实验独立地重复了4次。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57784/57784fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</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

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 ...

a-simplified-efficient-method-to-isolate-primary-human-keratinocytes

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

10.0K 次观看.  Shandong University. 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。

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

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.

Here, we present a protocol to generate a 3D organotypic melanoma spheroid skin model that recapitulates both the architecture and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention.

3D 脊髓黑色素瘤球形皮肤模型

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased ...

科研

癌症研究

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.

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. ...

发育生物学

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.

建立高通量表皮球体培养系统，以模拟角膜细胞干细胞可塑性

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human ...

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.

成人从人体皮肤的上皮干细胞的分离和培养

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

生物学

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.

天真成人干细胞的分离从初级人类成纤维细胞培养

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Generating Primary Cultures for Keratinocyte Live Cell Imaging

Tracking stem cells and committed progenitor behavior at the single-cell level in human skin has been challenging both in vivo and in vitro.&#160;Live-cell imaging has allowed for significant advances in the ability to identify differences between keratinocyte stem cells and committed progenitor behavior. Live-cell imaging is an evolving and somewhat challenging method to study keratinocyte behavior in vitro. This protocol has been developed to culture keratinocytes at low seeding density, enabling relatively long-term time-lapse photography and monitoring of individual cell behavior. Passage 0 keratinocytes are grown at clonal density, and time-lapse photography allows documentation of individual cell divisions and the time of their occurrence. For maximum biological relevance, freshly isolated human keratinocytes are placed in vitro. This approach focuses on proliferation. However, this protocol can be adapted for use in other live cell imaging applications measuring individual cell behavior, such as measuring cell migration, wound healing, and motility.

This protocol outlines the culture of freshly obtained (passage 0) keratinocytes for use in live cell imaging.

生成用于角质形成细胞活细胞成像的原代培养物

Tracking stem cells and committed progenitor behavior at the single-cell level in human skin has been challenging both in vivo and in ...

生物工程

Isolation, Culture, and Characterization of Primary Schwann Cells, Keratinocytes, and Fibroblasts from Human Foreskin

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.

从人包皮中分离、培养和表征原代施旺细胞、角质形成细胞和成纤维细胞

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid ...

神经科学

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.

来自新生儿和成年鼠皮肤的原代小鼠角化细胞的分离和培养

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).

小鼠表皮角质细胞的分离及其体外克隆培养物

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

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.

从成人小鼠腭中分离和培养原代口腔角质形成细胞

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