Name: skin punch biopsy explant culture for derivation of primary human fibroblasts
Uploaded: 2013-07-07T13:00:00
Description: Watch this Scientific Journal Video about Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts at JoVE.com

这个协议是由加州再生医学研究所（CIRM，TR1-01246）和帕金森联盟的发展。

皮肤穿刺活检获得使用标准程序（ 例如采用4mm圆Visipunch仪器获得）应保持在完全DMEM 20％FBS媒体冰上。一旦样品在实验室里，已经到达处理活检尽快。 1。制备皮肤穿刺活检 步骤1.1-1.3的内部进行生物安全柜 <ol><li>预先准备：0.1％明胶的6孔板的各孔中加入1毫升。设置板预留30-60分钟。吸出的明胶溶液，然后向每孔中加入800μl的完整的DM...

<ol>
	<li>Nguyen, H. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LRRK2+mutant+iPSC-derived+DA+neurons+demonstrate+increased+susceptibility+to+oxidative+stress.">LRRK2 mutant iPSC-derived DA neurons demonstrate increased susceptibility to oxidative stress.</a> Cell Stem Cell. 8, 267-280 (2011).</li><li>Mak, S. K., Tewari, D., Tetrud, J. W., Langston, J. W., Schule, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunction+in+skin+fibroblasts+from+a+Parkinson's+disease+patient+with+an+alpha-synuclein+triplication.">Mitochondrial dysfunction in skin fibroblasts from a Parkinson's disease patient with an alpha-synuclein triplication.</a> Journal of Parkinson's Disease. 1, 175-183 (2011).</li><li>Punch Zuber, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=biopsy+of+the+skin.">biopsy of the skin.</a> Am. Fam. Physician. 65, 1155-1164 (2002).</li><li>Takashima, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+fibroblast+cultures.">Establishment of fibroblast cultures.</a> Curr. Protoc. Cell Biol. Chapter 2, Unit 2, 1 (2001).</li><li>Kuroda, K., Tajima, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HSP47+is+a+useful+marker+for+skin+fibroblasts+in+formalin-fixed,+paraffin+embedded+tissue+specimens.">HSP47 is a useful marker for skin fibroblasts in formalin-fixed, paraffin embedded tissue specimens.</a> J. Cutan. Pathol. , 241-246 (2004).</li></ol>

这个协议中，可以得到相对纯培养的皮肤成纤维细胞。成纤维细胞特征的形态学特征拉长，纺锤状细胞体，圆形至椭圆形细胞核，和成纤维细胞生长汇合时，对齐和捆绑。媒体支持如角质形成细胞需要额外补充和生长因子的，或有丝分裂活性，而其他的细胞群中，这允许相对较纯的成纤维细胞培养的成纤维细胞的生长。 随后成纤维细胞传代与胰蛋白酶在1:3至1:5的比例?...

<table border="1">
<tbody>
 <tr>
 <td>Name of Equipment</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 </tr>
 <tr>
 <td>Dissecting microscope</td>
 <td>Leica</td>
 <td>S6E</td>
 </tr>
 <tr>
 <td>10 cm Tissue Culture Petri dish</td>
 <td>VWR</td>
 <td>25382-166</td>
 </tr>
 <tr>
 <td>6-well Tissue Culture plate </td>
 <td>VWR</td>
 <td>73520-906</td>
 </tr>
 <tr>
 <td>Stainless steel disposable sterile scalpels (2), blade No#15, Miltex</td>
 <td>VWR</td>
 <td>21909-660</td>
 </tr>
 <tr>
 <td>Sterile pointed-tip forceps</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>50 mL Conical tubes</td>
 <td>VWR</td>
 <td>21008-940</td>
 </tr>
 <tr>
 <td>2 mL Serological Pipettes</td>
 <td>VWR</td>
 <td>89130-894</td>
 </tr>
 <tr>
 <td>5 mL Serological Pipettes</td>
 <td>VWR</td>
 <td>89130-896</td>
 </tr>
 <tr>
 <td>10 mL Serological Pipettes</td>
 <td>VWR</td>
 <td>89130-898</td>
 </tr>
 <tr>
 <td>Pasteur Pipettes</td>
 <td>VWR</td>
 <td>14672-380</td>
 </tr>
 </tbody>
</table>
Table A. Table of specific reagents and equipments.
 <table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent</td>
 <td>Company </td>
 <td>Catalogue Number</td>
 </tr>
 <tr>
 <td>1X DMEM: High Glucose</td>
 <td>Invitrogen/Gibco</td>
 <td>11960-069</td>
 </tr>
 <tr>
 <td>20% Fetal Bovine Serum</td>
 <td>Invitrogen/Gibco</td>
 <td>26140-079</td>
 </tr>
 <tr>
 <td>1X L-Glutamine</td>
 <td>Invitrogen/Gibco</td>
 <td>25030-164</td>
 </tr>
 <tr>
 <td>1X MEM Non-essential Amino Acids </td>
 <td>Invitrogen/Gibco</td>
 <td>11140-076</td>
 </tr>
 <tr>
 <td>1X Penicillin/Streptomycin</td>
 <td>Invitrogen/Gibco</td>
 <td>15140-163</td>
 </tr>
 </tbody>
 </table>
Table B. Reagents.

skin punch biopsy explant culture for derivation of primary human fibroblasts

组织和细胞系来自个人与疾病的理想来源，以研究疾病相关的细胞表型。已成功应用于病人在本协议中的成纤维细胞在诱导多能干细胞疾病模型的推导。这些成纤维细胞的早期通道也可以用于基于细胞的功能分析研究特定的疾病的途径，机制2和随后的药物筛选方法。超过酶的程序提交的协议的优点1）再现性的技术从来自年龄较大的病人， 例如，患者的影响与帕金森氏病，2）在更具挑战性的的方法在技术上简单的方法使用酶处理的组织的小金额，和3 ）的时间代价：这个协议需要15-20分钟，可以立即进行活检后到达。酶处理可能需要长达4个小时，并有问题过量，降低细胞活力和随后附着的细胞时，没有妥善处理。本协议描述的解剖和准备一个4毫米的推导成纤维细胞培养人体皮肤活检，并具有非常高的成功率，处理病人源性组织样品时，这是非常重要的。在这种文化中，角质形成细胞活检组织内迁移出后的第一周准备。后的第一个产物，角质细胞，成纤维细胞出现7-10天。与20％FBS高糖DMEM培养基，有利于在角质形成细胞和成纤维细胞，成纤维细胞生长长满角质细胞。已稀释后第2代角质形成细胞产生相对均匀的成纤维细胞培养成纤维细胞的标记SERPINH1（HSP-47）表达。使用这种方法，所产生15-20万成纤维细胞可以在4-8周后细胞银行。皮肤剥离大约需要15-20分钟，细胞，然后每天一次监测在显微镜下，和媒体改变附件和细胞的产物，后每隔2-3天。

角质形成细胞生长的活检件只要48小时后，解剖。第一成纤维细胞的生长，可以观察到约1个星期后处理。一旦井已达到汇合，成纤维细胞传代两个通道达到三个150公分瓶（T150）和细胞冷冻保存。我们用此方法15-20万细胞生成。成纤维细胞是阳性的反SERPINH1（也被称为热休克蛋白47）（ 图1），这是定位于内质网的胶原特异性分子伴侣。 HSP47的皮肤成纤维细胞胶原的生物合成（黑田等?...

Watch this Scientific Journal Video about Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts at JoVE.com

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

成纤维细胞植体培养协议，从人体皮肤打孔活检是一种技术上的强大和简单的方法来获取皮肤细胞银行约15-20万个细胞在低通道数的4-8周内。

推导人类纤维原细胞的皮肤穿刺活检植文化

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts ...

Research

JoVE Journal

Medicine

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

从人类乳腺成纤维细胞癌相关的成纤维细胞的实验发电（CAFS）

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

科研

医学

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

利用人类纤维原细胞线粒体表型的监测领域的帕金森氏病

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

In vitro Organoid Culture of Primary Mouse Colon Tumors

In vitro Organoid Culture of Primary Mouse Colon Tumors

在体外化培养基层小鼠结肠肿瘤

Several  human and murine colon cancer cell lines have been established, physiologic  integrity of colon tumors such as multiple cell layers, basal-apical ...

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

三维组织培养模型研究人类原发性骨髓和恶性肿瘤

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Primary Culture of Human Vestibular Schwannomas

人体前庭神经鞘瘤的原代培养

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in ...

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#246;m Technique

Human Skeletal Muscle Biopsy Procedures Using the Modified BergstrÃƒÆ’Ã‚Â¶m Technique

人骨骼肌活检手术使用修改伯格斯坦技术

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

从整体组织和活检建立人类上皮Enteroids和Colonoids的

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

人的<em&gt;股外侧肌</em&gt;骨骼肌活检使用韦尔 - Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and ...

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

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 ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

人垂体腺瘤细胞的三维海藻酸钠珠文化

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...