作者希望感谢安妮特布拉克拉斯穆森和汀穆勒的技术支持

在接受整形手术的健康成人志愿者身上采集的皮肤样本需要得到所在机构伦理委员会的批准。该议定书得到了丹麦 Midtjylland 区域伦理委员会的批准 (M-20110027)。这里描述的方法来自 Maciaq et al.的类似研究10和刘和 Karasek11。
1. 从人的皮肤中分离角质形成细胞
<ol>
	<li>首先提出以下解决方案:50 毫升溶液中0.25% 胰蛋白酶和0.1% 葡萄糖在 DPBS。混合和过滤消毒 (0.2 µm) 的解决方案。准备10毫升的 RPMI-1640 + 2% FBS 溶液, 50 毫升的 DPBS 和角质形成细胞无血清培养基 (KSFM)。添加 KSFM 补充剂和250µL 庆大霉素到500毫升的 KSFM。在使用前预热溶液至37° c。</li>
	<li>从健康的成人志愿者那里收集皮肤, 接受整形手术。所使用的皮肤样本约10厘米 x 15 厘米, 但可以在大小上有所不同。通过在含有冷却元件的聚苯乙烯泡沫塑料盒中运输皮肤样品, 使皮肤保持凉爽。如有必要, 皮肤样品可在4° c 过夜贮存。</li>
	<li>使用无菌剪刀、手术刀和镊子, 从皮肤底部取出脂肪。</li>
	<li>将皮肤部分 (大约10厘米 x 15 厘米) 系在一个使用针头的盘子上面的无菌盖子上。首先, 用干燥的消毒纱布清洁皮肤。然后用70% 乙醇消毒的纱布垫清洁皮肤。</li>
	<li>使用足部刨床, 切开皮肤部分的上层 (表皮), 并将其放入9厘米的培养皿中。然后, 立即添加25毫升的 DPBS/胰蛋白酶/葡萄糖溶液 (准备在步骤 1.1) 的培养皿。孵育30分钟, 在37° c。</li>
	<li>使用吸管从培养皿中除去 DPBS/胰蛋白酶/葡萄糖溶液, 并添加10毫升的 RPMI-1640 + 2% FBS, 以使胰蛋白酶钝化。</li>
	<li>使用两个钳, 现在将表皮细胞释放到培养基中, 通过轻轻地刮和搅动皮肤部分的表皮和真皮室。</li>
	<li>过滤表皮细胞悬浮通过金属过滤器 (1 毫米孔大小) 和收集在50毫升管子。将其余的皮肤部分添加到50毫升管, 其中含有10毫升的 RPMI-1640, 没有 FBS 和漩涡十年代. 通过金属过滤器将悬浮液过滤成含有表皮细胞悬浮液的50毫升管。将 DPBS 添加到总容量为50毫升。</li>
	<li>在室温下将细胞悬浮液离心10分钟, 450 x g。</li>
	<li>在大约10毫升的37° c KSFM, 根据细胞颗粒的大小去除上清和重表皮细胞颗粒。在显微镜下使用台盼蓝染色法对细胞进行计数。从 10 cm x 15 cm 皮肤剖面获得的单元格数约为 50-100 x 106单元格。对每 75 cm2培养烧瓶, 将 8 x 106单元格与12毫升37° KSFM 传输到一起。轻轻摇动培养瓶, 确保细胞均匀分布。</li>
	<li>在37° c 恒温箱中孵育角质形成细胞, 100% 湿度和 5% CO2。2天三次, 每周更换培养基。传代细胞时, 文化是 70-80% 汇合, 这需要大约2周, 取决于增殖率的角质形成。</li>
</ol>
2. 角质形成细胞的传代
<ol>
	<li>将适当数量的0.05% 胰蛋白酶-EDTA 溶液加热到37° c 的恒温箱中。使用4.5 毫升0.05% 胰蛋白酶-EDTA 溶液为75厘米2培养烧瓶。</li>
	<li>从细胞中取出培养基, 并在细胞中添加 4.5 mL/75 cm2培养烧瓶 5ml 0.05% 胰蛋白酶-EDTA 溶液。将培养烧瓶放在孵化箱中 (37 ° c), 在大约5分钟后, 在显微镜下检查细胞是否开始松动。</li>
	<li>当大约50% 的细胞松动时, 轻轻地将培养瓶放在手上, 松开剩余的细胞。然后, 为了使胰蛋白酶钝化, 将6毫升的 RPMI-1640 + 2% FBS (37 ° c) 添加到 75 cm2培养烧瓶中, 并将该细胞悬浮液转移到50毫升管。</li>
	<li>冲洗3毫升的 RPMI 1640 + 2% FBS 的培养烧瓶, 并添加到50毫升管。在室温下离心10分钟, 450 x 克。
	<ol>
		<li>重10毫升 KSFM (37 ° c) 的颗粒细胞。将单元格计数并将 3 x 106单元格转换为 150 cm2培养瓶, 并与20毫升的 KSFM (37 ° c) 一起进行计算。轻轻摇动培养瓶, 确保细胞均匀分布。当培养 80-90% 汇合时, 冻结细胞, 这需要大约4-6 天, 这取决于角质形成的增殖率 (见下面的 3, 冻结协议)。扩大角质形成细胞以产生适当的冷冻储存。</li>
	</ol>
	</li>
</ol>
3. 冷冻角质形成细胞
<ol>
	<li>将适当数量的0.05% 胰蛋白酶-EDTA 溶液加热到37° c 的恒温箱中。使用9毫升0.05% 胰蛋白酶-EDTA 溶液为150厘米2培养烧瓶。</li>
	<li>从细胞中取出培养基, 并在细胞中添加 9 mL/150 cm2培养烧瓶 5ml 0.05% 胰蛋白酶-EDTA 溶液。将培养烧瓶放在孵化箱中 (37 ° c), 在大约5分钟后, 在显微镜下检查细胞是否开始松动。</li>
	<li>当大约50% 的细胞松动时, 轻轻地将培养瓶放在手上, 以便松开剩余的细胞。然后, 为了使胰蛋白酶钝化, 将12毫升的 RPMI-1640 + 2% FBS (37 ° c) 添加到 150 cm2培养烧瓶中, 并将细胞悬浮液吸入50毫升管。</li>
	<li>冲洗6毫升的 RPMI 1640 + 2% FBS 的培养烧瓶, 并添加到50毫升管。离心管为10分钟在 450 x g 在4° c。制备 ice-cold 细胞冷冻培养基 (KSFM + 10% 亚砜)。</li>
	<li>重 KSFM + 10% 亚甲基亚砜中的细胞团, 计数细胞, 并冻结细胞在密度为 6 x 106细胞/毫升使用标准慢冷冻冷冻方法12。</li>
	<li>将角质形成细胞储存在液氮中 (蒸气相), 直至使用。</li>
</ol>
4. 解冻和培养冰冻角质形成细胞
<ol>
	<li>在37° c 水浴中迅速解冻适当的冷冻瓶。用70% 的乙醇清洁 cryovial 的外部。将适当数量的细胞 (大约1.5万细胞/cm2) 转移到培养烧瓶中, 并立即将5ml 生长培养基 (KSFM) 添加到培养瓶中。 根据实验的设置, 可以使用任何大小的培养烧瓶。</li>
	<li>使用5毫升培养基为 25 cm2培养瓶。</li></ol>添加了培养烧瓶的细胞数量可能会有所不同, 因为细胞的生长会因捐献者而异。孵育37° c 恒温箱中的细胞, 100% 湿度和 5% CO2。
	<li>2天和三次, 每周使用新鲜 5ml KSFM。 注意: 我们的数据表明, 在这里所描述的协议所隔离的细胞中, 平均有95% 是阳性的角质-标记 cytokeratine-1413。</li>

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.作者没有利益冲突。

在这里, 我们描述了如何很容易地分离成人皮肤角质形成细胞, 以及如何培养他们在体外。该模型可以广泛应用于表皮细胞生物学的研究, 对研究皮肤疾病的研究有一定的参考价值。
这里所描述的协议的一些优点是, 与从接受包皮环切手术的新生儿包皮中分离出来的角质形成细胞相比, 成年患者的原始人角质产生者与男性和女性都是分离的。可以包括任何年龄≥18年。因?...

皮肤是人体最大的器官, 是抵御外界环境的屏障。皮肤由两个主要层组成: 真皮和表皮, 表皮构成皮肤最外层。表皮中最丰富的细胞类型是角质形成细胞, 它包含95% 以上的胞质1,2。角质形成细胞保持在不同阶段的分化在表皮, 并组织成基底, 棘, 颗粒, 和 cornified 层, 对应的特定阶段的差异3。角质形成细胞的主要功能是提供表皮的结构完整性, 从而产生一个完整的屏障, 外部世界。
角质形成细胞也代表了第一行防御病原体在皮肤, 因此发挥重要作用的先天免疫反应4,5。角质形成细胞对外界刺激的暴露导致胞内信号传导通路的活化, 随后产生多种炎症介质, 包括细胞因子、趋化因子和抗菌肽。这些角质形成细胞衍生的蛋白质参与炎症反应, 通过招募和激活免疫细胞, 如树突状组织, 嗜中性细胞, 和特定 T 淋巴细胞6,7。因此, 由于角质形成细胞在许多生物过程中扮演着重要的角色, 这里提出的技术背后的基本原理是生成一个体外模型来研究皮肤生物学。从新生儿包皮中获得的原代角质细胞培养物通常用于研究皮肤生物学8,9。然而, 根据这里所描述的技术, 来自两性的角质形成细胞得到了更高的生物多样性。
在这里, 我们提出了一个详细的协议, 从成人皮肤分离和生成原人角质形成细胞, 包括维持和冷冻的角质。该方法的总目标是生成主要的人角质形成细胞, 可以作为一个模型来研究皮肤生物学体外。

<table><tbody><tr><td>KSFM</td><td>ThermoFisher Scientific</td><td>17005-034</td><td>Cell culture medium</td></tr><tr><td>KSFM supplements</td><td>ThermoFisher Scientific</td><td>37000-015</td><td>Supplements for KSFM</td></tr><tr><td>DPBS</td><td>ThermoFisher Scientific</td><td>14190-144</td><td>DPBS without Calcium and Magnesium</td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D8418</td><td>Dimethyl sulfoxid</td></tr><tr><td>Gentamycin</td><td>ThermoFisher Scientific</td><td>15710-049</td><td>Cell culture medium additive</td></tr><tr><td>Sterilization filter</td><td>Sartorius</td><td>16534</td><td>Syringe filter with a pore size of 0.2 &amp;micro;m</td></tr><tr><td>Trypsin</td><td>Sigma-Aldrich</td><td>T7409</td><td>Used to trypsinize cells</td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G7528</td><td>-</td></tr><tr><td>RPMI-1640</td><td>ThermoFisher Scientific</td><td>61870-010</td><td>-</td></tr><tr><td>FBS</td><td>ThermoFisher Scientific</td><td>16000044</td><td>Used to inactivate trypsin</td></tr><tr><td>Forceps</td><td>-</td><td>-</td><td>Forceps from any company can be used</td></tr><tr><td>Scissors</td><td>-</td><td>-</td><td>Scissors from any company can be used</td></tr><tr><td>Scalpel</td><td>Swann Morton</td><td>0501</td><td>Scalpels from any company can be used</td></tr><tr><td>70% ethanol</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Gauze pads</td><td>NOBAMED</td><td>875420</td><td>Gauze pads from any provider can be used</td></tr><tr><td>Foot planer</td><td>Credo Solingen</td><td>1510</td><td>Foot planer from any provider can be used</td></tr><tr><td>Petri dishes</td><td>TPP</td><td>93100</td><td>Petri dishes from any provider can be used</td></tr><tr><td>Metal filter</td><td>-</td><td>-</td><td>In-house 1 mm hole size metal filter</td></tr><tr><td>75 cm&lt;sup&gt;2&lt;/sup&gt; culture flasks</td><td>NUNC</td><td>156499</td><td>-</td></tr><tr><td>150 cm&lt;sup&gt;2&lt;/sup&gt; culture flasks</td><td>TTP</td><td>90151</td><td>-</td></tr><tr><td>0.05% Trypsin-EDTA solution</td><td>ThermoFisher Scientific</td><td>25300-062</td><td>Used to trypsinize cells when passaging</td></tr></tbody></table>

generation and culturing of primary human keratinocytes from adult skin

角质形成细胞的主要功能是提供表皮的结构完整性, 从而保持对外部世界的机械屏障。此外, 角质形成细胞扮演一个重要的角色, 在启动, 维护和调节表皮免疫反应的一部分, 是先天免疫系统反应抗原刺激的快速, 非特异性的方式。在这里, 我们描述了一个从成人皮肤分离的初级人类角质形成的协议, 并证明这些细胞反应钙诱导的终端分化, 通过增加表达的分化标记 involucrin。此外, 我们表明, 孤立的角质形成细胞是响应 IL-1β-induced 活化的胞内信号通路, 通过 p38 MAPK 通路的活化测量。我们一起介绍了一种从成人皮肤中分离和培养原代角质形成细胞的方法。由于角质形成细胞是表皮中的主要胞型, 因此该方法对皮肤生物学的分子机制 (体外) 的研究是有益的。

钙诱导的末端分化 人角质形成细胞接受治疗后, 钙14,15,16。主要的人角质形成细胞被隔离和培养, 如上述协议所述。当大约 50-60% 汇合, 细胞被刺激了与钙 (1.2 毫米) 或车和细胞的图片在天 0, 1 和2被采取了。图 1显示了在钙刺激下观察到的角质形成细胞的形态学变化。
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Watch this Scientific Journal Video about Generation and Culturing of Primary Human Keratinocytes from Adult Skin at JoVE.com

Generation and Culturing of Primary Human Keratinocytes from Adult Skin

人类皮肤作为抵御外部环境的第一条防线。我们提出了一种从成人皮肤中分离原代角质形成细胞的方法。这些孤立的角质形成细胞是有用的许多实验设置, 是一个非常适合的模型研究的分子机制在皮肤生物学体外.

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. ...

generation-culturing-primary-human-keratinocytes-from-adult

Research

JoVE Journal

Developmental Biology

16.2K 次观看.  Aarhus University Hospital. 人类皮肤作为抵御外部环境的第一条防线。我们提出了一种从成人皮肤中分离原代角质形成细胞的方法。这些孤立的角质形成细胞是有用的许多实验设置, 是一个非常适合的模型研究的分子机制在皮肤生物学体外.

视频: Generation and Culturing of Primary Human Keratinocytes from Adult Skin

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

转基因器官皮肤培养使用灭活人真皮代

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

科研

发育生物学

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

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

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

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 ...

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

Cultivating a Three-dimensional Reconstructed Human Epidermis at a Large Scale

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process and the characterization of the model is described. Neonatal primary keratinocytes are grown submerged on permeable polycarbonate inserts and lifted to the air-liquid interface three days after seeding. After fourteen days of stimulation with defined growth factors and ascorbic acid in high calcium culture medium, the model is fully differentiated. Histological analysis revealed a completely stratified epidermis, mimicking the morphology of native human skin. To characterize the model and its barrier functions, protein levels and localization specific for early-stage keratinocyte differentiation (i.e., keratin 10), late-stage differentiation (i.e., involucrin, loricrin, and filaggrin) and tissue adhesion (i.e., desmoglein 1), were assessed by immunofluorescence. The tissue barrier integrity was further evaluated by measuring transepithelial electrical resistance. Reconstructed human epidermis was responsive to proinflammatory stimuli (i.e., lipopolysaccharide and tumor necrosis factor alpha), leading to increased cytokine release (i.e., interleukin 1 alpha and interleukin 8). This protocol represents a straightforward and reproducible in vitro method to cultivate reconstructed human epidermis as a tool to assess environmental effects and a broad range of skin-related studies.

This protocol describes a straightforward method to cultivate three-dimensional reconstituted human epidermis in a reproducible and robust manner. Additionally, it characterizes the structure-function relationship of the epidermal barrier model. The biological responses of the reconstituted human epidermis upon proinflammatory stimuli are also presented.

大规模培育三维重建人类表皮

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process ...

生物工程

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 ...

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 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 ...

Human Skin Keratinocyte Isolation: A Method to Culture Primary Keratinocytes from Skin Samples

Johansen, C. et al. <a href="https://www.jove.com/v/56863"> Generation and Culturing of Primary Human Keratinocytes from Adult Skin</a>. J. Vis. Exp. (2017)
This video describes the isolation of keratinocytes&#8212;the most prominent cells of human skin&#39;s epidermal layer, by employing a combination of enzymatic digestion and mechanical disruption methods. This method helps in the preferential growth of keratinocytes over other skin cell types using a selective growth medium.

人皮肤角质形成细胞分离:一种从皮肤样本中培养原代角质形成细胞的方法

Johansen, C. et al.  Generation and Culturing of Primary Human Keratinocytes from Adult Skin. J. Vis. Exp. (2017)
This video describes the isolation of ...