Name: engineering a bilayered hydrogel to control asc differentiation
Uploaded: 2012-05-25T13:00:00
Description: Watch this Scientific Journal Video about Engineering a Bilayered Hydrogel to Control ASC Differentiation at JoVE.com

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1。分离脂肪干细胞（ASCs）1，5 注：所有的程序，在室温下进行，除非另有说明。 <ol><li>分离大鼠肾周和附睾脂肪用无菌汉克的缓冲盐溶液，含1％胎牛血清（FBS），（5先前所描述的HBSS中）。这项研究已进行符合动物福利法“，实施动物福利条例，并按照实验动物护理和使用指南”的原则。 </li><li>百果组织和转移到1-2克25毫升含有500克50毫升管?...

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	<li>Natesan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bilayer+construct+controls+adipose-derived+stem+cell+differentiation+into+endothelial+cells+and+pericytes+without+growth+factor+stimulation.">A bilayer construct controls adipose-derived stem cell differentiation into endothelial cells and pericytes without growth factor stimulation.</a> Tissue Eng. Part A. 17, 941-953 (2011).</li><li>Nuttelman, C. R., Tripodi, M. C., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+hydrogel+niches+that+promote+hMSC+viability.">Synthetic hydrogel niches that promote hMSC viability.</a> Matrix Biol. 24, 208-218 (2005).</li><li>Benoit, 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=Integrin-linked+kinase+production+prevents+anoikis+in+human+mesenchymal+stem+cells.">Integrin-linked kinase production prevents anoikis in human mesenchymal stem cells.</a> J Biomed Mater Res A. 81, 259-268 (2007).</li><li>Willerth, S. M., Sakiyama-Elbert, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+stem+cells+and+biomaterial+scaffolds+for+constructing+tissues+and+cell+delivery.">Combining stem cells and biomaterial scaffolds for constructing tissues and cell delivery.</a> , (2008).</li><li>Natesan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-derived+stem+cell+delivery+into+collagen+gels+using+chitosan+microspheres.">Adipose-derived stem cell delivery into collagen gels using chitosan microspheres.</a> Tissue Eng. Part A. 16, 1369-1384 (2010).</li><li>Bubnis, W. A., Ofner, M. 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+determination+of+epsilon-amino+groups+in+soluble+and+poorly+soluble+proteinaceous+materials+by+a+spectrophotometric+method+using+trinitrobenzenesulfonic+acid.">The determination of epsilon-amino groups in soluble and poorly soluble proteinaceous materials by a spectrophotometric method using trinitrobenzenesulfonic acid.</a> Anal. Biochem. 207, 129-133 (1992).</li><li>Zhang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+PEGylated+fibrin+patch+for+mesenchymal+stem+cell+delivery.">A PEGylated fibrin patch for mesenchymal stem cell delivery.</a> Tissue Eng. 12, 9-19 (2006).</li><li>Bornstein, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstituted+rattail+collagen+used+as+substrate+for+tissue+cultures+on+coverslips+in+Maximow+slides+and+roller+tubes.">Reconstituted rattail collagen used as substrate for tissue cultures on coverslips in Maximow slides and roller tubes.</a> Lab Invest. 7, 134-137 (1958).</li><li>Zhang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+differentiation+of+bone+marrow+stem+cells+is+directed+by+a+tunable+three-dimensional+matrix.">Vascular differentiation of bone marrow stem cells is directed by a tunable three-dimensional matrix.</a> Acta Biomater. 6, 3395-3403 (2010).</li><li>Rochon, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+human+epithelial+cells+regulate+the+size+and+morphology+of+tissue-engineered+capillaries.">Normal human epithelial cells regulate the size and morphology of tissue-engineered capillaries.</a> Tissue Eng. Part A. 16, 1457-1468 (2010).</li><li>Liu, H., Collins, S. F., Suggs, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+culture+for+expansion+and+differentiation+of+mouse+embryonic+stem+cells.">Three-dimensional culture for expansion and differentiation of mouse embryonic stem cells.</a> Biomaterials. 27, 6004-6014 (2006).</li></ol>

ASCs的是众所周知的，他们易于隔离和向多种细胞类型分化的能力。在这个手稿中描述的技术，我们能够利用这些细胞暴露多个biomatrices同时ASCs的可塑性。细胞迁移远离他们的CSM基地，并进入其周围的细胞外环境，细胞采取从断头台上的提示，并可以保持“干细胞”（胶原蛋白），或向血管和血管支持细胞类型（纤维蛋白）被诱导分化。由于我们的实验室是在皮肤及软组织伤口愈合的兴趣，我们?...

<table border="1"><tbody><tr><td> 试剂/设备名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>汉克斯平衡盐溶液（HBSS中） </td><td> Gibco公司</td><td> 14175 </td><td>耗材</td></tr><tr><td>胎牛血清</td><td>胎牛血清</td><td> SH30071.03 </td><td>耗材</td></tr><tr><td>二型胶原酶</td><td> Sigma-Aldrich公司</td><td> C6685 </td><td>耗材</td></tr><tr><td> 70微米尼龙网过滤器</td><td> BD公司</td><td> 352350 </td><td>耗材</td></tr><tr><td> 100μm的尼龙网过滤器</td><td> BD公司</td><td> 352360 </td><td>耗材</td></tr><tr><td> MesenPRO生长介质系统</td><td> Invitrogen公司</td><td> 12746-012 </td><td>耗材</td></tr><tr><td> L-谷氨酰胺</TD&gt; <td> Gibco公司</td><td> 25030 </td><td>耗材</td></tr><tr><td>氯化钙2·2H 2 O </td><td>西格玛</td><td> C8106 </td><td>耗材</td></tr><tr><td>的T75组织培养瓶</td><td> BD公司</td><td> 137787 </td><td>耗材</td></tr><tr><td>壳聚糖</td><td> Sigma-Aldrich公司</td><td> 448869 </td><td>耗材</td></tr><tr><td>醋酸</td><td> Sigma-Aldrich公司</td><td> 320099 </td><td>耗材</td></tr><tr><td>正辛醇</td><td> Acros Organics公司</td><td> 150630025 </td><td>耗材</td></tr><tr><td>山梨醇单声道，油酸</td><td> Sigma-Aldrich公司</td><td> S6760 </td><td>耗材</td></tr><tr><td>氢氧化钾</td><td> Sigma-Aldrich公司</td><td> P1767 </td><td>耗材</td></tr><tr><td>丙酮</td><td> Fisher Scientific则</td><td> L - 4859 </td><td>耗材</td></tr><tr><td>乙醇</td><td> Sigma-Aldrich公司</td><td> 270741 </td><td>耗材</td></tr><tr><td>硝基苯酸</td><td> Sigma-Aldrich公司</td><td> P2297 </td><td>耗材</td></tr><tr><td>盐酸</td><td> Sigma-Aldrich公司</td><td> 320331 </td><td>耗材</td></tr><tr><td>乙醚</td><td> Sigma-Aldrich公司</td><td> 472-484 </td><td>耗材</td></tr><tr><td> 8微米的细胞培养板插入</td><td> BD公司</td><td> 353097 </td><td>耗材</td></tr><tr><td> 1.5毫升离心管</td><td>费舍尔</td><td> 05-408-129 </td><td>耗材</td></tr><tr><td> MTT法试剂</td><td> Invitrogen公司</td><td> M6494 </td><td>耗材</td></tr><tr><td>二甲基亚砜</td><td> Sigma-Aldrich公司</td><td> D8779 </td><td>耗材</td></tr><tr><td> qtracker细胞标记试剂盒（问跟踪655） </td><td>分子探针</td><td> Q2502PMP </td><td>耗材</td></tr><tr><td> 1型胶原</td><td> travigen </td><td> 3447-020-01 </td><td>耗材</td></tr><tr><td>氢氧化钠</td><td> Sigma-Aldrich公司</td><td> S8045 </td><td>耗材</td></tr><tr><td> 12孔细胞培养板</td><td> BD公司</td><td> 353043 </td><td>耗材</td></tr><tr><td>纤维蛋白原</td><td>西格玛</td><td> F3879 </td><td>耗材</td></tr><tr><td>凝血酶</td><td>西格玛</td><td> T6884 </td><td>耗材</td></tr><tr><td> benztriazole衍生的聚乙烯</td><td> sunbio </td><td> DE-034GS </td><td>耗材</td></tr><tr><td>平板Tris缓冲（pH值7.6） </td><td>西格玛</td><td> T5030 </td><td>耗材</td></tr><tr><td>离心分离</td><td> Eppendorf公司</td><td> 5417R </td><td>马巴贝西pment </td></tr><tr><td>轨道摇床</td><td>新不伦瑞克省Scienctific </td><td> C24的</td><td>设备</td></tr><tr><td>湿润的孵化器与空气，5％的CO 2 </td><td> Thermo Scientific的</td><td> 370型</td><td>设备</td></tr><tr><td>架空搅拌器</td><td>伊嘉</td><td> visc6000 </td><td>设备</td></tr><tr><td>磁力搅拌器</td><td>康宁</td><td> PC-210 </td><td>设备</td></tr><tr><td>真空干燥器</td><td> - </td><td> - </td><td>设备</td></tr><tr><td>粒度分析仪</td><td>马尔文</td><td> STP2000 Spraytec </td><td>设备</td></tr><tr><td>水浴</td><td> Fisher Scientific则</td><td> isotemp210 </td><td>设备</td></tr><tr><td>分光光度计</td><td>贝克曼</td><td>贝克曼DU 800UV/Visible分光光度计</td><td>设备</td></tr><tr><td>涡旋</td><td> diagger </td><td> 3030A </td><td>设备</td></tr><tr><td>酶标仪</td><td>分子器件</td><td> SpectraMax M2的</td><td>设备</td></tr><tr><td>光/荧光显微镜</td><td>奥林巴斯</td><td> IX71 </td><td>设备</td></tr><tr><td>共聚焦显微镜</td><td>奥林巴斯</td><td> FV-500激光共聚焦显微镜</td><td>设备</td></tr><tr><td>扫描电子显微镜</td><td>卡尔蔡司显微成像</td><td>狮子座435副总裁</td><td>设备</td></tr><tr><td>透射电子显微镜</td><td> JEOL </td><td> JEOL 1230 </td><td>设备</td></tr></tbody></table>

engineering a bilayered hydrogel to control asc differentiation

多年来的天然聚合物获得了更多，因为它们的宿主的生物相容性和交互能力与细胞在体外和体内的重要性，认为在再生医学中的承诺的一个研究领域，是新型生物材料和干细胞的组合使用。在组织工程领域的一个基本战略是指导细胞的功能，使用三维支架（如脱细胞外基质，水凝胶，微/纳米颗粒）。这项技术已经从发现，细胞需要一个基板后，他们能够坚持，增殖，并表达他们分化 ​​的细胞表型和功能2-3。最近，它也已被确定，细胞不仅使用这些基板的坚持，但也互动，并采取从矩阵基板（例如，细胞外基质，细胞外基质）的线索。因此，细胞和支架有一个相互连接，为控制组织发展，组织，和最终功能。脂肪干细胞（ASCs）是间质，非造血干细胞存在于脂肪组织，可以表现出多谱系分化的细胞现成的源（即前血管内皮细胞和周细胞）作为。我们的假设是，脂肪干细胞可以向简单的合作，培养他们的不同表型，同时在双层矩阵1。我们的实验室专注于皮肤伤口愈合。为此，我们建立了从天然生物材料的复合材料基体，纤维蛋白，胶原蛋白，壳聚糖，可以模仿特定皮肤伤口愈合的ECM环境的特点和功能。

Watch this Scientific Journal Video about Engineering a Bilayered Hydrogel to Control ASC Differentiation at JoVE.com

Engineering a Bilayered Hydrogel to Control ASC Differentiation

该协议着重于利用干细胞的固有能力，其周围的细胞外基质的线索，被诱导分化成多个表型。这种方法手稿延伸我们描述一个利用双层水凝胶的模型，PEG-纤维蛋白和胶原蛋白的组成和特性，同时脂肪干细胞分化<sup&gt; 1</sup&gt;。

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