作者要感谢费德里卡Zanzottera博士对促进执行流式细胞分析研究。

伦理陈述:自协议的临床应用程序需要使用患者的皮肤自体组织，其在体外表征同源皮肤组织在艾米利亚-罗马涅地区的皮肤银行下列国家规定的采伐，加工指引临床使用之前进行和分发用于移植的组织（CNT 2013）。 1.临床应用生物综合楼注:该协议是基于临床的使用Rigeneracons（组织干扰物）和Rigenera机（组织干扰物系统）（ 图1A）。组织破坏剂是能够扰乱小片使用由六角形刀片和过滤的细胞和细胞外基质的成分具有约50微米的截止提供一网格组织的人体组织的生物医学干扰物。 <ol><li>通过双向收集患者的skinsamplesOPSY冲头（ 图1B）和分解加入1ml生理盐水，每件以获得自体微移植物6,7,9（或见步骤2.1）。 </li><li>放置1毫升胶原海绵的微移植物的（ 图1C）toform生物复合物用于临床应用。 </li><li>培养另一1毫升上在6ml补充有10％胎牛血清的DMEM培养基中的存在胶原海绵的微移植物的在37℃在5％CO 2的潮湿气氛中。 </li><li>以下培养3天后，固定生物复合物用0.3％多聚甲醛在室温10分钟。倾直接特定分配器到样品的石蜡。得到的切片用厚度为5μm的切片，并在玻璃载玻片放直接。 </li><li>浸泡5微米石蜡切片中用于组织学分析的玻璃，含有15 - 20毫升二甲苯（市售 - 间 - 二甲苯（40混合 - 65％），对二甲苯（20％），邻二甲苯（20％ ）和ETH基苯（6 - 20％）和甲苯，三甲基苯，苯酚，噻吩，吡啶和硫化氢）的每个3分钟的痕迹。 </li><li>沉浸在降低等级的乙醇的切片（100％至70％）（100％乙醇1小时，95％的乙醇1小时后，80％的乙醇1小时，70％的乙醇进行1小时），然后去离子水进行去paraffinizing和再水化的章节。 </li><li>染色切片与1 - 2毫升1克/ L Ematoxylin为1 - 2分钟，随后在水中漂洗，以除去任何Ematoxylin盈余。 </li><li>染色为4的部分 - 与曙红Y醇溶液5分钟，在浓度与70％乙醇混合的1％，而在用水稀释。 </li><li>使用1 - 2毫升曙红Y为每张幻灯片部分和流动自来水下冲洗。 </li><li>浸入部分中增加的乙醇等级（见步骤1.6），最后，在二甲苯中的通道1小时，盖玻片与基于安装mediumand放大100倍（ 图1D）在光学显微镜下观察之后。 </li> &lt;/ OL&gt; 2.收集，解体并在组织的体外分析<ol><li>分别为0.6毫米1毫米或2毫米的4种不同的多器官和/或多的行李箱区域的厚度用皮，走自主皮肤组织，乳头去epidermized真皮（DED）或真皮网状层（真皮）从40到55年的范围内组织的捐助者，下面就采收，加工和销售用于移植组织国家规则（CNT 2013年）。 <ol><li>轻轻冲洗在0.9％NaCl溶液的所有样品将它们在培养皿中在轨道摇动器5分钟。 </li><li>使用5毫米活检穿孔，创建样品它是均匀的，从皮肤组织，扣减和真皮直径和分解之前称重所有组织标本。 </li><li>插入皮肤组织，扣减或分别真皮，在组织破坏剂八个，三个或四个均匀样品，加入1.5毫升的解聚盐溶液。 </li><li>执行不同对于所有组织样品解聚的时间在表1中所指示的。 </li><li>使用从完整的组织样本作为对照衍生冲床活检的相应数字。 </li><li>机械解聚后，吸含有微移植物和地点分别在一个12孔板的单个孔的每个样品的盐溶液。为进行完整控制冲床活检相同的协议。 </li><li>添加补充有10％胎牛血清（FBS）和抗生素，以每个样品的RPMI 1640培养基1ml中。 </li><li>立即评估细胞生存力。到每个孔中含有微移植物（分别由皮肤组织，扣减或真皮八个，三个或四个均匀样品同时分解获得）添加含有0.5毫克/毫升的MTT（3- [介质1毫升4,5-二甲基吡啶-2-基] -2,5-二苯基溴化）溶液，并在5％CO 2 /空气的气氛中，在37℃孵育3小时。为进行完整控制相同的协议冲床活检。 </li><li>温育后，除去所有含培养基的MTT，并添加到每个样品1毫升二甲基亚砜（DMSO）中10分钟。 </li><li>转移的每个样品和DMSO在反应杯并使用分光光度计在570纳米处光密度（OD）读取。计算细胞存活率作为吸光度在570nm和该比在解聚之前使用组织的克数（克）的重量。为进行完整控制冲床活检相同的协议。 </li></ol></li><li>机械分解后，吸出含有皮肤组织，单个供体的扣减和真皮样品衍生微移植盐溶液。 <ol><li>分别将每个样品中的12孔平板的单个孔或在用于分别细胞活力测试和形态分析的培养烧瓶中。 </li><li>培养微移植物加入1ml（12孔板）或5毫升（培养瓶）的RPMI补充有10％FBS的1640培养基和抗生素在37℃在5％CO 2 / Al的气氛r表示24小时或7天。 </li><li>后24小时或7天（重复步骤2.1.8-2.1.10）评估细胞活力。 </li><li> 24小时后，并在烧瓶中培养7天后用光学显微镜进行形态分析评估细胞悬浮液的存在。 </li><li>分析真皮的样品阳性的间质和造血细胞标志物，包括CD146，CD34和CD45抗原通过FACS分析6。 </li><li>在层流罩种子每个微接枝和各组织样本的相应小片段不完全分解的（由皮肤组织，分别扣减或真皮八个，三个或四个均匀样品同时分解获得）（&gt; 50微米的尺寸后在含有5％羊血肉汤100μl的哥伦比亚琼脂平板分解处理）。 </li><li>孵育在37℃的板三天，以评估不育上哥伦比亚琼脂板上进行微生物分析11。 </li></ol></li></ol>

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作者安东尼奥·格拉齐亚诺是人类脑电波SRL，生产和市场Rigenera系统的科学主任。笔者莱蒂齐亚已找到是人类脑电波SRL科学分部的合作者

这一初步研究表明，通过该协议得到微移植物可与胶原海绵结合，如已经报道了其它临床应用中，以优化微移植物的植入物9的效力，10，特别是，本研究中报道的生物的能力-complexes，通过微移植物和胶原海绵构成的，之后，从临床应用30天佐剂的腿病变的伤口愈​​合。此外， 在体外结果提供的证据有关此协议的有效性来分解三个不同的皮肤组织，保持可观的细胞存活率?...

在过去的几年中，一些新的方法，已经开发了生物技术在手术过程中，得到的自体细胞的悬浮液的领域中，以治疗急性和慢性皮肤损伤提供一步法治疗。而且，从创伤，糖尿病，感染和其他原因造成的急性，但主要是慢性伤口的管理，仍然具有挑战性。有越来越多的证据表明，慢性伤口已成为严重的全球性健康问题，引起全世界对1的医疗保健系统巨大的财政负担。 为了增加在皮肤损伤的治疗成功率，缺乏广泛操纵（包括蜂窝增强）和无菌条件下的维护是必要的，以创建可以在的受损区域被立即施加一个细胞悬浮液患者中，从而避免在洁净室较长的处理，如细胞工厂。圣从小皮肤活检，研磨，离心等分离方法（ 例如，酶或机械）arting，经常用于获得一个细胞悬浮液，其可在生长培养基中培养。所有的这些方法通常需要执行的很长一段时间，强调细胞结构，并导致降低细胞生存力的。另一个显著方面是获得准备好由临床医师可使用自体的细胞悬浮液，例如，以修复受损的区域。此外，众所周知，自体组织移植物存活的过户手续于通过感应和传导2,3的原则接收站点，最终生存下来。理想的移植组织应该随时可用并具有低抗原性和供区的发病率4。 这些证据的基础上，本研究的第一个目的是创建适合自体生物复合物在组织修复的临床应用。为此，我们描述了一个新的方法，以从被这个协议分类的皮肤组织开始自体微移植。案例呈现在本文中也描述为临床应用该协议结合胶原蛋白海绵获得自体微移植。这种方法已被报道为在人组织5的机械分解效率，并已用于临床移植物和真皮组织6,7的再生以及用于在口腔颌面外科8-10结缔组织再生疗法。 此外，本研究的第二个目的是皮肤组织的本协议其分解后的生物学特性。为此目的，皮肤组织的不同的同源样品来自不同的多器官的躯干区衍生和/或多组织捐赠者进行了处理上的收获，加工下列国家的规则和分配在艾米利亚 - 罗马涅地区的皮肤银行移植组织（CNT 2013年）。 病例报告: 显示由于车祸复杂外伤35岁的女病人被送往医院安科纳的重症监护病房。病人表现的腿感染，由于一个开放的伤口，并用外固定稳定复合骨折。两个病灶清除，实施时间与伤口成为负压治疗（VAC疗法）后的清洁和骨膜出现了健康，我们就从恢复两个月后，应用的协议。这个系统分解后，得到了微移植物被用来创建生物复合物胶原海绵这些建议后来为了植入，调查其对病灶修复功效。

<table><tbody><tr><td>Rigenera Machine</td><td>Human Brain Wave</td><td>79210R</td><td></td></tr><tr><td>Rigeneracons</td><td>Human Brain Wave</td><td>79450S</td><td></td></tr><tr><td>MTT</td><td>Roche Diagnostic GmbH</td><td>11465007001</td><td></td></tr><tr><td>RPMI medium</td><td>PBI International</td><td>733-2292</td><td></td></tr><tr><td>DMEM medium</td><td>PBI International</td><td>F 0415</td><td></td></tr><tr><td>Antibiotics</td><td>Biological Industries PBI</td><td>03-038-1 &amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Antibodies for FACS Analysis</td><td>eBiosciences</td><td>code 12-1469 for CD146-P; code 17-0349&amp;nbsp; for CD34-APC&amp;nbsp;</td><td></td></tr><tr><td>Columbia agar</td><td>BioMerieux Company</td><td>43041</td><td></td></tr><tr><td>Condress&amp;reg;</td><td>Abiogen Pharma</td><td></td><td>collagen sponge used for bio-complexes</td></tr><tr><td>DMSO&amp;nbsp;</td><td>Bioniche Pharma USA LLC, Lake Forest, IL</td><td></td><td></td></tr><tr><td>NaCl solution</td><td>Fresenius Kabi, Bad Homburg, Germany</td><td></td><td></td></tr><tr><td>Xylene&amp;nbsp;</td><td>Carlo Erba</td><td></td><td></td></tr></tbody></table>

tissue characterization after a new disaggregation method for skin micro-grafts generation

几种新的方法，已经开发了生物技术在手术过程中，得到的自体细胞的悬浮液的领域中，为了对急性和慢性皮肤损害提供一个步骤的治疗方法。而且，从创伤，糖尿病，感染和其他原因引起的慢性而且急性伤口的管理，仍然具有挑战性。在这项研究中，我们描述了一个新的方法来创建从单个患者的皮肤组织及其临床应用自体微移植。此外，还进行体外皮肤，去epidermized真皮（DED）和多器官和/或多组织捐助者的真皮源性皮肤组织的生物学特性。所有组织被这个新协议分类的，使我们能够得到可行的微移植物。特别是，我们报道了这一创新协议是能够创建自体微移植物和胶原海绵准备上皮损要施加由生物复合物。在克里尼上的腿病变自体生物复合物的校准应用也有报告，表示再epitalization过程和病变的柔软性两者的改进。此外，我们的体外模型表明，该系统机械解聚后的细胞活力保持在一段时间内培养可达七（7）天。我们还观察到，通过流式细胞术分析，从解聚获得的细胞的池是由几个细胞类型，包括间质干细胞，即施加在组织再生和修复的方法中的一个关键的作用，对它们的高的再生潜力。最后，我们表现出体外 ，在琼脂培养的菜时，此过程中保持微移植的不育。总之，我们的结论是这种新的再生方法可以是一个有希望的工具，临床医师在一个步骤上可行，无菌的，并且准备使用，可以单独或组合使用最常见的生物scaffo施加微移植物获得LDS。

在这个初步研究，第一个目的是探讨人类自体微移植与生物支持相结合，如胶原蛋白的生产生物复合物准备使用的能力。这些生物复合物在患者中植入 引起的车祸中（ 图2A）腿部病变和30天后（ 图2B）的组织修复相关的完整的再上皮中观察到。此外，临床随访上升5个月（ 图2C）后显示出已损坏的区域的良好?...

Watch this Scientific Journal Video about Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation at JoVE.com

Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation

该协议描述了一种新的方法来分解人体组织和​​创建自体微移植，与胶原蛋白海绵相结合，引起人体生物复合物准备皮损的治疗使用。另外，该系统保持在机械解聚后不同时间的微移植物的细胞生存力。

后皮肤微移植生成一个新的方法解聚表征的组织

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one ...

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21.5K 次观看.  Burns Centre and Emilia Romagna Regional Skin Bank. 该协议描述了一种新的方法来分解人体组织和​​创建自体微移植，与胶原蛋白海绵相结合，引起人体生物复合物准备皮损的治疗使用。另外，该系统保持在机械解聚后不同时间的微移植物的细胞生存力。 

视频: Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation

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

Isolation of Papillary and Reticular Fibroblasts from Human Skin by Fluorescence-activated Cell Sorting

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have traditionally been attributed to the superficial papillary or lower reticular dermis according to their histological localization. In mouse dermis, papillary and reticular fibroblasts originate from two different lineages with diverging functions regarding physiological and pathological processes and a distinct cell surface marker expression profile by which they can be distinguished.
Importantly, evidence from explant cultures from superficial and lower dermal layers suggest that at least two functionally distinct dermal fibroblasts lineages exist in human skin dermis as well. However, unlike for mouse skin, cell surface markers enabling the discrimination of different fibroblast subsets have not yet been established for human skin. We developed a novel protocol for the isolation of human papillary and reticular fibroblast populations via fluorescence-activated cell sorting (FACS) using the two cell surface markers Fibroblast Activation Protein (FAP) and Thymocyte antigen 1 (Thy1)/CD90. This method enables the isolation of pure fibroblast subsets without in vitro manipulation, which was shown to affect gene expression, thus permitting accurate functional analysis of human dermal fibroblast subsets in regard to tissue homeostasis or disease pathology.

This manuscript describes a FACS-based protocol for isolation of papillary and reticular fibroblasts from human skin. It circumvents in vitro culture which was inevitable with the commonly used isolation protocol via explant cultures. The emanating fibroblast subsets are functionally distinct and display differential gene expression and localization within the dermis.

荧光活化细胞分选分离人皮肤状和网状成纤维细胞

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have ...

遗传学

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

在压缩的胶原组织糜:含细胞Biotransplant单上演重建修复

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

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 ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

从表型分析和人体皮肤淋巴细胞分离<em&gt;离体</em&gt;细胞培养

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

免疫与感染

Apparatus for Harvesting Tissue Microcolumns

This manuscript describes the production process for a laboratory apparatus, made from off-the-shelf components, that can be used to collect microcolumns of full-thickness skin tissue. The small size of the microcolumns allows donor sites to heal quickly without causing donor site scarring, while harvesting full-thickness tissue enables the incorporation of all cellular and extracellular components of skin tissue, including those associated with deeper dermal regions and the adnexal skin structures, which have yet to be successfully reproduced using conventional tissue engineering techniques. The microcolumns can be applied directly into skin wounds to augment healing, or they can be used as the autologous cell/tissue source for other tissue engineering approaches. The harvesting needles are made by modifying standard hypodermic needles, and they can be used alone for harvesting small amounts of tissue or coupled with a simple suction-based collection system (also made from commonly available laboratory supplies) for high-volume harvesting to facilitate studies in large animal models.

Here we describe a protocol for producing harvesting needles that can be used to collect full-thickness skin tissue without causing donor site scarring. The needles can be combined with a simple collection system to achieve high-volume harvesting.

水样组织的采集装置

This manuscript describes the production process for a laboratory apparatus, made from off-the-shelf components, that can be used to collect microcolumns ...

Decellularization for the Preparation of Highly Preserved Human Acellular Skin Matrix for Regenerative Medicine

Extracellular matrix (ECM) provides biophysical and biochemical stimuli to support self-renewal, proliferation, survival, and differentiation of surrounding cells due to its content of diverse bioactive molecules. Due to these characteristics, the ECM has been recently considered a promising candidate for the creation of biological scaffolds to boost tissue regeneration. Emerging studies have demonstrated that decellularized human tissues could resemble the native ECM in their structural and biochemical profiles, preserving the three-dimensional (3D) architecture and the content of fundamental biological molecules. Hence, decellularized ECM can be employed to promote tissue remodeling, repair, and functional reconstruction of many organs. Selecting the appropriate decellularization procedure is crucial to obtain acellular tissues that retain the characteristics of the ideal microenvironment for cells. 
The protocol described here provides a detailed step-by-step description of the decellularization method to obtain a reproducible and effective cell-free biological ECM. Skin fragments from patients undergoing plastic surgery were scaled down and decellularized using a combination of sodium dodecylsulfate (SDS), Triton X-100, and antibiotics. To promote the regular and homogeneous transport of the solution through the samples, they were enclosed in embedding cassettes to ensure protection from mechanical insults. After the decellularization procedure, the snow-white color of skin fragments indicated complete and successful decellularization. Additionally, decellularized samples showed an intact and well-preserved architecture. The results suggest that the proposed decellularization method was effective, fast, and reproducible and protected samples from architectural damages.

Decellularized human skin is suitable for tissue regeneration. A major issue of decellularization is the preservation of the native architecture, along with the appropriate content of structural proteins, glycosaminoglycans (GAGs), and growth factors. The method proposed allows fast and effective decellularization, producing decellularized skin with well-preserved native features.

Extracellular matrix (ECM) provides biophysical and biochemical stimuli to support self-renewal, proliferation, survival, and differentiation of ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

在毛发再生的上皮 - 间充质信号的快速遗传分析

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

生物学

Xenograft Skin Model to Manipulate Human Immune Responses In Vivo

The human skin xenograft model, in which human donor skin is transplanted onto an immunodeficient mouse host, is an important option for translational research in skin immunology. Murine and human skin differ substantially in anatomy and immune cell composition. Therefore, traditional mouse models have limitations for dermatological research and drug discovery. However, successful xenotransplants are technically challenging and require optimal specimen and mouse graft site preparation for graft and host survival. The present protocol provides an optimized technique for transplanting human skin onto mice and discusses necessary considerations for downstream experimental aims. This report describes the appropriate preparation of a human donor skin sample, assembly of a surgical setup, mouse and surgical site preparation, skin transplantation, and post-surgical monitoring. Adherence to these methods allows for maintenance of xenografts for over 6 weeks post-surgery. The techniques outlined below allow maximum grafting efficiency due to the development of engineering controls, sterile technique, and pre- and post-surgical conditioning. Appropriate performance of the xenograft model results in long-lived human skin graft samples for experimental characterization of human skin and preclinical testing of compounds in vivo.

Xenograft Skin Model to Manipulate Human Immune Responses In Vivo

The present protocol describes how to graft human skin onto non-obese diabetic (NOD)-scid interleukin-2 gamma chain receptor (NSG) mice. A detailed description of the preparation of human skin for transplant, preparation of mice for transplant, transplantation of split-thickness human skin, and post-transplantation recovery procedure are included in the report.

异种移植皮肤模型在体内操纵人类免疫反应

The human skin xenograft model, in which human donor skin is transplanted onto an immunodeficient mouse host, is an important option for translational ...

后皮肤微移植生成一个新的方法解聚表征的组织

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