Name: generating primary fibroblast cultures from mouse ear and tail tissues
Uploaded: 2016-01-10T13:00:00
Description: Watch this Scientific Journal Video about Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues at JoVE.com

This work was supported by the NRF grant HUJ-CREATE - Cellular and Molecular Mechanisms of Inflammation.

小鼠饲养在无病原体条件符合机构的指导方针，直到安乐死（机构动物护理和使用委员会（IACUC）的指导方针，在新加坡国立大学和国家咨询委员会的实验动物研究（NACLAR）的指导方针）。 1.鼠标<ol><li>订购一个鼠标适当的遗传背景。这个协议是基于组织从一个C57BL / 6小鼠衍生的。 </li></ol> 2.准备完全培养基<ol><li> 10％胎牛血清（FCS），50μM2-巯基乙醇，100微米的天冬酰胺?...

<ol>
	<li>Fitzpatrick, L. E., McDevitt, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-derived+matrices+for+tissue+engineering+and+regenerative+medicine+applications.">Cell-derived matrices for tissue engineering and regenerative medicine applications.</a> Biomater Sci. 3 (1), 12-24 (2015).</li><li>Elenbaas, B., 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=Human+breast+cancer+cells+generated+by+oncogenic+transformation+of+primary+epithelial+cells.">Human breast cancer cells generated by oncogenic transformation of primary epithelial cells.</a> Genes Dev. 15 (1), 50-65 (2001).</li><li>Stansley, B., Post, J., Hensley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+review+of+cell+culture+systems+for+the+study+of+microglial+biology+in+Alzheimer's+disease.">A comparative review of cell culture systems for the study of microglial biology in Alzheimer's disease.</a> J Neuroinflammation. 9 (1), 115 (2012).</li><li>Lim, J., Dobson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+transfection+of+HUVEC+and+MEF+cells+using+DNA+complexes+with+magnetic+nanoparticles+in+an+oscillating+field.">Improved transfection of HUVEC and MEF cells using DNA complexes with magnetic nanoparticles in an oscillating field.</a> J Genet. 91 (2), 223-227 (2012).</li><li>Li, M., 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=High-efficiency+transduction+of+fibroblasts+and+mesenchymal+stem+cells+by+tyrosine-mutant+AAV2+vectors+for+their+potential+use+in+cellular+therapy.">High-efficiency transduction of fibroblasts and mesenchymal stem cells by tyrosine-mutant AAV2 vectors for their potential use in cellular therapy.</a> Hum Gene Ther. 21 (11), 1527-1543 (2010).</li><li>Patel, M., Yang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+reprogramming+somatic+cells+to+induced+pluripotent+stem+cells.">Advances in reprogramming somatic cells to induced pluripotent stem cells.</a> Stem Cell Rev. 6 (3), 367-380 (2010).</li><li>Newman, A. C., Nakatsu, M. N., Chou, W., Gershon, P. D., Hughes, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+requirement+for+fibroblasts+in+angiogenesis:+fibroblast-derived+matrix+proteins+are+essential+for+endothelial+cell+luman+formation.">The requirement for fibroblasts in angiogenesis: fibroblast-derived matrix proteins are essential for endothelial cell luman formation.</a> Mol Biol Cell. 22 (20), 3791-3800 (2011).</li><li>Ohlund, D., Elyada, E., Tuveson, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+heterogeneity+in+the+cancer+wound.">Fibroblast heterogeneity in the cancer wound.</a> J Exp Med. 211 (8), 1503-1523 (2014).</li><li>Guo, S., Dipietro, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+affecting+wound+healing.">Factors affecting wound healing.</a> J Dent Res. 89 (3), 219-229 (2010).</li><li>Werner, S., Krieg, T., Smola, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keratinocyte-fibroblast+interactions+in+wound+healing.">Keratinocyte-fibroblast interactions in wound healing.</a> J Invest Dermatol. 127 (5), 998-1008 (2007).</li><li>Shen, Y. J., 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=Genome-derived+cytosolic+DNA+mediates+type+I+interferon-dependent+rejection+of+B+cell+lymphoma+cells.">Genome-derived cytosolic DNA mediates type I interferon-dependent rejection of B cell lymphoma cells.</a> Cell Rep. 11 (3), 460-473 (2015).</li><li>Seluanov, A., Vaidya, A., Gorbunova, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+primary+adult+fibroblast+cultures+from+rodents.">Establishing primary adult fibroblast cultures from rodents.</a> J Vis Exp. (44), (2010).</li><li>Baglole, C. J., 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=Isolation+and+phenotypic+characterization+of+lung+fibroblasts.">Isolation and phenotypic characterization of lung fibroblasts.</a> Methods Mol Med. 117, 115-127 (2005).</li><li>Alt, E., 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=Fibroblasts+share+mesenchymal+phenotypes+with+stem+cells,+but+lack+their+differentiation+and+colony-forming+potential.">Fibroblasts share mesenchymal phenotypes with stem cells, but lack their differentiation and colony-forming potential.</a> Biol Cell. 103 (4), 197-208 (2011).</li><li>Kuilman, T., Michaloglou, C., Mooi, W. J., Peeper, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+essence+of+senescence.">The essence of senescence.</a> Genes Dev. 24 (22), 2463-2479 (2010).</li><li>Lander, M. R., Moll, B., Rowe, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+procedure+for+culture+of+cells+from+mouse+tail+biopsies:+brief+communication.">A procedure for culture of cells from mouse tail biopsies: brief communication.</a> J Natl Cancer Inst. 60 (2), 477-478 (1978).</li><li>Moore, C. B., Allen, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+ear+fibroblast+derivation+from+mice.">Primary ear fibroblast derivation from mice.</a> Methods Mol Biol. (1031), 65-70 (2013).</li><li>Liu, J., 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=Generation+of+stable+pluripotent+stem+cells+from+NOD+mouse+tail-tip+fibroblasts.">Generation of stable pluripotent stem cells from NOD mouse tail-tip fibroblasts.</a> Diabetes. 60 (5), 1393-1398 (2011).</li></ol>

在这里，我们提供了一种简单，廉价且快速的实验步骤建立从耳朵和小鼠的尾巴初级成纤维细胞培养物。提取应导致粘附和迅速分裂成纤维细胞内后3天的隔离组织。原代细胞的一个重要的限制是衰老，一个永久生长停滞15。使用的协议，成纤维细胞培养可传代5-6次之前的成纤维细胞变得衰老，通过细胞的扁平化表示，尺寸增加（2-3倍增加）和故障扩大。 当执行的成纤...

主细胞来源于体外条件下活组织进行培养。它通常假设原代细胞更接近生理状态和其原始比永生化或肿瘤细胞系1的组织的遗传背景。出于这个原因，原代细胞代表用于研究生物学问题2,3的有用模型。然而，不同于无限增长建立的细胞系，原代细胞最终经历衰老中培养并需要经常重新建立。 常用的原代细胞包括成纤维细胞，上皮细胞，内皮细胞，T细胞，B细胞，骨髓衍生的巨噬细胞（BMDM）和骨髓来源的树突细胞（BMDC）。成纤维细胞通常用作原代细胞培养模型。它们提供超过其他初级细胞关键优势。细胞培养物容易建立，容易地维持和不需要purifica用前，培养细胞。他们有快速的初始扩散和专门的媒体或活化协议没有要求。成纤维细胞可以使用的生物，化学和物理协议4,5-有效地转染。有一种可能性，以存储耳朵之前建立的细胞培养物达10天室温。成纤维细胞培养有利于胞质突起的可视化和适于重新编程成诱导多能干细胞（iPS）6。 成纤维细胞是结缔组织的重要细胞分泌胶原蛋白和细胞外基质7。它们所提供的结构框架在许多组织8和播放在伤口愈合和组织修复9,10-必不可少的作用。 在这里，我们描述了一个简单而快速（&lt;4小时）协议来建立从耳朵成纤维细胞培养和小鼠11尾。该协议需要最少的鼠标经验收获组织（相对于其它协议12,13），并且可以用于建立从存储在介质在RT至10天耳朵培养物。

<table border="0" cellpadding="0" cellspacing="0" width="642">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI-1640</td>
			<td style="width:140px;">HyClone</td>
			<td style="width:119px;">SH30027.01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Calf Serum</td>
			<td style="width:140px;">HyClone</td>
			<td style="width:119px;">SV30160.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2-mercaptoethanol</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:119px;">M3148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Asparagine</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:119px;">A4159</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glutamine</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:119px;">G8540</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin/Streptomycin</td>
			<td style="width:140px;">HyClone</td>
			<td style="width:119px;">SV30010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:140px;">Merck Millipore</td>
			<td style="width:119px;">107017</td>
			<td>Absolute for analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Collagenase D</td>
			<td style="width:140px;">Roche Diagnostics</td>
			<td style="width:119px;">11088866001</td>
			<td>From Clostridium histolyticum, lyophilized, non-sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pronase protease</td>
			<td style="width:140px;">Merck Millipore</td>
			<td style="width:119px;">53702</td>
			<td>From Streptomyces griseus&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris buffer (pH 8)</td>
			<td style="width:140px;">1st BASE</td>
			<td style="width:119px;">1415</td>
			<td>Ultra pure grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.5M EDTA (pH 8)</td>
			<td style="width:140px;">1st BASE</td>
			<td style="width:119px;">BUF-1053</td>
			<td>Biotechnology grade</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">10X Phosphate Buffered Saline (PBS)</td>
			<td style="width:140px;">1st BASE</td>
			<td style="width:119px;">BUF-2040-10X4L</td>
			<td>Ultra pure grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin-EDTA solution 10X</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:119px;">59418C-100ML</td>
			<td>0.5% trypsin, 0.2% EDTA, trypsin gamma irradiated by SER-TAIN process, without phenol red, in saline</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Amphotericin B</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:119px;">A2492-20ml</td>
			<td>250 &#956;g/ml in deionized water, sterile-filtered</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scissors</td>
			<td style="width:140px;">Aesculap</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Forcep</td>
			<td style="width:140px;">Aesculap</td>
			<td style="width:119px;">AE-BD312R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.2 &#956;M syringe filter</td>
			<td style="width:140px;">Sartorius Stedim</td>
			<td style="width:119px;">16534</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">70 &#956;M cell strainer</td>
			<td style="width:140px;">SPL</td>
			<td style="width:119px;">93070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Syringe plunger</td>
			<td style="width:140px;">Terumo</td>
			<td style="width:119px;">SS+10L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cryovial tube</td>
			<td style="width:140px;">NUNC</td>
			<td style="width:119px;">368362</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1.7 ml microcentrifuge tube</td>
			<td style="width:140px;">Axygen</td>
			<td style="width:119px;">MCT-175-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10 cm cell culture dish</td>
			<td style="width:140px;">Greiner</td>
			<td style="width:119px;">664160</td>
			<td>Cell culture treated dish&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">15 ml conical bottom tube</td>
			<td style="width:140px;">Greiner</td>
			<td style="width:119px;">188271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50 ml conical bottom tube</td>
			<td style="width:140px;">Greiner</td>
			<td style="width:119px;">227261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Water bath</td>
			<td style="width:140px;">GFL</td>
			<td style="width:119px;">1002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge</td>
			<td style="width:140px;">Eppendorf</td>
			<td style="width:119px;">5810R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Incubation shaker</td>
			<td style="width:140px;">Sartorius Stedim</td>
			<td style="width:119px;"></td>
			<td>Certomat-BS1</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Zeiss Axiovert 25 light microscope</td>
			<td style="width:140px;">Carl Zeiss AG</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

generating primary fibroblast cultures from mouse ear and tail tissues

原代细胞是直接从组织来源和被认为是更具有代表性的细胞在体内比建立的细胞系的生理状态。然而，原代细胞培养，通常具有有限的寿命，需要频繁重新建立。成纤维细胞是原代细胞的一个方便的来源。在这里，我们讨论一个简单而快速的实验程序，以建立从耳朵和老鼠尾巴初级成纤维细胞培养。该协议可以用于建立初级成纤维细胞培养从存储在RT至10天耳朵。当协议是经过精心接着，污染物是不太可能发生尽管使用储存延长的时间在某些情况下，非无菌的组织。成纤维细胞增殖迅速在培养和接受复制衰老之前可扩展到相当数量。

来自组织的结果的成纤维细胞的一个显著量组织碎片的提取（ 图1）。在对比组织碎片，纤维母细胞附着到1天，3培养的间组织培养塑料表面。成纤维细胞培养物的培养基可以安全地变更上培养，其中应显著减少在培养（图2）的碎片本水平的3天。的成纤维细胞显示的细长形态和一个清晰可见的细胞质（图1和2）。有丝分裂的细胞应该从培养的第3?...

Watch this Scientific Journal Video about Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues at JoVE.com

Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues

我们描述用于产生从耳朵和小鼠的尾部的成纤维细胞初级一个简单而快速的实验程序。该过程不需要特殊的动物训练和可用于成纤维细胞培养的产生从存储在RT至10天耳朵。

从鼠耳和尾组织生成主成纤维细胞培养

Primary cells are derived directly from tissue and are thought to be more representative of the physiological state of cells in vivo than established cell ...

Research

JoVE Journal

Developmental Biology

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

在成年小鼠V-SVZ细胞的神经分析的稳定，高效的基因修饰干细胞自主和非自主的影响

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

科研

发育生物学

Murine Dermal Fibroblast Isolation by FACS

小鼠皮肤成纤维隔离用流式细胞仪

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast ...

Isolation of CD 90+ Fibroblast/Myofibroblasts from Human Frozen Gastrointestinal Specimens

从人类冷冻胃肠标本分离CD 90+成纤维细胞/肌成纤维细胞


Fibroblasts/myofibroblasts (MFs) have been gaining increasing attention for their role in pathogenesis and their contributions to both wound healing and ...

Using Primary Neurosphere Cultures to Study Primary Cilia

使用主神经球文化研究纤毛

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, ...

Generation and Culturing of Primary Human Keratinocytes from Adult Skin

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. ...

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

成人小鼠成纤维细胞对红细胞生成子细胞的直接线重编程

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell ...

Tissue Preparation and Immunostaining of Mouse Craniofacial Tissues and Undecalcified Bone

小鼠颅面组织和未切除骨骼的组织制备及免疫染色

Tissue immunostaining provides highly specific and reliable detection of proteins of interest within a given tissue. Here we describe a complete and ...

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

小鼠骨骼肌肉外泄原发性肌基器的分离和分化

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to ...

Clonal Genetic Tracing using the Confetti Mouse to Study Mineralized Tissues

使用纸屑小鼠研究矿化组织的克隆遗传追踪

Labeling an individual cell in the body to monitor which cell types it can give rise to and track its migration through the organism or determine its ...

Microwaving and Fluorophore-Tyramide for Multiplex Immunostaining on Mouse Adrenals &#8722; Using Unconjugated Primary Antibodies from the Same Host Species

Microwaving and Fluorophore-Tyramide for Multiplex Immunostaining on Mouse Adrenals ÃƒÂ¢Ã‹â€ ' Using Unconjugated Primary Antibodies from the Same Host Species

微挥和荧光素-提拉美化物，用于小鼠肾上腺的多重免疫染色 – 使用来自同一宿主物种的未结合原抗体

Immunostaining is widely used in biomedical research to show the cellular expression pattern of a given protein. Multiplex immunostaining allows labeling ...