Name: generation of genetically modified organotypic skin cultures using devitalized human dermis
Uploaded: 2015-12-14T14:00:00.000+00:00
Duration: 9 min 16 s
Description: Watch this Scientific Journal Video about Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis at JoVE.com

这项工作是supportedby美国癌症协会的研究学者格兰特（RSG-12-148-01-DDC）和CIRM基础生物学奖（RB4-05779）。

根据加州大学圣迭戈分校的研究伦理委员会的指导方针进行了人体皮肤的协议。人的皮肤可以从废弃的手术样本中获得，或从皮肤银行购买（皮肤银行被列在材料/设备表 ）。其中皮肤是从主或施主的年龄衍生的位置不是本实验只要基底膜区蛋白（胶原蛋白/层粘连蛋白）在真皮临界不会降解。 1.准备失活的人真皮的<ol><li>在接收到人体皮肤时，将组织在组织培养罩解冻（〜3-5分钟）。根据皮肤的大小，放置解冻组织在一个无菌的一次性50ml锥形管或125毫升瓶装。从皮肤银行皮肤的典型大小为0.75平方英尺。 </li><li>填充容器75％满，支持4倍青霉素和链霉素（笔/链球菌）的1X PBS。剧烈摇晃5分钟和处置的液体。重复此步骤3次以上。 </li><li>在最后一次洗涤后，将组织转移到新的试管/瓶，加上新的4倍青霉素/链霉素/ PBS中以填充容器的75％。孵育在组织培养箱此混合物在37℃下进行2周，以允许从表皮真皮的分离。 </li><li>在无菌条件下，使用镊子剥离表皮从真皮。真皮是全白，而表皮会有着色根据供体（ 图1A）的比赛。根据该机构的协议处理人体组织丢弃表皮。 </li><li>将在管或瓶真皮和4倍笔/链球菌稀释于1×PBS中剧烈振荡（5分钟洗4次）清洗。在最后一次洗涤后，将真皮转移到新的试管/瓶在4倍青霉素/链霉素稀释于1×PBS中，并储存在4℃直至准备使用。真皮可以储存在4℃下长达一年。 </li></OL&gt; 2.遗传修饰小学的人角质形成细胞注:生长在完全DMEM培养基[DMEM + 10％胎牛血清（FBS）+青霉素/链霉素]，制造病毒感染的原代人角质形成细胞用嗜凤细胞。病毒包装基因编码蛋白质如的gag-pol和信封被稳定地整合到凤细胞基因组中时，用逆转录病毒载体转染其允许病毒的生产。凤凰细胞可被转染的高效率，可用于高病毒效价的生产。从这个包装线生产的病毒也可以感染多种哺乳动物细胞，包括人。 <ol><li>第1天:转染前一天，种子凤细胞在6孔板中以每孔80万细胞在完全DMEM培养基中的密度。 </li><li>第2天:转染的当天，用3微克的逆转录病毒载体的每孔。等分试样3微克逆转录病毒载体导入1.5ml试管。使用100μ组合;升的DMEM加6微升转染试剂的每3微克载体的构建。拌6微升转染试剂用在1.5ml管100微升DMEM中。 （对所使用的逆转录病毒载体中列出的设备/材料表）。 <ol><li>孵育5分钟，并添加106微升混合物到预等分管含有3微克的逆转录病毒载体的构建。混合并在室温下孵育30分钟。添加这种混合物全部滴凤凰细胞的每一个孔。 </li></ol></li><li>第3天:转染后的第二天，从凤凰细胞取出媒体和加入2毫升新鲜完整的DMEM培养基。在同一天，种子初级人角质形成细胞到6孔板中，每孔75000个细胞的密度。保持在角质形成细胞无血清培养基（KCSFM）抗生素（青霉素/链霉素）的角质形成细胞。 注:原代人角质形成细胞购买。细胞可以从多家供应商（在材料/设备表中列出）购买。 </li><li>第4天:HAR从转染的凤细胞，其现在包含病毒颗粒归属介质。通过使用注射器0.45μm的过滤器传递介质（以除去任何污染的凤细胞），并放置2 ml的到角质形成细胞（前一天接种在75000个细胞/孔:参见步骤2.3）。加入聚凝胺（5微克/毫升）向媒体帮助调解感染过程。病毒滴度是〜1×10 7 TU /毫升。 <ol><li>旋6孔板在离心机中，在200×g离心1小时，在室温。旋转后，取出含有病毒的媒体和添加KCSFM前，用1×PBS洗一次细胞。 </li></ol></li><li>第5天:感染同一批次的角质形成细胞的再按照步骤2.4。 注意:选择使用药物的角化细胞是否存在的逆转录病毒载体的选择标记和扩大用于下游分析或应用，例如器官型培养使用。 </li></ol> 3.设置器官皮肤文化<ol><li>构建从实验室中常用的材料或盒录像带器官可以通过金属加工店进行定制（ 图2A - F）。从3.5厘米组织培养板使这些磁带盒，使用烧灼来从一个3.5厘米培养皿（ 图2A-B）的盖子的中心除去1.0厘米×1.0厘米的正方形。为此无菌条件下。 <ol><li>附加方形栓用透明指甲油（ 图2C）纸盒（3.5 cm培养皿的盖子）的底部。允许5分钟的指甲油干，翻转磁带上，使其搁在方形栓（ 图2D）。放置盒插入直径6cm培养皿。 注:方形栓可以从各种手工艺品商店购买。 </li></ol></li><li>制备角质形成细胞生长培养基（KGM）由66％的DMEM，22％的Ham的F12，100单位/毫升青霉素，100微克/ ml链霉素，10％FBS，2.67微克/毫升腺嘌呤，40 N克/ ml氢化可的松，10纳克/毫升的霍乱毒素，4.94微克/毫升的胰岛素，5微克/毫升转铁蛋白，1.36纳克/毫升三碘左旋甲腺氨酸，10纳克/毫升的EGF，和10微克/毫升盐酸环丙沙​​星。用0.22微米的过滤和储存过滤介质在4°C，直到准备使用。 </li><li>取真皮出在4x青霉素/链霉素+ PBS溶液和在KGM中洗2次，然后孵育在KGM中，在37℃下在使用前两天。 </li><li>切割用手术刀真皮至1.5厘米×1.5厘米大小的块，然后放置到器官盒。放置真皮朝上真皮的顶部。真皮的顶部将是进入与角质形成细胞（ 图1B）接触的一侧。 </li><li>将预切真皮到器官盒朝上（图3A）的真皮的顶侧的方孔。 </li><li>翻转整个器官盒含有真皮过使用镊子（图3B）。冰上解冻的细胞外基质的解决方案。使用针头和注射器绘制出100微升每盒细胞外基质的解决方案。加入5滴到真皮（光面）的底部，并轻微振荡以确保整个真皮（图3B）的均匀分布。 </li><li>允许用于商业细胞外基质溶液5分钟后，完全固化（图3C）。凝固后，使用镊子翻动录像带回去。添加4毫升KGM到直径6cm培养皿。 </li><li>放置0.5-1百万遗传修饰的角质形成细胞在含有真皮（图3D）的器官型盒。 <ol><li> Trypsinize角化细胞通过将4 ml的0.05％胰蛋白酶的10个到厘米板5分钟，骤冷用4ml完全DMEM介质。 </li><li>计数使用血球的角化细胞，然后降速，在200×g离心5分钟。重悬0.5-1百万个细胞在90μ（取决于可用的细胞数）升KGM和放置所有单元格到真皮层。 注意:它放置细胞相同数量的上真皮同样的实验中，以确保出现在再生表皮的厚度的任何差异不是由于在初始细胞数的差异是很重要的。在真皮层放置少于0.5万个细胞可导致分层穷人和分化。 </li></ol></li><li>隔日变化对器官文化KGM媒体。嘉实组织对真皮的角质形成细胞放置后1-14天。表皮完整的分层和分化通常在5天后发生。 </li></ol>

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遗传操作在人皮肤器官型培养提供许多优点常用研究2D培养细胞以及小鼠模型。 2D培养缺乏完整的组织和器官中发现的三维细胞 - 细胞和细胞 - 细胞外基质相互作用。最近的研究还发现，与三维培养的细胞显示多基因表达的相似性对原代人皮肤肿瘤16的二维和三维培养的皮肤癌细胞之间的巨大差异。人体器官皮肤的文化也有过小鼠模型的几个优点。小鼠模型是耗时且昂贵的产生以及人类和?...

人类表皮是一个分层上皮，它可通过细胞外基质被称为基底膜zone.The表皮不仅作为不可渗透的屏障，以防止水分的损失，而且作为防御的第一道防线，以保护连接到下面的真皮身体从国外和有毒物质1。基底层，这是表皮的最深处层，包含表皮干细胞和祖细胞的产生的已分化后代，形成了表皮2的其余部分。表皮祖细胞分化它们迁移向上，以形成被称为棘层3分化的细胞的第一层。在棘层，细胞打开角蛋白1和10的表达，其然后提供强度来承受物理应力为表皮的分化层。作为棘层细胞进一步分化，它们向上移动在表皮为米颗粒层其特征是，形成的透明角质和片状颗粒以及其组装下面质膜结构蛋白。随着细胞继续在分化过程中，质膜下方的蛋白质交联到对方，而层状颗粒从细胞挤出 ​​，以形成富含脂质屏障称为角质层4。 疾病涉及在表皮生长和分化的影响〜人口5的20％的改变。因此，了解该方法的机制是非常重要的。因为许多这些疾病的表现是在细胞-细胞或细胞-基质接触队伍，器官型培养其中人表皮复原在3D环境中已创建6-10。这些方法通常包括使用接种在细胞外基质的主要或转化角化细胞如失活的人类的真皮层，基质胶或胶原蛋白。 要明白，在表皮生长和分化的重要的基因调控机制，角化细胞可以遗传通过逆转录病毒载体操纵以拦截或过表达的基因在二维培养，然后在三维重构。这些方法已被广泛用于表征涉及表皮干细胞和祖细胞的自我更新和分化以及进展为肿瘤11-21基因。这里，提供了深入协议，关于如何通过利用逆转录病毒的改变在表皮器官型培养的基因表达。

<table><tbody><tr><td>Human skin</td><td>New York Firefighters Skin Bank</td><td>&lt;a href=&quot;http://www.cornellsurgery.org/pro/services/burn-surgery/skin-bank.html&quot;&gt;http://www.cornellsurgery.org/pro/services/burn-surgery/skin-bank.html&lt;/a&gt;</td><td></td></tr><tr><td>PEN/STREP</td><td>GIBCO</td><td>15140-122</td><td></td></tr><tr><td>amphotropic phoenix cell lines</td><td>ATCC</td><td>CRL-3213</td><td></td></tr><tr><td>FUGENE 6 transfection reagent</td><td>Promega</td><td>E2691</td><td></td></tr><tr><td>Keratinocyte Media (KCSFM)</td><td>Life Technologies</td><td>17005042</td><td></td></tr><tr><td>DMEM</td><td>GIBCO</td><td>11995</td><td></td></tr><tr><td>Ham&#039;s F12</td><td>Cambrex</td><td>12-615F</td><td></td></tr><tr><td>FBS</td><td>GIBCO</td><td>10437-028</td><td></td></tr><tr><td>Adenine</td><td>Sigma</td><td>A-9795</td><td></td></tr><tr><td>Cholera Toxin</td><td>Sigma</td><td>&amp;nbsp;C-8052</td><td></td></tr><tr><td>Hydrocortisone</td><td>Calbiochem</td><td>3896</td><td></td></tr><tr><td>Insulin</td><td>Sigma</td><td>I-1882</td><td></td></tr><tr><td>EGF</td><td>Invitrogen</td><td>13247-051</td><td></td></tr><tr><td>Transferrin&amp;nbsp;</td><td>Sigma</td><td>T-0665</td><td></td></tr><tr><td>Ciprofloxacin Hydrochloride</td><td>Serologicals</td><td>89-001-1</td><td></td></tr><tr><td>cautery</td><td>Bovie Medical Corporation</td><td>AA01</td><td></td></tr><tr><td>Matrigel</td><td>Corning</td><td>354234</td><td></td></tr><tr><td>Keratin 1 antibody</td><td>Biolegend</td><td>PRB-149P</td><td></td></tr><tr><td>square pegs</td><td>Arts and crafts stores</td><td></td><td></td></tr><tr><td>human neonatal keratinocytes</td><td>ATCC</td><td>PCS-200-010</td><td></td></tr><tr><td>human neonatal keratinocytes</td><td>Cell Applications</td><td>102K-05n</td><td></td></tr><tr><td>MSCV retroviral vector</td><td>Clontech</td><td>634401</td><td></td></tr><tr><td>LZRS retroviral vector</td><td>Addgene</td><td></td><td></td></tr><tr><td>pSuper.Retro.Puro Retroviral vector</td><td>Oligoengine</td><td>VEC-PRT-0002&amp;nbsp;</td><td></td></tr><tr><td>hexadimethrine bromide&amp;nbsp;</td><td>Sigma</td><td>H9268-5G</td><td></td></tr></tbody></table>

generation of genetically modified organotypic skin cultures using devitalized human dermis

器官型培养允许它模仿功能和其在体内的组织对应的生理3D环境进行细胞-细胞接触和细胞-基质相互作用至关重要的重构。这是通过皮肤器官培养忠实地概括了表皮分化和分层方案例证。原代人表皮角质形成细胞是通过逆转录病毒基因在那里可以很容易地过度表达或撞倒的基因操纵。这些遗传修饰的角质形成细胞随后可以用来再生人表皮在器官型培养物的皮肤提供了强大的模型来研究遗传途径冲击表皮生长，分化和疾病进展。这里介绍的协议描述方法以制备灭活人真皮以及遗传操作初级人角质形成细胞，以便产生皮肤器官培养物。再生人体皮肤可downstr使用EAM的应用，如基因表达分析，免疫染色，和染色质免疫沉淀，随后通过高通量测序。因此，这些代转基因器官皮肤培养物将允许基因是保持皮肤稳态的关键的确定。

在产生器官型人皮肤的第一步是从真皮除去表皮。皮肤在37℃下在4×青霉素/链霉素/ PBS两周孵化应该允许真皮从表皮（图1A）的分离。如果分离表皮和真皮层，很难然后将组织在37℃于4笔/链球菌/ PBS一个星期，然后再次尝试剥离使用镊子。 一个关键的灭活真皮再生人表皮的是，确保了角质形成细胞被放置在真皮的正确的一面。真皮具有顶部和底部。真皮的顶部是?...

Watch this Scientific Journal Video about Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis at JoVE.com

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

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 ...

generation-genetically-modified-organotypic-skin-cultures-using

Research

JoVE Journal

Developmental Biology

11.2K 次观看.  UCSD Stem Cell Program, University of California, San Diego. 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. 

视频: Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

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

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

三维全层皮肤的生成等效和自动伤人

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

生物工程

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

从脐带血源性多能干细胞中提取三维皮肤有机细胞

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls.
The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as &#34;bio-scaffolds&#34; for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications.
In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

We present a protocol to obtain cell-derived matrices rich in extracellular matrix proteins, using macromolecular crowders (MMC). In addition, we present a protocol which incorporates MMC in 3D organotypic skin co-culture generation, which reduces culture time while maintaining maturity of construct.

提高2D和3D皮肤<em&gt;体外</em&gt;模型使用大分子拥挤

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

禽胚胎皮肤外植体培养方案的全面回顾

Using Avian Skin Explants to Study Tissue Patterning and Organogenesis

The developing avian skin during embryogenesis is a unique model that can provide valuable insights into tissue patterning. Here three variations on skin explant cultures to examine different aspects of skin development are described. First, ex vivo organ cultures and manipulations offer researchers opportunities to observe and study the development of feather buds directly. Skin explant culture can grow for 7 days enabling direct analysis of cellular behavior and 4D imaging at intervals during this growth period. This also allows for physical and molecular manipulations of culture conditions to visualize tissue response. For example, growth factor-coated beads can be applied locally to induce changes in feather patterning in a limited area. Alternatively, viral transduction can be delivered globally in the culture media to up or downregulate gene expression. Second, the skin recombination protocol allows researchers to investigate tissue interactions between the epidermis and mesenchyme that are derived from different skin regions, different life stages, or different species. This affords an opportunity to test the time window in which the epithelium is competent to respond to signals and its ability to form different skin appendages in response to signals from different mesenchymal sources. Third, skin reconstitution using dissociated dermal cells overlaid with intact epithelium resets skin development and enables the study of the initial processes of periodic patterning. This approach also enhances our ability to manipulate gene expression among the dissociated cells before creating the reconstituted skin explant. This paper provides the three culture protocols and exemplary experiments to demonstrate their utility.

作者聚焦:深入了解当前的实验性禽皮外植体方法

Here we describe protocols for three types of avian embryonic skin explant cultures that can be used to examine tissue interactions, 4D imaging timelapse movie (3D plus time), global or local perturbation of molecular function, and systems biology characterization.

使用禽皮肤外植体研究组织模式和器官发生

The developing avian skin during embryogenesis is a unique model that can provide valuable insights into tissue patterning. Here three variations on skin ...

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 ...

Generation of Self-assembled Vascularized Human Skin Equivalents

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to study skin development, wound healing, and grafting techniques. Many HSEs continue to lack vasculature and are additionally analyzed through post-culture histological sectioning which limits volumetric assessment of the structure. Presented here is a straightforward protocol utilizing accessible materials to generate vascularized human skin equivalents (VHSE); further described are volumetric imaging and quantification techniques of these constructs. Briefly, VHSEs are constructed in 12 well culture inserts in which dermal and epidermal cells are seeded into rat tail collagen type I gel. The dermal compartment is made up of fibroblast and endothelial cells dispersed throughout collagen gel. The epidermal compartment is made up of keratinocytes (skin epithelial cells) that differentiate at the air-liquid interface. Importantly, these methods are customizable based on needs of the researcher, with results demonstrating VHSE generation with two different fibroblast cell types: human dermal fibroblasts (hDF) and human lung fibroblasts (IMR90s). VHSEs were developed, imaged through confocal microscopy, and volumetrically analyzed using computational software at 4- and 8-week timepoints. An optimized process to fix, stain, image, and clear VHSEs for volumetric examination is described. This comprehensive model, imaging, and analysis techniques are readily customizable to the specific research needs of individual labs with or without prior HSE experience.

The goal of this protocol is to describe the generation and volumetric analysis of vascularized human skin equivalents using accessible and simple techniques for long term culture. To the extent possible, the rationale for steps is described to allow researchers the ability to customize based on their research needs.

自组装血管化人体皮肤等价物的生成

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to ...

转基因器官皮肤培养使用灭活人真皮代

摘要

探索更多视频

此视频中的章节

3D 脊髓黑色素瘤球形皮肤模型

成人皮肤角质形成细胞的生成与培养

三维全层皮肤的生成等效和自动伤人

从脐带血源性多能干细胞中提取三维皮肤有机细胞

提高2D和3D皮肤

使用禽皮肤外植体研究组织模式和器官发生

使用禽皮肤外植体研究组织模式和器官发生

使用禽皮肤外植体研究组织模式和器官发生

大规模培育三维重建人类表皮

自组装血管化人体皮肤等价物的生成