这项工作是由NIH / NCI资助R01CA - 118916

1。从人体皮肤中提取的上皮干细胞<ol><li>开始之前隔离上皮干细胞的过程，需要准备各自的媒体和试剂（见表1）。 </li><li>收集新鲜成人换装程序或打孔活检人类头皮，然后孵化的DMEM / 10％FBS / Dispase（4毫克/毫升）一夜之间在4 ° C。潜伏期为2-4小时，在37 ° C也是有效的。皮肤件应为1厘米，最大宽度允许酶渗透。 </li><li>消毒的培养皿转移到皮肤，从皮肤拉断每个头发抓住附近的皮肤表面发干，拉动坚决和顺利。选择基于他们在解剖显微镜下的形态休止阶段的卵泡，切出的凸起区域（图1A）转移卵泡到15毫升的消毒管。生长期毛囊也可以用来切上部1 / 3的卵泡，如果获得毛囊“隆起”的地区（图1B）。 </li><li>孵育15-20分钟，在室温下晃动定期在0.05％胰酶- EDTA（GIBCO）和Versene（0.53毫米EDTA 4Na，GIBCO公司）（1:1）混合的孤立的卵泡片段，添加4毫升的DMEM + 10 ％胎牛血清停止反应，5分钟800转的旋转。此外，毛囊组织片段植可以放在没有胰蛋白酶消化，从单一的毛囊细胞培养文化。 </li><li>弃上清小心（〜0.2-0.5毫升保存，以避免失去细胞），并重新暂停在1毫升KCM公司（角质形成细胞的培养基），无表皮生长因子。 </li></ol> 2。文化小学的上皮干细胞<ol><li>前培养的上皮干细胞，丝裂霉素C - 3T3 - 6300320细胞（饲养层细胞），应准备如下处理。 3T3 - 6300320细胞培养的DMEM与10％FBS治疗前。删除培养媒体，用PBS洗细胞两次，治疗与15μg/ mL的丝裂霉素C的DMEM无血清在2小时37 ° C，5％的CO 2的细胞。删除丝裂霉素C的DMEM，用PBS洗两次，细胞已经准备好使用。 （丝裂霉素C处理的3T3 - 6300320细胞还可以准备以前保存于-80 ° C.解冻和种子细胞培养初级上皮干细胞前一天，于37孵育℃，5％的CO 2。 150,000-200,000 6孔板，每孔细胞3T3 - 6300320建议。 </li><li>步骤1.5种子孤立毛囊干丝裂霉素细胞的C - KCM公司3T3 - 6300320细胞没有表皮生长因子在37℃过夜治疗℃，5％CO 2，含有表皮生长因子- KCM公司改为第二天。细胞是生长在37 ° C在潮湿的大气中含有5％的CO 2 。所有的角质形成细胞是美联储含有表皮生长因子与KCM公司每2天，并成长为14-20天。每天检查细胞在显微镜下。毛囊干细胞会形成菌落（图2A）。毛囊植后，将形成10-14天（图2B）的副产物。 </li></ol> 3。通道上皮干细胞<ol><li>用PBS洗涤细胞一次。卸下Versene（预热至RT）治疗饲养层细胞3T3 - 6300320。孵育文化Versene抛出5分钟，然后轻轻摇动和吸饲养层细胞。 </li><li>毛囊干细胞Versene或PBS一次清洗。 </li><li>加入预热（至37 ° C，临界）胰蛋白酶- EDTA（2X），并在37 ° C时7分钟或更长的时间，必要时对毛囊干细胞的分离。最长不超过15分钟是最好的。 </li><li>添加KCM公司W / O型表皮生长因子，以阻止胰蛋白酶，轻轻吸管分散细胞，在200克旋转下来，5分钟。 </li><li>与KCM公司暂停细胞无表皮生长因子，计数和丝裂霉素C处理的3T3 - 6300320细胞层上的重制版。 </li></ol> 4。不朽上皮干细胞原代细胞，甚至成人干细胞，达到串行通道和文化个月后的衰老。基层干细胞永生化是一种有效的的方式来缓解这个问题。 <ol><li>板小学在饲养层上的1通过上皮干细胞（3T3 - 6300320）（〜35万细胞/ 6孔板）和2天的文化。 </li><li>文化PA317 LXSN 16E6E7细胞株有一天播种后用DMEM含10％胎牛血清的主要上皮干细胞。 （此生产线生产amphotropic的逆转录病毒LXSN16E6E7编码的HPV16型E6和E7开放读码框架，可用于稳定感染和不朽多种细胞类型）。 </li><li>治疗2小时，用丝裂霉素C（3T3 - 6300320细胞相同的协议）LXSN 16E6E7 PA317细胞系。加丝裂霉素C治疗PA317 LXSN 16E6E7细胞株主上皮干细胞，共培养6天与表皮生长因子KCM公司。与表皮生长因子媒体续订新鲜KCM公司每2天。 </li><li>删除3T3 - 6300320 16E6E7 LXSN PA317细胞与Versene。添加丝裂霉素C处理3T3 - 6300320 NHP细胞（新霉素，潮霉素，嘌呤霉素抗性〜20细胞，每孔）的上皮干细胞。细胞被选中下添加0.2毫克/毫升的G418（GIBCO）itional 6天。续订与EGF媒体每隔2天的新鲜G418包含KCM公司。 尚存的上皮干细胞将是永生的干细胞。不PA317 LXSN 16E6E7细胞共同培养的主上皮干细胞的井应设置控制G418筛选，在这口井的所有细胞被杀死后经G418选择6天。 </li></ol> 5。代表性的成果通过皮肤上皮干细胞和永生化上皮干细胞的早期形式组成严密的殖民地饲养层细胞包围的小角质形成细胞（图2A）。如果在无饲养层，无血清媒体定义的补充的培养，干细胞会分散，不能形成紧殖民地（他们成长为单个细胞和小群）。永生上皮干细胞的维持稳定的表型为12个月的连续传代，但往往比主细胞形成更紧密的殖民地。他们没有形成锚地独立的殖民地，在软琼脂实验表明，这些永生细胞所不具备的特点癌细胞。小学和永生化上皮干细胞表达毛囊干细胞标记的细胞角蛋白15（图3A），并能分化成表皮，毛囊和皮脂腺的谱系（图3B - D）。 <img src="/files/ftp_upload/2561/2561fig1.jpg" alt="图1" /> 图1。弹拨的毛发。 dispase治疗皮肤采摘后，毛囊会出现一个细胞周围的头发底部球无论是俱乐部的休止期头发（一）或（B）与上皮细胞周围的头发长度的袖子生长期毛发。休止期隆起地区形成了一个独特的微解剖形态学地区，而生长期隆起明显。生长期灯泡包含矩阵角质形成细胞，占过境放大，也可以分离和培养的细胞。 <img src="/files/ftp_upload/2561/2561fig2.jpg" alt="图2" /> 图2干细胞培养。 （一）上皮干细胞，从皮肤形成紧上皮殖民地（通过电子邮件指定）时，KCM公司培养的饲养层细胞（由F指定）。 （二）毛囊植，引起上皮的副产物。休止期隆起地区（指定由T）连接到的菜和上皮细胞集落一起饲养层细胞包围。 <img src="/files/ftp_upload/2561/2561fig3.jpg" alt="图3" /> 图3。干细胞的分化。 （一）干细胞群，维持上皮标志物如细胞角蛋白15的表达。 （二）皮肤上皮干细胞可分化K6Hf表达确定的沿毛囊血统。 （三）被培养的细胞可以去epidermalized真皮或其他矩阵在气 - 液界面上，将形成一个与分层表皮角化层，颗粒层，棘层。 （四）干细胞可诱导沿油红积极球皮脂腺血统。

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细胞的提取和培养方法是惊人的轻便和可重复性。我们产生了几十个人在广泛的年龄范围，包括遗传性皮肤缺损18患者的上皮干细胞培养。最好是组织收获的一天开始的过程，但是细胞会继续生存在冰上媒体好几天了，如果需要，促进通宵航运。弃置换装皮肤产生数百个可行的毛囊细胞提取单细胞悬液。对于更有限的组织，如冲床从头皮活检，，外植体的文化可能会更有效，因为细胞胰酶...

角质形成细胞的媒体（KCM） [DMEM和火腿小号F12（GIBCO公司，3:1），腺嘌呤（Sigma公司，180毫米），10％胎牛血清（GIBCO公司），霍乱毒素（ICN，0.1纳米），青霉素/链霉素（GIBCO公司，为100 U / ml的100毫克/毫升，分别），氢化可的松（Sigma公司，0.4毫克/毫升，1.1毫米），T/T3（转GIBCO公司;，5微克/毫升，649纳米和三碘- L -甲状腺素，西格玛2纳米）胰岛素（Sigma公司，5毫克/毫升，862纳米），EGF（Sigma公司，10毫微克/毫升，1.6海里），pH值7.2]

isolation and culture of adult epithelial stem cells from human skin

所有组织自我更新的动态平衡，是依赖于成人干细胞。由于未分化的干细胞进行不对称分裂，它们所产生的子细胞，保留干细胞表型和过境扩增细胞（助教细胞），移植的干细胞小生境，进行快速增殖和晚期分化重新填充组织。 在表皮，毛囊和肠上皮干细胞已被确定为细胞在体外增殖潜能高，慢骑自行车的标签保留细胞在 体内 1-3 。成年后，组织特异性干细胞再生的组织在其居住，在正常的生理营业额，以及在压力4-5倍。此外，干细胞一般认为是多有力，拥有的能力上升到多种类型的细胞内组织6。例如，啮齿类动物毛囊干细胞可以生成表皮，皮脂腺和毛囊7-9。我们已经表明，从人类毛囊隆起地区干细胞表现出多潜能 10 。 干细胞已经成为在生物医学研究的宝贵工具，由于其作为体外研究发育生物学，分化，肿瘤的发生和其可能的治疗用途的系统实用工具。成人上皮干细胞很可能将在如外胚层发育不良，monilethrix，瑟顿综合征，Menkes病，遗传性大疱性表皮松解症和alopecias 11-13等疾病的治疗非常有用。此外，如烧伤，慢性伤口和溃疡等皮肤问题，将有利于干细胞相关治疗 14,15 。鉴于潜在的成年细胞重新编程为国家多能干（iPS细胞）16,17，可以提供方便和可扩展的成人干细胞在人体皮肤细胞诱导和下游治疗的疾病范围广泛，包括一个非常宝贵的资源糖尿病和帕金森氏病。

Watch this Scientific Journal Video about Isolation and Culture of Adult Epithelial Stem Cells from Human Skin at JoVE.com

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

隔离可行的成人上皮干细胞的人体皮肤快速，可靠的方法是描述。该方法利用酶消化，皮肤胶原质基质，毛囊和隔离的单细胞悬液或细胞培养的组织碎片采摘。

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 ...

isolation-and-culture-of-adult-epithelial-stem-cells-from-human-skin

Research

JoVE Journal

Biology

28.3K 次观看.  University of Massachusetts Medical School. 隔离可行的成人上皮干细胞的人体皮肤快速，可靠的方法是描述。该方法利用酶消化，皮肤胶原质基质，毛囊和隔离的单细胞悬液或细胞培养的组织碎片采摘。

视频: Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

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

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 distinct pools of SCs, which are located in the permanent portion of the HF, a region known as the bulge. These multipotent bulge SCs were initially identified as slow cycling label retaining cells; however, their isolation has been made feasible after identification of specific cell markers, such as CD34 and keratin 15 (K15). Here, we describe a robust method for isolating bulge SCs and epidermal keratinocytes from mouse HFs utilizing fluorescence activated cell-sorting (FACS) technology. Isolated hair follicle SCs (HFSCs) can be utilized in various in vivo grafting models and are a valuable in vitro model for studying the mechanisms that govern multipotency, quiescence and activation.

An ideal model for studying adult stem cell biology is the mouse hair follicle. Here we present a protocol for isolating different populations of hair follicles stem cells and epidermal keratinocytes, employing enzymatic digestion of mouse dorsal skin followed by FACS analysis.

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 ...

科研

发育生物学

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

从人类毛囊神经嵴干细胞的分离和培养的

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

神经科学

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. ...

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 of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

使用非整合方法分离成人人体皮肤纤维细胞与诱导多能干细胞的生成

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

生物工程

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 ...

生物学

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 ...

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 ...